Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.     

Tonic autoinhibition contributes to the heterogeneity of evoked dopamine release in the rat striatum

Electrically evoked dopamine release as measured by voltammetry in the rat striatum is heterogeneous in both amplitude and temporal profile. Previous studies have attributed this heterogeneity to variations in the density of dopamine (DA) terminals at the recording site. We reach the alternate conclusion that the heterogeneity of evoked DA release derives from variations in the extent to which DA terminals are autoinhibited. We demonstrate that low-amplitude, slow evoked DA responses occur even though recording electrodes are close to DA terminals. Moreover, the D₂ agonist and antagonist, quinpirole and raclopride, respectively, affect the slow responses in a manner consistent with the known functions of pre-synaptic D₂ autoreceptors. Recording sites that exhibit autoinhibited responses are prevalent in the dorsal striatum. Autoinhibition preceded electrical stimulation, which is consistent with our prior reports that the striatum contains a tonic pool of extracellular DA at basal concentrations that exceed the affinity of D₂ receptors. We conclude that the striatum contains DA terminals operating on multiple time courses, determined at least in part by the local variation in autoinhibition. Thus, we provide direct, real-time observations of the functional consequence of tonic and phasic DAergic signaling in vivo .     

Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma

Although microdialysis is widely used to sample endogenous and exogenous substances in vivo, interpretation of the results obtained by this technique remains controversial. The goal of the present study was to examine recent criticism of microdialysis in the specific case of dopamine (DA) measurements in the brain extracellular microenvironment. The apparent steady-state basal extracellular concentration and extraction fraction of DA were determined in anesthetized rat striatum by the concentration difference (no-net-flux) microdialysis technique. A rate constant for extracellular clearance of DA calculated from the extraction fraction was smaller than the previously determined estimate by fast-scan cyclic voltammetry for cellular uptake of DA. Because the relatively small size of the voltammetric microsensor produces little tissue damage, the discrepancy between the uptake rate constants may be a consequence of trauma from microdialysis probe implantation. The trauma layer has previously been identified by histology and proposed to distort measurements of extracellular DA levels by the no-net-flux method. To address this issue, an existing quantitative mathematical model for microdialysis was modified to incorporate a traumatized tissue layer interposed between the probe and surrounding normal tissue. The tissue layers are hypothesized to differ in their rates of neurotransmitter release and uptake. A post-implantation traumatized layer with reduced uptake and no release can reconcile the discrepancy between DA uptake measured by microdialysis and voltammetry. The model predicts that this trauma layer would cause the DA extraction fraction obtained from microdialysis in vivo calibration techniques, such as no-net-flux, to differ from the DA relative recovery and lead to an underestimation of the DA extracellular concentration in the surrounding normal tissue.     

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.     

An inverse correlation between the apparent rate of dopamine clearance and tonic autoinhibition in subdomains of the rat striatum: a possible role of transporter-mediated dopamine efflux

The dopaminergic terminal field in the rat striatum is compartmentalized into sub-domains that exhibit distinct dynamics of electrically evoked dopamine release. The fast striatal domains, where dopamine release is predominantly vesicular, exhibit conventional dopaminergic activity. However, vesicular dopamine release is tonically autoinhibited in the slow domains, which suggests that dopamine reaches the autoreceptors via a non-vesicular route. Hence, it appears that the domains use distinct mechanisms to regulate the basal dopamine concentration available to activate, or not, pre-synaptic autoinhibitory receptors. However, direct detection of local variations in tonic extracellular dopamine concentrations is not yet possible. So, the present study employed voltammetry to test the hypothesis that the apparent rate of dopamine clearance from the extracellular space should be domain-dependent. The apparent rate of dopamine clearance is equal to the difference in the rates of dopamine release and uptake that determine extracellular dopamine concentrations. This study confirms that the apparent rate of dopamine clearance is slower in the slow striatal domains where vesicular dopamine release is tonically autoinhibited. These findings support the view that the basal concentration in slow domains is maintained by a non-vesicular release process, possibly transporter-mediated efflux.     

Nicotine increases dopamine clearance in medial prefrontal cortex in rats raised in an enriched environment

Environmental enrichment results in differential behavioral and neurochemical responsiveness to nicotine. The present study investigates dopamine clearance (CL_DA) in striatum and medial prefrontal cortex (mPFC) using in vivo voltammetry in rats raised in enriched (EC) or impoverished conditions (IC) and administered nicotine (0.4 mg/kg) or saline. Baseline CL_DA in striatum or mPFC was not different between EC and IC. Across repeated DA application, striatal CL_DA increased in saline-control EC and IC. CL_DA increased in mPFC in saline-control IC; CL_DA did not change in saline-control EC. Thus, enrichment differentially alters dynamic responses of the dopamine transporter (DAT) to repeated DA application in mPFC, but not in striatum. In EC, nicotine increased mPFC CL_DA compared to saline-control, but had no effect on CL_DA in IC; nicotine had no effect in striatum in EC or IC. Compared to respective saline-controls, nicotine increased dihydroxyphenylacetic acid content in striatum and mPFC in EC, but not in IC. Nicotine also had no effect on DA content in striatum or mPFC in EC or IC. Results indicate that enrichment eliminated the dynamic response of mPFC DAT to repeated DA application in saline-control and augmented the nicotine-induced increase in DAT function in mPFC, but not in striatum.     

Unc13A dynamically stabilizes vesicle priming at synaptic release sites for short-term facilitation and homeostatic potentiation

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Ischemic flow threshold for striatal dopamine release in rats

To determine the level of cerebral blood flow reduction which causes striatal dopamine release, extracellular dopamine and cerebral blood flow was simultaneously determined using in vivo brain dialysis and a hydrogen clearance method, respectively, in the striatum of spontaneously hypertensive rats, before and during experimental cerebral ischemia. The ischemic flow threshold for neurotransmitter dopamine release was found to be 20% of the resting value or 8–10 ml/100g/min of cerebral blood flow, being similar to those for energy and membrane failures.     

Optogenetic versus electrical stimulation of dopamine terminals in the nucleus accumbens reveals local modulation of presynaptic release

The nucleus accumbens is highly heterogeneous, integrating regionally distinct afferent projections and accumbal interneurons, resulting in diverse local microenvironments. Dopamine (DA) neuron terminals similarly express a heterogeneous collection of terminal receptors that modulate DA signaling. Cyclic voltammetry is often used to probe DA terminal dynamics in brain slice preparations; however, this method traditionally requires electrical stimulation to induce DA release. Electrical stimulation excites all of the neuronal processes in the stimulation field, potentially introducing simultaneous, multi‐synaptic modulation of DA terminal release. We used optogenetics to selectively stimulate DA terminals and used voltammetry to compare DA responses from electrical and optical stimulation of the same area of tissue around a recording electrode. We found that with multiple pulse stimulation trains, optically stimulated DA release increasingly exceeded that of electrical stimulation. Furthermore, electrical stimulation produced inhibition of DA release across longer duration stimulations. The GABA_B antagonist, CGP 55845, increased electrically stimulated DA release significantly more than light stimulated release. The nicotinic acetylcholine receptor antagonist, dihydro‐β‐erythroidine hydrobromide, inhibited single pulse electrically stimulated DA release while having no effect on optically stimulated DA release. Our results demonstrate that electrical stimulation introduces local multi‐synaptic modulation of DA release that is absent with optogenetically targeted stimulation.

     

Non-genomic modulation of dopamine release by bisphenol-A in PC12 cells

An endocrine disruptor chemical, bisphenol-A (BPA), is reported to have several short-term actions in various tissues and/or cells; however, the mechanisms of these actions have not been fully elucidated. We investigated short-term actions evoked by BPA in pheochromocytoma PC12 cells. BPA elicited dopamine release in PC12 cells in a dose-dependent manner. A selective N-type calcium channel antagonist (omega-conotoxin GVIA) and a ryanodine receptor blocker (ryanodine) inhibited the BPA-induced dopamine release. The expression of ryanodine receptor mRNA was detected by RT-PCR in PC12 cells. Subsequently, in order to prove whether membrane receptors participate in BPA-evoked dopamine release, a guanine nucleotide-binding protein inhibitor [guanosine 5'-(beta-thio) diphosphate], cyclic AMP antagonist (Rp-cAMPS) or protein kinase A inhibitor (H7 or H89) was added to PC12 cells prior to BPA-treatment. All of these agents suppressed BPA-evoked dopamine release, indicating that multiple signaling pathways may be involved in BPA-evoked dopamine release in PC12 cells. In conclusion, we demonstrated that BPA induced dopamine release in a non-genomic manner through guanine nucleotide-binding protein and N-type calcium channels. These findings illustrate a novel function of BPA and suggest that exposure to BPA influences the function of dopaminergic neurons.     

In vivo monitoring of brain neurotransmitter release for the assessment of neuroendocrine interactions

Summary 1. The neurotransmitter mechanisms regulating neuroendocrine processes have been traditionally inferred from the effects of drugs purportedly acting through specific transmitter systems. The direct appraisal of changes in endogenous neuromediators had to rely initially on analyses of brain samples obtained post-morten.2. Currently, a more physiological assessment is available through the monitoring ot the extracellular levels of neurotransmitters and their metabolites in discrete brain areas of living animals. Two methodologies, namely in vivo voltammetry and microdialysis, are being increasingly used for this purpose. This article summarizes their principles, relative merits, and limitations and presents some relevant applications.3. Thus, microdialysis data show a differential response in the amphetamine-induced dopamine release in the nucleus accumbens in adult male and female rats castrated prepuberally. Given their high time-resolution, in vivo electrochemistry techniques seem especially suited for studying the fast, non-genomic effects of steroid hormones. This is illustrated by the voltammetric detection of a rapid release of dopamine in the corpus striatum induced by progesterone in males.4. These methodologies should be regarded as complementary tools for the assessment of the neurochemical correlates of neuroendocrine interactions.     

Characterization of the striatal dopaminergic neurotransmission in MEN2B mice with elevated cerebral tissue dopamine

The Ret receptor tyrosine kinase is the common signaling receptor for the glial cell line-derived neurotrophic factor (GDNF) family ligands. The Met918Thr mutation leads to constitutive activation of Ret and is responsible for dominantly inherited cancer syndrome MEN2B. Previously, we found that the mice carrying the mutation (MEN2B mice) have profoundly increased tissue dopamine (DA) concentrations in the striatum as well as increased striatal levels of tyrosine hydroxylase (TH) and dopamine transporter. The aim of this study was to characterize the striatal dopaminergic neurotransmission in MEN2B mice and to clarify the mechanisms by which they compensate their over-production of DA. We found that tyrosine hydroxylase activity and DA synthesis are increased in MEN2B mice. Augmented effects of α-methyl-para-tyrosine (αMT, an inhibitor of TH) and tetrabenazine (VMAT2 blocker) on DA levels suggest that also storage of DA is increased in MEN2B mice. There was no difference in the basal extracellular DA concentrations or potassium-evoked DA release between the genotypes. The effects of cocaine and haloperidol were also similar between the genotypes as assessed by in vivo microdialysis. However, with in vivo voltammetry we found increase in stimulated DA release in MEN2B mice and detailed analysis of DA overflow showed that uptake of DA was also enhanced in MEN2B mice. Thus, our data show that enhanced synthesis of DA leading to increased storage and releasable pools in pre-synaptic terminals in MEN2B mice apparently also leads to increased DA release, which in turn is compensated by higher dopamine transporter activity.     

Selective stimulation of serotonin2c receptors blocks the enhancement of striatal and accumbal dopamine release induced by nicotine administration

The effects of acute and repeated nicotine administration on the extracellular levels of dopamine (DA) in the corpus striatum and the nucleus accumbens were studied in conscious, freely moving rats by in vivo microdialysis. Acute intraperitoneal (i.p.) injection of nicotine (1 mg/kg) increased DA outflow both in the corpus striatum and the nucleus accumbens. Repeated daily injection of nicotine (1 mg/kg, i.p.) for 10 consecutive days caused a significant increase in basal DA outflow both in the corpus striatum and the nucleus accumbens. Acute challenge with nicotine (1 mg/kg, i.p.) in animals treated repeatedly with this drug enhanced DA extracellular levels in both brain areas. However, the effect of nicotine was potentiated in the nucleus accumbens, but not in the corpus striatum. To test the hypothesis that stimulation of 5-HT (5-hydroxytryptamine, serotonin)(2C) receptors could affect nicotine-induced DA release, the selective 5-HT(2C) receptor agonist RO 60-0175 was used. Pretreatment with RO 60-0175 (1 and 3 mg/kg, i.p.) dose-dependently prevented the enhancement in DA release elicited by acute nicotine in the corpus striatum, but was devoid of any significant effect in the nucleus accumbens. RO 60-0175 (1 and 3 mg/kg, i.p.) dose-dependently reduced the stimulatory effect on striatal and accumbal DA release induced by an acute challenge with nicotine (1 mg/kg, i.p.) in rats treated repeatedly with this alkaloid. However, only the effect of 3 mg/kg RO 60-0175 reached statistical significance. The inhibitory effect of RO 60-0175 on DA release induced by nicotine in the corpus striatum and the nucleus accumbens was completely prevented by SB 242084 (0.5 mg/kg, i.p.) and SB 243213 (0.5 mg/kg, i.p.), two selective antagonists of 5-HT(2C) receptors. It is concluded that selective activation of 5-HT(2C) receptors can block the stimulatory action of nicotine on central DA function, an effect that might be relevant for the reported antiaddictive properties of RO 60-0175.     

Regulation of nucleus accumbens dopamine release by the dorsal raphe nucleus in the rat

The effects of microinfusingl-glutamate, serotonin (5-HT), (±)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH DPAT; a 5-HT_1A agonist), and muscimol (a GABA_A agonist) into the dorsal raphe nucleus on the extracellular levels of 5-HT, dopamine (DA) and their metabolites in the nucleus accumbens were studied in unanesthetized, freely moving, adult male Wistar rats, using the technique of microdialysis coupled with small-bore HPLC. Administration of 0.75 gl-glutamate produced a 25–50% increase (P<0.05) in the extracellular levels of both 5-HT and DA. On the other hand, infusion of 8-OH DPAT and, to a lesser extent, 5-HT produced a significant (P<0.05) decrease in the extracellular levels of both 5-HT and DA. Muscimol (0.25 or 0.50 g) had little effect on the extracellular concentrations of 5-HT or DA following its administration. In general, the extracellular levels of the major metabolites of 5-HT and DA in the nucleus accumbens were not altered by microinfusion of any of the agents. The data indicate that (a) the 5-HT neurons projecting to the nucleus accumbens from the dorsal raphe nucleus can be activated by excitatory amino acid receptors and inhibited by stimulation of 5-HT_1A autoreceptors, and (b) the dorsal raphe nucleus 5-HT neuronal system may regulate the ventral tegmental area DA projection to the nucleus accumbens.Special issue dedicated to Dr. Morris H. Aprison     

Adenosine modulation of synaptosomal dopamine release.

The effects of adenosine and its analog 2-chloroadenosine on release of preloaded [³H]-dopamine from striatal synaptosomes was explored. Both adenosine and 2-chloroadenosine were found to decrease the amount of dopamine released both by depolarization (with KCl) and by amphetamine. Addition of exogenous adenosine deaminase enhanced dopamine release above controls, and blockade of the endogenous adenosine deaminase activity with deoxycoformycin resulted in a decrease in dopamine release. The methylxanthines, believed to be adenosine antagonists, inhibited dopamine release by an unknown mechanism, and hence it was impossible to evaluate antagonism of the inhibitory effects of adenosine by these agents. The depolarization-induced release of dopamine appeared to be more sensitive to the actions of adenosine than was the amphetamine-induced release. The data obtained so far seem to indicate that adenosine is capable of modulating the release of transmitter substances in brain tissue in a manner analogous to that which has been observed in the peripheral nervous system.     

Dopamine release mediated by the dopamine transporter, facts and consequences

Spontaneous and/or stimulated neural activity of the nigrostriatal dopamine (DA) pathway makes amines run out from the neurons. This DA dynamic follows a rather complex path, running in or out the terminals, and flushing or diffusing into the extracellular space. The location of this leakage is not limited to the axon terminals; it also occurs from the cell bodies and dendrites. This molecular release mechanism was, for a long time, considered as being produced, in part, by the exocytosis of previously stored vesicles. The DA carrier protein (DAT, DA transporter) embedded in the DA cell membrane is known to clear previously released amines through an inward DA influx. The DAT also appears to be an active vector of amine release. Particular local conditions and the presence of numerous psychostimulant substances are able to trigger an outward efflux of DA through the DAT. This process, delivering slowly large amounts of amine could play a major regulatory role in extracellular DA homeostasis.     

The ontogeny of apomorphine-induced alterations of neostriatal dopamine release: Effects on potassium-evoked release

The effects of apomorphine (0.05, 0.1, and 1.0 mg/kg, s.c.) on K⁺-evoked dopamine release were studied through the use of in vivo microdialysis in the neostriatum of developing and adult rats. Fifteen-minute samples were collected from urethane-anesthetized rats 5, 10–11, 21–22, 35–36 days of age, and adults, and quantified by high performance liquid chromatography with electrochemical detection. Apomorphine attenuated K⁺-evoked dopamine release in all age groups, suggesting that the dopamine autoreceptor modulating release in the neostriatum is functional by 5 days of age. A dose-response effect of apomorphine was observed in all age groups except at 5 and 10 days of age. Absolute levels of extracellular dopamine were significantly lower at 5 and 10 days of age compared with the other ages, and the effectiveness of a high-K⁺ artificial cerebrospinal fluid to evoke dopamine release increased with age.     

Changes in the kinetics of dopamine release and uptake have differential effects on the spatial distribution of extracellular dopamine concentration in rat striatum

The objective of this study was to examine whether the limited diffusion distance of dopamine in rat striatum produces spatial heterogeneity in the extracellular dopamine concentration on a dimensional scale of a few micrometers. Such heterogeneity would be significant because it would imply that the concentration of dopamine at a given receptor depends on the receptor's ultrastructural location. Spatially resolved measurements of extracellular dopamine were performed in the striatum of chloral hydrate-anesthetized rats with carbon fiber microdisk electrodes. Dopamine was monitored during electrical stimulation of the nigrostriatal pathway before and after administration of drugs that selectively affect the kinetics of evoked dopamine release and dopamine uptake. The effects of nomifensine (20 mg/kg), L-DOPA (250 mg/kg), and alpha-methyl-p-tyrosine (250 mg/kg) on the amplitude of the stimulation responses were examined. The outcome of these experiments was compared with predictions derived from a mathematical model that combines diffusion with the kinetics of release and uptake. The results demonstrate that the extracellular dopamine concentration is spatially heterogeneous on a micrometer scale and that changing the kinetics of dopamine release and uptake has different effects on this spatial distribution. The impact of these results on brain neurochemistry is considered.