L-dopa administration enhances exocytotic dopamine release in vivo in the rat striatum.

Peripheral administration of L-3,4-dihydroxyphenylalanine (L-DOPA) methylester increased extracellular levels of DOPA and dopamine (DA) in the rat striatum monitored by in vivo brain microdialysis. The increase in DA levels persisted after inhibition of DA reuptake by nomifensine. Administration of blockers of voltage-dependent Na+ (tetrodotoxin) or Ca2+ (NKY-722) channels through the dialysis membrane completely eliminated the increase in DA levels. These results demonstrate that the L-DOPA-induced DA release is exocytotic in nature and hence, derived from neurons in the striatum.     

Enhancement of Dopamine Release In Vivo from the Rat Striatum by Dialytic Perfusion of 6R-l-erythro-5,6,7,8-Tetrahydrobiopterin

We have previously reported that intracerebroventricular administration of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4), a cofactor for tyrosine hydroxylase, enhances biosynthesis of 3,4-dihydroxyphenylethylamine (dopamine) in the rat brain. In the present study, we have more precisely examined the effects of 6R-BH4 on dopamine release in vivo from the rat striatum using brain microdialysis. The amount of dopamine collected in striatal dialysates was determined using HPLC with electrochemical detection after purification with an alumina batch method. When the striatum was dialyzed with Ringer solution containing various concentrations of 6R-BH4 (0.25, 0.5, and 1.0 mM), dopamine levels in striatal dialysates increased in a concentration-dependent manner. Biopterin had little effect on dopamine levels in dialysates. The 6R-BH4-induced increase in dopamine levels in dialysates was abolished after pretreatment with tetrodotoxin (50 microM) added to the perfusion fluid, but after pretreatment with nomifensine (100 mg/kg, intraperitoneal injection), an inhibitor of dopamine uptake mechanism, a larger increase was observed. After inhibition of tyrosine hydroxylase by pretreatment with alpha-methyl-p-tyrosine (250 mg/kg, intraperitoneal injection), most of the increase persisted. These results suggest that 6R-BH4 has a dopamine-releasing action, which is not dependent on biosynthesis of dopamine.     

Dopamine transporters are involved in the onset of hypoxia-induced dopamine efflux in striatum as revealed by in vivo microdialysis

Although many studies have revealed alterations in neurotransmission during ischaemia, few works have been devoted to the neurochemical effects of mild hypoxia, a situation encountered during life in altitude or in several pathologies. In that context, the present work was undertaken to determine the in vivo mechanisms underlying the striatal dopamine efflux induced by mild hypoxaemic hypoxia. For that purpose, the extracellular concentrations of dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid were simultaneously measured using brain microdialysis during acute hypoxic exposure (10% O₂, 1 h) in awake rats. Hypoxia induced a +80% increase in dopamine. Application of the dopamine transporters inhibitor, nomifensine (10 μM), just before the hypoxia prevented the rise in dopamine during the early part of hypoxia; in contrast the application of nomifensine after the beginning of hypoxia, failed to alter the increase in dopamine. Application of the voltage-dependent Na⁺ channel blocker tetrodotoxin abolished the increase in dopamine, whether administered just before or after the beginning of hypoxia. These data show that the neurochemical mechanisms of the dopamine efflux may change over the course of the hypoxic exposure, dopamine transporters being involved only at the beginning of hypoxia.     

Dopaminergic regulation of theta activity of septohippocampal neuron in the awake rabbit

The effects of dopamine reuptake blocker nomifensine and nonselective antagonist of dopamine receptors haloperidol on the theta rhythmicity of the medial septal neurons and hippocampal EEG were investigated in the rabbit. Bilateral intracerebroventricular infusion of nomifensine (9 micrograms in each ventriculus) produced an increase in both the rate of firing and the theta modulation of medial septal neurons; the theta power of the hippocampal EEG also augmented. The degree of neuronal theta stability (time constant of damping, tao theta) significantly increased. The frequency of rhythmic bursts in the neuronal firing also substantially elevated. The amplitude, regularity and frequency of theta waves in the hippocampal EEG also increased. The antagonist haloperidol (12.5 mg) caused the opposite effect. The theta activity of medial septal neurons and the theta power of the hippocampal EEG decreased after haloperidol injection. Theta rhythmicity of septal neurons significantly diminished, the rate of rhythmic bursts in the neuronal firing also decreased, although not substantially. The theta amplitude and regularity in the hippocampal EEG also decreased. Effects of both drugs built up rapidly and then gradually attenuated. Nomifensine infusion against the background of exposure to haloperidol provoked neither increasing neuronal firing rate, nor elevating theta activity. These finding suggest that dopaminergic system produces activation of the septohippocampal system in situations that require selective attention to functionally important information.     

Metabolic Responses of the Dopaminergic System during Hypoxia in Newborn Brain

The purpose of this review is to describe the relationship between the dopamine and amino acid neurotransmitter systems and cortical oxygen pressure during different levels of cerebral hypoxia using newborn piglets as an animal model, adding new data from our laboratory. The extracellular dopamine increases as the oxygen pressure in the cortex decreases. The relationship between oxygen pressure and dopamine levels is the same whether the hypoxia is induced by reduced F_iO₂ (high-flow hypoxia) or by hypocapnia-induced cerebral vasoconstriction (low-flow hypoxia). Thus it appears that, particularly in mild hypoxia, the extracellular level of dopamine depends primarily on the oxygen concentration in the tissue with minimal influence of parameters such as blood flow and pH. There is no "oxygen reserve" in the brain of newborn piglets and the extracellular levels of dopamine in the striatum increase almost linearly with decrease in oxygen pressure, with even small decreases in oxygen pressure resulting in increased dopamine levels. In contrast, the changes in extracellular concentrations of the excitatory amino acids glutamate and aspartate are variable and transient. In a majority of 2- to 5 day-old piglets even very low oxygen pressures in the brain did not result in significant alterations in the extracellular levels of glutamate and aspartate. These changes in the dopaminergic system may contribute directly and indirectly to the neuronal damage that occurs during hypoxic/ischemic insult and reoxygenation in newborn brain, particularly in the striatum. A variety of mechanisms are discussed by which dopamine, in particular extracellular dopamine, can increase cellular toxicity.     

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)     

Chronic treatment with a dopamine uptake blocker changes dopamine and acetylcholine but not glutamate and GABA concentrations in prefrontal cortex, striatum and nucleus accumbens of the awake rat

The present study was aimed to investigate the effects of a chronic treatment with the dopamine uptake blocker nomifensine on the in vivo extracellular concentrations of dopamine, acetylcholine, glutamate and GABA in the prefrontal cortex, striatum and nucleus accumbens. Male Wistar rats received intraperitoneal (i.p.) daily injections of nomifensine (10 mg/kg) or saline for 22 days. Microdialysis experiments were performed on days 1, 8, 15 and 22 of treatment to evaluate the effects of the injection of nomifensine or saline. Motor activity of the animals was monitored during microdialysis experiments. Injections of nomifensine increased extracellular concentration of dopamine in striatum and nucleus accumbens, but not in prefrontal cortex. Acetylcholine concentrations in striatum but not in nucleus accumbens were increased by nomifensine on days 15 and 22 of treatment. In prefrontal cortex, nomifensine increased acetylcholine levels without differences among days. No changes were found on glutamate and GABA concentrations in the three areas studied. Injections of nomifensine also increased spontaneous motor activity and stereotyped behaviour without differences among days. These results show that systemic chronic treatment with a dopamine uptake blocker produces differential effects on extracellular concentrations of dopamine and acetylcholine, but not glutamate and GABA, in different areas of the brain.     

Locally infused taurine, GABA and homotaurine alter differently the striatal extracellular concentrations of dopamine and its metabolites in rats

Summary We studiedin vivo the effects of locally infused taurine (50, 150, and 450 mM) on the striatal dopamine and its metabolites in comparison with those of GABA and homotaurine, a GABA_A receptor agonist, in freely moving rats. The extracellular dopamine concentration was elevated maximally 2.5-, 2- and 4-fold by taurine, GABA and homotaurine, respectively. At 150 mM concentration, at which the maximum effects occurred, homotaurine increased the extracellular dopamine more than taurine or GABA. When taurine and GABA were infused simultaneously with tetrodotoxin the output of dopamine did not differ from that in the presence of tetrodotoxin alone. In comparison, tetrodotoxin did not inhibit the increase in extracellular dopamine caused by homotaurine. Furthermore, omission of calcium from the perfusion fluid inhibited the increase of extracellular dopamine caused by GABA. However, it did not block the increase of dopamine caused by taurine or homotaurine. The present study suggests that the effects of intrastriatal taurine, GABA and homotaurine on the striatal extracellular dopamine differ. Thus, these amino acids seem to affect the striatal dopaminergic neurons via more than one mechanism.     

EFFECTS OF ELECTRICAL STIMULATION OF THE NIGROSTRIATAL PATHWAY OF THE RAT ON DOPAMINE METABOLISM

Abstract— Electrical stimulation of the nigrostriatal dopaminergic pathway in rat brain elicited a frequency and current intensity-dependent increased in the formation of homovanillic acid in the basal ganglia. The accumulation of the acid in probenecid-treated animals was constant over 1 h, when maximally stimulated at 25 Hz and 300 μA. Dopamine levels remained unchanged during stimulation. When prior to stimulation the inhibitor of catecholamine synthesis α-methyl- p -tyrosine methyl ester was administered, dopamine levels declined biphasically. Tyrosine and nomifensine, a dopamine uptake inhibitor, and apomorphine had no major effect on the formation of homovanillic acid, whilst α-methyl- p -tyrosine prevented its formation. Our data suggest that dopamine in the striatum is compartmentalized and that the newly-synthesized amine is released and converted to homovanillic acid. Apomorphine decreases dopamine flux only when dopaminergic neurons are at rest. When depolarized neither access of the precursor nor reuptake seem to influence the conversion of dopamine to homovanillic acid.     

Voltammetric study of the control of striatal dopamine release by glutamate

The central dopamine systems are involved in several aspects of normal brain function and are implicated in a number of human disorders. Hence, it is important to understand the mechanisms that control dopamine release in the brain. The striatum of the rat receives both dopaminergic and glutamatergic projections that synaptically target striatal neurons but not each other. Nevertheless, these afferents do form frequent appositional contacts, which has engendered interest in the question of whether they communicate with each other despite the absence of a direct synaptic connection. In this study, we used voltammetry in conjunction with carbon fiber microelectrodes in anesthetized rats to further examine the effect of the ionotropic glutamate antagonist, kynurenate, on extracellular dopamine levels in the striatum. Intrastriatal infusions of kynurenate decreased extracellular dopamine levels, suggesting that glutamate acts locally within the striatum via ionotropic receptors to regulate the basal extracellular dopamine concentration. Infusion of tetrodotoxin into the medial forebrain bundle or the striatum did not alter the voltammetric response to the intrastriatal kynurenate infusions, suggesting that glutamate receptors control a non-vesicular release process that contributes to the basal extracellular dopamine level. However, systemic administration of the dopamine uptake inhibitor, nomifensine (20 mg/kg i.p.), markedly decreased the amplitude of the response to kynurenate infusions, suggesting that the dopamine transporter mediates non-vesicular dopamine release. Collectively, these findings are consistent with the idea that endogenous glutamate acts locally within the striatum via ionotropic receptors to control a tonic, impulse-independent, transporter-mediated mode of dopamine release. Although numerous prior in vitro studies had suggested that such a process might exist, it has not previously been clearly demonstrated in an in vivo experiment.     

Combined Microdialysis and Fos Immunohistochemistry for the Estimation of Dopamine Neurotransmission in the Rat Caudate-Putamen

Extracellular dopamine (DA) concentrations estimated by transcerebral dialysis and D1-dependent c-fos expression, as demonstrated by Fos immunohistochemistry, were studied after blockade of DA reuptake by GBR-12909. Rats implanted with dialysis probes in the dorsal caudate-putamen did not show any Fos-positive neuronal labeling in the implanted area or in the rest of the caudate-putamen. Administration of GBR-12909 dose-dependently increased DA output in dialysates and resulted in the appearance in the caudate-putamen of Fos-positive neurons whose density was related to the dose of GBR-12909 and to the increase in extracellular concentrations of DA. The D1 antagonist SCH-23390 blocked GBR-12909-induced activation of Fos while potentiating the stimulation of DA output. The results show that following blockade of DA reuptake by GBR-12909, the induction of Fos is related to stimulation of D1 receptors by extracellular DA. Combination of brain dialysis with Fos immunohistochemistry might provide a method for estimating the functional significance of extracellular DA as measured by brain microdialysis.     

Involvement of Nitric Oxide in Dopaminergic Transmission in Rat Striatum: An In Vivo Electrochemical Study

Abstract: In vivo electrochemical detection with a Nafion-coated carbon fiber working electrode, which provides information on the spatial and temporal dynamics of dopamine overflow, was used to investigate the involvement of nitric oxide (NO) in the dopaminergic transmission in the striatum of urethane-anesthetized Sprague-Dawley rats. A mixture of N -methyl- d -aspartate (NMDA) and nomifensine, a dopamine uptake blocker, was locally pressure-ejected to elicit a transient dopamine overflow from the dopamine-containing nerve terminals in the striatum. Local application of N ^ω-nitro- l -arginine methyl ester ( l -NAME), which blocks endogenous NO formation, increased the magnitude of dopamine release evoked by a subsequent NMDA and nomifensine application but resulted in no significant alteration in the time course. Furthermore, microejection of l -arginine, an NO precursor, or sodium nitroprusside (SNP), an NO generator, did not cause detectable changes in dopamine level in the striatal extracellular space. However, NMDA-induced dopamine release was profoundly inhibited with l -arginine or SNP pretreatment. In addition, NO affects dopamine uptake in rat striatum. Exogenous dopamine applied through a micropipette, reversibly and reproducibly, elicited an electrochemical signal. The time course of these signals was significantly prolonged by l -NAME treatment. These data suggest that NO is diversely involved in regulating dopaminergic transmission in rat striatum.     

Nomifensine amplifies subsecond dopamine signals in the ventral striatum of freely-moving rats

The present experiment examined the effect of the dopamine transporter blocker nomifensine on subsecond fluctuations in dopamine concentrations, or dopamine transients, in the nucleus accumbens and olfactory tubercle. Extracellular dopamine was measured in real time using fast-scan cyclic voltammetry at micron-dimension carbon fibers in freely-moving rats. Dopamine transients occurred spontaneously throughout the ventral striatum in the absence of apparent sensory input or change in behavioral response. The frequency of dopamine transients increased at the presentation of salient stimuli to the rat (food, novel odors and unexpected noises). Administration of 7 mg/kg nomifensine amplified spontaneous dopamine transients by increasing both amplitude and duration, consistent with its known action at the dopamine transporter and emphasizing the dopaminergic origin of the signals. Moreover, nomifensine increased the frequency of detected dopamine transients, both during baseline conditions and at the presentation of stimuli, but more profoundly in the nucleus accumbens than in the olfactory tubercle. This difference was not explained by nomifensine effects on the kinetics of dopamine release and uptake, as its effects on electrically-evoked dopamine signals were similar in both regions. These findings demonstrate the heterogeneity of dopamine transients in the ventral striatum and establish that nomifensine elevates the tone of rapid dopamine signals in the brain.     

Mechanism of action of methylmercury on in vivo striatal dopamine release. Possible involvement of dopamine transporter

Methylmercury (MeHg) produces significant increases in the spontaneous output of dopamine (DA) from rat striatal tissue. The mechanism through MeHg produces such increase in the extracellular DA levels could be due to increased DA release or decreased DA uptake into DA terminals. One of the aims of this study was to investigate the role of DA transporter (DAT) in the MeHg-induced DA release. Coinfusion of 400 microM MeHg and nomifensine (50 microM) or amphetamine (50 microM) produced increases in the release of DA similar to those produced by nomifensine and amphetamine alone. In the same way, MeHg-induced DA release was not attenuated under Ca(2+)-free conditions or after pretreatment with reserpine (10 mg/kg i.p.) or tetrodotoxin (TTX), suggesting that the DA release was independent of calcium and vesicular stores, as well as it was not affected by the blockade of voltage sensitive sodium channels. Thus, to investigate whether depolarization of dopaminergic terminal was able to affect MeHg-induced DA release, we infused 75 mM KCl through the dialysis membrane. Our results clearly showed a decrease induced by MeHg in the KCl-evoked DA release. Taken together, these results suggest that MeHg induces release of DA via transporter-dependent, calcium- and vesicular-independent mechanism and it decreases the KCl-evoked DA release.     

In vivo characterization of somatodendritic dopamine release in the substantia nigra of 6-hydroxydopamine-lesioned rats

We investigated the effect of an injection of 6-hydroxydopamine (6-OHDA) into the rat medial forebrain bundle (MFB) on the degeneration and the function of the dopaminergic cell bodies in the substantia nigra (SN) 3 and 5 weeks after lesioning. After injection of 6-OHDA into the MFB a complete loss of dopamine content was apparent in the striatum 3 weeks after lesioning. In the SN the amount of tyrosine hydroxylase-immunoreactive dopamine cells decreased gradually, with a near-complete lesion (> 90%) obtained only after 5 weeks, indicating that neurodegeneration of the nigral cells was still ongoing when total dopamine denervation of the striatum had already been achieved. Baseline dialysate and extracellular dopamine levels in the SN, as determined by in vivo microdialysis, were not altered by the lesion. A combination of compensatory changes of the remaining neurones and dopamine originating from the ventral tegmental area may maintain extracellular dopamine at near-normal levels. In both intact and lesioned rats, the somatodendritic release was about 60% tetrodotoxin (TTX) dependent. Possibly two pools contribute to the basal dopamine levels in the SN: a fast sodium channel-dependent portion and a TTX-insensitive one originating from diffusion of dopamine. Amphetamine-evoked dopamine release and release after injection of the selective dopamine reuptake blocker GBR 12909 were attenuated after a near-complete denervation of the SN (5 weeks after lesioning). So, despite a 90% dopamine cell loss in the SN 5 weeks after an MFB lesion, extracellular dopamine levels in the SN are kept at near-normal levels. However, the response to a pharmacological challenge is severely disrupted.     

多巴胺重摄取的分子机制

多巴胺转运体(Dopamine transporter,DAT)位于多巴胺能神经元表面,主要负责将细胞外的多巴胺重摄取至多巴胺能神经元内,控制细胞外多巴胺的浓度,进而影响多巴胺的信号强度和时长。多巴胺转运体与注意力缺陷多动症、抑郁、成瘾等中枢神经系统的功能异常相关。多巴胺转运体的重摄取功能受多种因素的调节,包括底物的浓度、自身位点的翻译后修饰、细胞内蛋白激酶的活性、细胞外的调节信号等。本文就近年来DAT的分子调节机制以及在脑疾病发病机制中作用的研究进展做一综述。     

The electrophysiological actions of dopamine and dopaminergic drugs on neurons of the substantia nigra pars compacta and ventral tegmental area.

The dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area play a crucial role in regulating movement and cognition respectively. Several lines of evidence suggest that a degeneration of dopaminergic cells in the substantia nigra produces the symptoms of Parkinson's disease. On the other hand, a hyperactivity of the dopaminergic transmission in the brain induces dyskinesia, dystonia and psychosis. It is also well established that the euphoric and rewarding responses evoked by drugs of addiction, such as amphetamine and cocaine, are mediated by central dopamine systems. Electrophysiological experiments which study the activity of single dopaminergic neurons in the ventral mesencephalon have shown that dopamine and dopaminergic drugs reduce the firing frequency of these cells. This is due to the stimulation of D2-D3 autoreceptors and to a hyperpolarization of the membrane produced by an increase in potassium conductance. In addition, substances which increase the release (amphetamine), the synthesis (levodopa) or block the uptake (cocaine, nomifensine, amineptine) of dopamine in the brain inhibit the firing activity of the dopaminergic cells throughout dopamine-mediated mechanisms. In this review, we will briefly examine the literature concerning the physiological and behavioural responses caused by dopamine and dopaminergic agents on the dopaminergic neurons of the ventral mesencephalon. Our conclusion suggests that the electrophysiological actions of dopamine and dopamine-related drugs on dopaminergic cells in the ventral mesencephalon might be indicative of the pharmacological effects of these agents on the brain.     

Effect of direct and indirect dopamine agonists on brain extracellular ascorbate levels in the striatum and nucleus accumbens of awake rats

Systemic administration of direct and indirect dopamine agonists resulted in increased extracellular ascorbic acid levels in the striatum and, to a lesser degree, in the nucleus accumbens as measured by in vivo voltammetry. Intraperitoneal d-amphetamine sulfate (5mg/kg) increased ascorbate concentrations in striatal extracellular fluid. Amphetamine also increased extracellular ascorbate levels in the nucleus accumbens although more gradually and to a lesser extent. Intraperitoneal phenethylamine hydrochloride (20 mg/kg) following pargyline hydrochloride pretreatment (20 mg/kg) increased extracellular ascorbate levels in the striatum significantly above the small increase seen in the nucleus accumbens. The direct acting dopamine agonists Ly-141865 and Ly-163502 when given i.p. at 1 mg/kg, resulted in increased extracellular ascorbate concentrations in both brain areas, again with a significantly greater effect in the striatum. These results indicate that brain extracellular ascorbate levels can be modulated by dopaminergic neuro-transmission and that this modulation is quantitatively different in different dopamine-containing brain structures.     

Evidence for a functional coupling between dopamine reuptake and tyrosine hydroxylation in striatal nerve terminals

In rat striatal synaptosomes incubated with [¹⁴C]tyrosine, the evolution of ¹⁴CO₂, taken as a measure of dopamine synthesis, was inhibited by exogenous dopamine and by the dopaminergic receptor agonist ADTN. The inhibition was not counteracted by dopaminergic receptor antagonists (haloperidol, sulpiride, pimozide or domperidone). Instead, it was prevented by dopamine uptake blockers, suggesting that dopamine and ADTN (a substrate of the dopamine carrier) acted once inside the nerve endings and not through activation of autoreceptors on their external membrane. The dopamine uptake inhibitors nomifensine, benztropine and cocaine increased ¹⁴CO₂ evolution from incubated striatal synaptosomes. Depolarization with KCl also increased dopamine synthesis and this action was potentiated when the reuptake of the released catecholamine was prevented by carrier blockers. The rate of dopamine synthesis was lowered when synaptosomal dopamine was raised upon incubation with monoamine oxidase inhibitors or with l-DOPA. The inhibition was counteracted by dopamine reuptake blockers. The data suggest that dopamine synthesis in striatal nerve endings is under the inhibitory control of the transmitter recaptured following release.