Neurotensin facilitates dopamine release in vitro from rat striatal slices

Neurotensin, (0.1-10 microM) stimulated the release of [3H]dopamine from rat striatal slices in a calcium-dependent manner, and potentiated the K+-evoked release of [3H]dopamine and endogenous dopamine. This effect was dose-dependent, (1 nM-1 microM) with an EC50 of approximately 10 nM, and was mediated by means of a receptor of similar structure-activity profile to those described in other tissues.     

The release of endogenous dopamine from rat striatum during incubation in vitro

In this study the conditions for quantitative estimation of the spontaneous and KCl-stimulated release of endogenous dopamine from rat striatum during in vitro incubation have been investigated. KCl concentrations of 15-35 mM produced increasing stimulation of dopamine release during 20 min of incubation in vitro, while KCl concentrations of 45 and 60 mM produced no further significant increase in release. In the presence of 4.7 or 20 mM KCl, dopamine release was linear throughout a 20 min period of incubation; in the presence of 25 or 60 mM KCl, however, dopamine release was not linear, reaching a maximum within 8 min of incubation. Three structurally unrelated inhibitors of monoamine uptake were shown to enhance the net spontaneous release of endogenous dopamine at low concentrations, but to inhibit release at high concentrations. The ethanol-induced enhancement of striatal dopamine release was used to demonstrate the importance of an appropriate concentration of an amine uptake inhibitor in the incubation media. It is concluded that the incubation of rat striatal tissues in vitro, coupled with amine analysis by high performance liquid chromatography with electrochemical detection, constitutes an effective technique for the quantitative estimation of endogenous dopamine release, providing appropriate concentrations of a reuptake inhibitor and a monoamine oxidase inhibitor are included in the incubation media. Where rates of KCl-stimulated amine release with time are to be determined, however, a low concentration of KCl must be used, or the tissues must be subjected to incubation for short periods of time.     

Release of endogenous dopamine from electrically stimulated slices of rat striatum

An experimental system is described for measuring the release of endogenous dopamine from electrically stimulated slices of rat striatum. Striatal slices were field-stimulated by two high frequency trains (S1 and S2) applied 10, 30 or 60 min apart. The quantities of dopamine released by the two stimuli were compared from slices incubated with and without dopamine's precursor, L-tyrosine. Sustained release of dopamine evoked by the two stimuli was shown to require the inclusion of tyrosine (50 microM) in the superfusate.     

Inhibition by melatonin of dopamine release from rat hypothalamus: regulation of calcium entry

The [Ca²⁺] dependency of dopamine release evoked by electrical field stimulation of hypothalamic tissue from female rats at the estrous stage was assessed in the presence of melatonin. At concentrations of 10⁻⁹–10⁻⁶ M, melatonin inhibited the [Ca²⁺]-dependent dopamine release.Melatonin reduced tissue uptake of ⁴⁵Ca²⁺ during stimulation either by an electrical field or by elevated K⁺ concentration. About 90% of this inhibition by melatonin of ⁴⁵Ca²⁺ uptake, as well as of dopamine release, was not observed in the presence of the calcium ionophore A23187. Hence, the inhibitory effect on dopamine release by melatonin may stem from the reduction of calcium entry into the presynaptic nerve endings.     

Electroconvulsive shock blocks the opioid-mediated inhibition of dopamine release in rat striatal slices

Summary The fractional release technique was applied to investigate the effects of single electroshock (ECS) on the opioid-mediated inhibition of dopamine release in rat striatal slices. Animals were submitted to ECS 24h before the experiments. The results demonstrate that pre-treatment with ECS suppresses the inhibition of dopamine release mediated by opioid receptors. These data suggest that single ECS treatment modifies the sensitivity of the opioid receptors located on the presynatic dopamine terminals in the rat striatum.     

Release of endogenous dopamine and cholecystokinin from rat striatal slices: effects of amphetamine and dopamine antagonists

The release of immunoreactive cholecystokinin (CCK) and dopamine was monitored simultaneously from superfused rat striatal slices. Exposure of the tissue to medium containing elevated of dopamine (10⁻⁷ and 10⁻⁶)M), the dopamine agonist pergolide (10⁻⁷, M), the D₂-antagonist sulpride (1 μM) or the D₁-antagonist (SCH 23390) had no significant effect on basal overflow or on evoked release of CCK. On the other hand, preincubation of striatal slices withd-amphetamine (10⁻⁵ M) enhanced basal and veratrine-stimulated dopamine release but markedly suppressed evoked CCK release. Sulpiride blocked this action of amphetamine whereas SCH 23390 was ineffective. The data suggests that whereas it is difficult to observe any effects of exogenous dopamine agonists or antagonists on evoked CCK release, endogenously released dopamine appears to interact with D₂-receptors to suppress evoked CCK release from rat striatal slices.     

Release of somatostatin from extra-hypothalamic rat brain slices: inhibition by dopamine and morphine

Calcium-dependent potassium-evoked release of somatostatin-like immuno-reactivity (SSLI) and preloaded [3H]noradrenaline ([3H]NA) could be demonstrated simultaneously from slices of rat cerebral cortex, globus pallidus and trigeminal nucleus. The release of [3H]NA from cortical slices differed from that of somatostatin (SS) in its K+-dependency, with the release of SS having a higher threshold. Both morphine (10 microM) and dopamine (50 microM) significantly inhibited the potassium-evoked release of SS from the cerebral cortex, without affecting its basal release. The effect of morphine was naloxone reversible.     

A simple in vitro technique to measure the release of endogenous dopamine and dihydroxyphenylacetic acid from striatal tissue using high performance liquid chromatography with electrochemical detection

An easily constructed, inexpensive and simple system is described for the superfusion of neural tissue. To characterize the system, the dynamics of endogenous dopamine and dihydroxyphenylacetic acid release from striatal tissue were studied before and after exposure to potassium, amphetamine or cocaine.     

Age-related decrease in apomorphine modulation of acetylcholine release from rat striatal slices

Release of [3H]acetylcholine ( [3H]ACh) was assessed in striatal slices from mature, middle-aged and senescent Wistar rats 8, 12 and 24 months of age, respectively. There was an age-related decline in basal release of [3H]ACh as a function of age which was correlated with a decline in accumulation of [3H]ACh. However, the most striking finding was the failure of apomorphine to inhibit KCl-induced [3H]ACh release in the senescent (24 months) animals. Striatal dopaminergic receptor losses in senescence apparently produce several subsequent changes in striatal function which ultimately result in the decline of motor-behavioral function.     

Postnatal development of functional dopamine, opioid and tachykinin receptors that regulate acetylcholine release from rat neostriatal slices. Effect of 6-hydroxydopamine lesion

In the present work we have studied the postnatal development of functional dopamine, opioid and tachykinin receptors, which regulate cholinergic activity in the neostriatum. The release of endogenous acetylcholine from rat striatal slices was measured using a chemiluminescent method. We have observed that the inhibition mediated by dopamine through D₂ receptors was not detectable until postnatal day 10, whereas the inhibition mediated by opioid receptors was detectable at postnatal day 15 for δ-receptors ([D-Pen², D-Pen⁵]-enkephalin) and at postnatal day 21 for μ-receptors ([D-Ala², Gly(ol)⁵]-enkephalin). Excitatory effect mediated by tachykinins through NK₁ ([Sar⁹, Met(O2)¹¹]-Substance P), NK₂ ([Nle¹⁰]-Neurokinin A_4–10), or NK₃ (senktide) receptors was already detectable at postnatal day 5.

In order to examine the influence of dopamine in the development of tachykinin and opioid systems in the neostriatum, we induced dopamine deficiency by intraventricular injection of 6-hydroxydopamine at postnatal day 3. We observed an increase in senktide-evoked acetylcholine release at postnatal day 30. The effect produced by [Sar⁹, Met(O₂¹¹]-Substance P and [Nle¹⁰]-Neurokinin A_4–10 was not modified. Furthermore, at postnatal day 35, we could observed that the two opioid receptor agonists have no effect.

Our results show that dopamine, tachykinins and opioids are already able to mediate the modulation of acetylcholine release in early stages of development with a different pattern of postnatal development. Furthermore, the integrity of a dopaminergic system plays an important role in the functional development of the neostriatal cholinergic neurons which are differentially modulated by opioids or tachykinins.     

Prenatal cocaine exposure decreases nigrostriatal dopamine release in vitro: effects of age and sex

The present study examined the effects of prenatal cocaine (PCOC) exposure, age, sex, and estrous phase on the functional development of nigrostriatal dopamine (DA) neurons. Striatal tissue was obtained from prepubescent and adult rats of both sexes after bidaily exposure to saline (1 ml/kg) or cocaine (20 mg/kg/ml saline) from embryonic days 15-21. Tissue levels, basal release, and electrically evoked (1 or 8 Hz) overflow of endogenous DA and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), as well as their efflux in response to superfusion with the DA transport blocker, nomifensine (10 microM), were measured from superfused striatal slices. Generally, these measures were highest in tissue from males and adults. Tissue DA and DOPAC levels and the rate of DA turnover were unaffected by PCOC exposure. Slices from PCOC-exposed juvenile and adult male rats exhibited significantly reduced basal and electrically evoked DA release at both stimulation intensities, in conjunction with higher levels of presynaptic DA reuptake. Female rats were largely spared from the effects of PCOC exposure, and measures did not vary with estrous phase. These findings demonstrate that the effects of PCOC exposure on various parameters of nigrostriatal DA neuronal function are not uniform across age, sex, or phases of the estrous cycle. These novel alterations in nigrostriatal DA transmission are in need of independent replication, but they may have profound implications for behavioral activities regulated by these neurons and, thus, may provide a basis for sex-selective effects of PCOC in exposed humans. Possible mechanisms of deleterious effects of PCOC exposure in select groups are discussed.     

Correlation of rates of calcium entry and endogenous dopamine release in mouse striatal synaptosomes

The time course of simultaneous Ca2+ entry and endogenous dopamine release was examined in mouse striatal synaptosomes depolarized by 30 mM KCl. Ca2+ entry and endogenous dopamine release exhibited fast and slow phase processes. The fastest rates occurred between 0 and 1 s. Ca2+ uptake and dopamine release dropped off quickly with 5-15 s rates at 13 and 10%, respectively, of the 0-1 s rate. Both processes were maintained at relatively high rates at the 1-3 and 3-5 s intervals suggesting mixed fast and slow phase processes. Uptake of Ca2+ and release of dopamine occurred in parallel over the entire 30 s measurement period; however, approximately 70% of the Ca2+ uptake and dopamine release occurred within the first 5 s following depolarization. A calculated ratio of Ca2+ entry versus dopamine release showed that approximately 1-2 Ca2+ ions were required to cause the release of one dopamine molecule. This ratio remained constant from 1 to 15 s following depolarization. Our results suggest that Ca2+ entry is coupled to endogenous dopamine release for both the fast and slow phase process.     

Effects of drugs interfering with sodium channels and calcium channels on the release of endogenous dopamine from superfused substantia nigra slices

The importance of voltage-dependent sodium channels and different types of voltage-sensitive calcium channels for depolarisation-induced release of endogenous dopamine from dendrites and cell bodies in superfused guinea pig substantia nigra slices was investigated. The stimulatory effect of veratridine (10 μM) on dopamine release was only marginally attenuated in Ca²⁺-free medium but was completely blocked by tetrodotoxin (1 μM) and by the dopamine reuptake inhibitor GBR 12909 (10 μM). Low extracellular concentration of Na⁺ stimulated the dopamine release. Potassium-evoked dopamine release was completely Ca²⁺-dependent, not blocked by GBR 12909 and partially blocked by tetrodotoxin. Nifedipine (20 μM), ω-conotoxin GVIA (0.5 μM), penfluridol (5 μM), and Ni²⁺ (20 μM) had no effect, amiloride (1 mM) attenuated and neomycin (350 μM), and ω-agatoxin IVA (1 μM) almost totally blocked the potassium-induced dopamine release. The results suggest that veratridine released dopamine mostly by reversing the dopamine transporter. High concentrations of potassium induced release of nigral dopamine by opening of voltage-sensitive calcium channels of P/Q type but not L-type, N-type and probably not T-type. The depolarisation evoked by high concentrations of potassium seems to open voltage-sensitive calcium channels both by the depolarisation induced by potassium per se and by the secondary depolarisation induced by opening of voltage-dependent sodium channels. Synapse 26:359–369, 1997. © 1997 Wiley-Liss Inc.     

In vivo release of endogenous dopamine from rat caudate putamen and nucleus accumbens by 40 mM potassium chloride

1. 1. The resting and K⁺-stimulated release rates of endogenous dopamine (DA) have been measured in vivo at four different sites in the rat caudate putamen and nucleus accumbens.

2. 2. A push-pull cannula was inserted into the brain sites chosen, and the tissue was perfused with artificial cerebrospinal fluid (CSF) containing 2.6 or 40 mM KC1. The DA content of the perfusates was determined by a radioenzymatic procedure.

3. 3. DA release was significantly increased above unstimulated levels by 40 nM KC1 in all areas tested. Neither unstimulated nor K⁺-stimulated release rates varied significantly among the regions examined.

4. 4. K⁺-stimulated DA release was not significantly diminished by perfusing the tissue with calcium-free medium, suggesting that release was probably supported by residual amounts of calcium in the tissue.

Real-time measurement of dopamine release in rat brain

Dopamine release at the submicromolar level has been observed in the striatum of an anesthetized rat on a millisecond time scale. Fast-scan cyclic voltammetry with Nafion-coated microelectrodes has been synchronized with electrical stimulation of the medial forebrain bundle, and synaptic overflow is observed following a burst of 15 impulses. The rapid appearance of dopamine following this stimulus indicates that the source of dopamine is very close (approximately 10 micron) to the electrode. The rapid disappearance of released dopamine reflects the potency of cellular uptake for dopamine. Inhibition of dopamine uptake with nomifensine allows the measurement of dopamine overflow as a result of a single stimulus impulse or with low-frequency stimulations, both comparable to physiological dopaminergic impulse flow.     

Magnesium ion augmentation of inhibitory effects of adenosine on dopamine release in the rat striatum

Abstract The effects of adenosine and magnesium ion (Mg²⁺) on striatal dopamine release were studied in awake rats by in vivo microdialysis. The mean striatal basal levels of dopamine release at Mg²⁺ free perfusate were 56.95 ± 5.30 fmol/sample (for 20 min). By varying the Mg²⁺ levels in perfusate from 0 mmol/L to 1, 10 or 40 mmol/L, the dopamine release was inhibited by Mg²⁺ in a level-dependent manner. Perfusion with modified Ringer's solution containing zero Mg²⁺ and from 5 to 50 μmol/L adenosine, non-selective adenosine agonist, as well as 0.1 μmol/L 2-chloro-N⁶-cyclopentyladenosine (CCPA), selective adenosine Al agonist, showed no effect on dopamine release. However, from 5 to 50 μmol/L adenosine and from 0.1 to 1 μmol/L CCPA plus Mg²⁺ (1 and 40 μmol/L) perfusion decreased the dopamine release. This inhibitory effect of adenosine and CCPA on striatal dopamine release was enhanced by an increase in extracellular Mg²⁺ levels. Levels of 50 μmol/L of 8-cyclopentyl-l,3-dimethylxanthine (CPT), a selective adenosine Al receptor antagonist, in perfusate increased the dopamine release under conditions both with and without Mg²⁺. This stimulatory effect of CPT on striatal dopamine release was reduced by an increase in extracellular Mg²⁺ levels. As a result, CPT antagonized the inhibitory effects of adenosine and CCPA on dopamine release under conditions of the presence and absence of Mg²⁺. These results suggest that the inhibition of striatal dopamine release by adenosine was mediated by adenosine Al receptor. This inhibition was intensified by Mg²⁺. This study also revealed that the concentrations of Mg²⁺, which ranged from physiological to supraphysiological, reduced the striatal dopamine release; furthermore it was found that the physiological concentration of Mg²⁺ potentiated the effects of adenosine agonists, but inhibited adenosine antagonist. Thus, the present study, using in vivo microdialysis preparations, suggests Mg²⁺ inhibits the calcium ion channels and enhances the adenosinergic function in the central nervous system.     

3-Mercaptopropionic acid inhibits GABA release from rat brain slices in vitro

3-Mercaptopropionic acid (3MP) (1 mM) inhibited the potassium-evoked release of endogenous GABA from slices of rat hippocampus and cerebral cortex in vitro. This did not appear to be due to an inhibition of GABA biosynthesis, since 3MP failed to affect the basal rate of GABA release or to accelerate the decline in the GABA content of tissue slices during prolonged exposure to 3MP (up to 120 min). 3MP, furthermore, inhibited the potassium-evoked release of [3H]GABA from preloaded brain slices, suggesting a direct inhibitory effect on GABA release. The threshold concentration was approximately 0.1 mM. 3MP at 1 mM failed to inhibit the potassium-evoked release of [3H]5-hydroxytryptamine, [3H]noradrenaline or somatostatin under similar conditions. The ability of 3MP to inhibit GABA release may contribute to the convulsant properties of this substance in vivo.     

Differences between the release of radiolabelled and endogenous dopamine from superfused rat brain slices: Effects of depolarizing stimuli, amphetamine and synthesis inhibition

Direct comparisons between radiolabelled and endogenous dopamine (DA) release from superfused rat brain slices have been made. Striatal slices were prelabelled with [3H]dopamine ([3H]DA), then superfused at 0.5 ml/min and the released catecholamines analyzed by HPLC with electrochemical detection and the radioactivity present in superfusate fractions also counted. Two successive 50 mM K+ pulses released similar amounts of endogenous DA from striatal slices, but the second pulse released 50% less [3H]DA than the first. A K+ gradient (5-53 mM) released relatively more [3H]DA compared to endogenous DA at lower K+ than at higher K+ concentrations. Blockade of DA synthesis in vitro by 50 microM a-methyl-p-tyrosine greatly reduced K+-induced endogenous DA release without any major effect on [3H]DA release. Amphetamine (10 microM) greatly increased both basal DA release and release induced by a 5 microM veratrine pulse, but its effects were 3-4 times greater on endogenous than on [3H]DA release. Although a-methyl-p-tyrosine reduced both basal and veratrine-stimulated endogenous DA release from non-prelabelled tissue by over 50% in either the presence or absence of amphetamine, it did not decrease endogenous DA release from prelabelled tissue. These studies indicate that labelled and endogenous amine release do not always occur in parallel, and that major causes of discrepancy between them may include the presence of a large newly-synthesized component in endogenous release and the uneven distribution of labelled amine within endogenous releasable pools. The results also suggest that the prelabelling process itself may alter the pools contributing to subsequent endogenous release. In the light of these studies, the assumption that labelled amine release provides an accurate marker for endogenous release should be reconsidered.     

Inhibition of fast phase calcium uptake and endogenous norepinephrine release in rat brain region synaptosomes by ethanol

The effects of ethanol on fast phase calcium (Ca2+) uptake and endogenous norepinephrine release were assessed simultaneously in KCl-depolarized synaptosomes isolated from rat hypothalamus, brainstem and cerebellum. Incubation of brain regional synaptosomes with ethanol resulted in a concentration-dependent inhibition of Ca2+ uptake after 1 s of depolarization. Hypothalamic synaptosomes were most sensitive to the inhibitory effect of ethanol on voltage-dependent Ca2+ uptake and brainstem synaptosomes were least sensitive. Endogenous norepinephrine release from synaptosomes was not altered by addition of ethanol in vitro at any of the concentrations examined (25-200 mM). Chronic ethanol administration resulted in an adaptation to the inhibitory effect of ethanol on Ca2+ uptake into hypothalamic synaptosomes but did not alter the inhibitory effect of ethanol on Ca2+ uptake into brainstem or cerebellar synaptosomes. Fast phase, voltage-dependent norepinephrine release was inhibited by ethanol added in vitro but only in synaptosomes isolated from hypothalami and cerebella of chronically treated animals. Brain regional norepinephrine concentrations were unaltered by chronic ethanol administration. These results suggest that chronic ethanol treatment may alter the coupling of Ca2+ entry with norepinephrine release in some noradrenergic neurons. Effects of ethanol on synaptosomal Ca2+ entry and norepinephrine release differ depending on the brain region.