Regional distribution of monoamines in the nucleus accumbens of the rat

Monoamine concentrations were low in the rostral area of the nucleus accumbens. Their distributions were not identical. Differences were observed in the medial area. DA concentrations were high in both medial and caudal areas. Noradrenaline (NA) and serotonin (5-HT) concentrations were considerably lower than the dopamine (DA) concentration. The NA concentration was highest in the caudal area of the nucleus accumbens and the (5-HT) concentration was highest in the ventrocaudal area. There was a rostrocaudal decrease in the 3,4-dihydroxyphenylacetic acid (DOPAC)/DA and 5-hydroxyindole-3-acetic acid (5-HIAA)/5-HT ratios. Uptake of [³H]DA and [¹⁴C]choline was lowest in the rostral area. The K⁺-stimulated release of [¹⁴C]acetylcholine (ACh) was also lowest rostrally, but there was no rostrocaudal difference in the K⁺-stimulated release of [³H]DA. These results provide further evidence of the heterogeneity of the nucleus accumbens.     

ACCUMULATION AND DISAPPEARANCE OF NORADRENALINE AND ITS MAJOR METABOLITES SYNTHESIZED FROM INTRAVENTRICULARLY INJECTED [3H]DOPAMINE IN THE RAT BRAIN

Abstract— A new combined ion-exchange and thin-layer-chromatographic procedure is described which separates and measures quantitatively, after intraventricular injection of [³H]dopamine (DA), the rat brain content of labelled noradrenaline (NA) and the following labelled noradrenaline metabolites: free 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG), conjugated MOPEG, free plus conjugated dihydroxyphenylethyleneglycol (DOPEG), vanillic mandelic acid (VMA) and normetanephrine (NM). Labelled dopamine and its metabolites were also measured. The time-course study performed from 5 min to 24 h after [³H]DA showed that MOPEG and DOPEG, mainly as conjugates, are major NA metabolites whereas VMA is a very insignificant NA metabolite in the rat brain. A very rapid initial increase of [³H]NM, free MOPEG and conjugated MOPEG was found during the time interval where the [³H]NA biosynthesis is very high (0–15 min). This combined with the finding that these metabolites stabilize at lower levels during the [³H]NA ‘storage phase’ (9–24 h) provides a strong indication that newly synthesized NA preferentially is metabolized. Our measurements of endogenous NA, free MOPEG and conjugated MOPEG provide additional support. The injections of various decreasing doses of [³H]DA (3·08–0·0010 μg) showed that the proportions of total [³H]MOPEG and total [³H]DOPEG to [³H]NA were constant after all [³H]DA doses investigated. This finding indicates that the [³H]NA synthesized in situ behaves as a tracer, even after injections of non-tracer doses of [³H]DA. The results seem thus to indicate that the present technique provides a powerful tool for the investigations on central noradrenaline metabolism.     

Release of ( 3 H)noradrenaline and ( 3 H)dopamine from field stimulated cerebral cortex slices. Effect of tyrosine hydroxylase and dopamine- -hydroxylase inhibition

Rat cerebral cortex slices were incubated in vitro with [³H]dopamine (DA) or [³H]noradrenaline (NA) (10^?7M), superfused by fresh buffer and stimulated by an electric field. The stimulation-induced overflow of [³H]DA and [³H]NA was determined. In slices from untreated rats about 16 ng [³H]NA/g tissue was formed from [³H]DA, corresponding to about 5 per cent of the endogenous NA concentration. Stimulation markedly enhanced the overflow of [³H]NA. The [³H]NA newly formed from [³H]DA was overflowing to a greater extent than [³H]NA previously taken up from the incubation medium, indicating a preferential release of newly synthesized transmitter. The stimulation-induced overflow of [³H]DA and [³H]NA was increased in slices of rats pretreated with a tyrosine hydroxylase inhibitor (H44/68). It seems that depletion of the endogenous NA stores of central NA neurons by tyrosine hydroxylase inhibition makes the [³H]cate-cholamines more available for release. Pretreatment of the rats with the DA-β-hydroxylase inhibitors FLA63 or FLA69 considerably diminished the formation of [³H]NA from [³H]DA. Stimulation markedly enhanced the overflow of [³H]DA indicating that DA can act as a ‘false transmitter’ in central NA neurons after DA-β-hydroxylase inhibition.     

The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool

The impact of syntaxin and SNAP-25 cleavage on [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca2+-dependent K+-induced [3H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [3H]NA and [3H]DA were differentially affected: both toxins blocked the former, dependent on external Ca2+, but not the latter, internal Ca2+-dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [3H]catecholamine release. More precisely, [3H]NA exocytosis induced by ionomycin was sensitive to toxins in the early phase of release but not later. The Ca2+-independent [3H]NA exocytosis evoked by hypertonic sucrose, thought to release from the readily releasable pool (RRP) of vesicles, was significantly reduced by BoNT/C1. Pre-treating synaptosomes with phorbol-12-myristate-13-acetate, to increase the RRP, enhanced the sensitivity to BoNT/C1 of [3H]NA release elicited by sucrose or ionomycin. Accordingly, cleavage of syntaxin was augmented by the phorbol-ester. To conclude, our results suggest that clostridial toxins selectively target exocytosis involving vesicles set into the RRP.     

Serotonin, dopamine, noradrenaline and their metabolites: levels in the brain of the house cricket (Acheta domesticus L.) during a 24-hour period and after administration of quipazine--a 5-HT2 receptor agonist.

1. The levels of 5-HT, DA, NA and DA metabolites (NADA, DOPAC) measured by HPLC (with electrochemical detection) in the brain of the house cricket did not change over a 24-hr period. The level of 5-HIAA, a 5-HT metabolite, was below the limit of detection. 2. The 5-HT and DOPAC levels decreased and NADA increased after quipazine injection but DA and NA levels did not change after it. 3. [3H]Ketanserin was used to identify serotonin receptors bound to sites in the house cricket brain with a KD of 5 nM and a concentration of Bmax 180 fmol/mg protein.     

Uptake and metabolism of catecholamines in the awake dog

The contributions of uptake, metabolism and excretion to the removal of circulating dopamine (D) have been studied in comparison to those of adrenaline (A) and noradrenaline (NA). In two experiments radioactive catecholamines were infused during 80 min in an awake dog. In the first experiment [14C]D and D, L-[3H]A was used and in the second experiment these catecholamines were infused together with D, L-[14C]NA. Renal excretion of 14C-radioactivity was almost equal in both experiments, as was the case with the accumulation of 14C-components in plasma, demonstrating that the uptake of D was comparable to that of NA. The removal of [14C]D, [14C]NA and [3H]A, by uptake was 50, 50 and 13.5% respectively after 1 h. The conversion by metabolism was 46, 46 and 81%. Renal excretion was 3.5, 2 and 0.5%. Thus only 0.5, 2 and 5% was left in the extracellular fluid (ECF). In a report on similar experiments in anaesthetized dogs much higher levels of unchanged NA in plasma were measured. Probably this is due to anaesthesia inhibiting uptake. In the pulmonary circulation 14C-radioactivity was extracted at a constant rate during infusion which can mainly be attributed to extraneuronal uptake of [14C]D and to neuronal uptake of [14C]NA. Besides extraneuronal uptake of [3H]A in the lung expiration of [3H]water may contribute to the pulmonary extraction.     

Release of Purines, Noradrenaline, and GABA from Rat Hippocampal Slices by Field Stimulation

Labelled adenine, noradrenaline (NA), and gamma-aminobutyric acid (GABA) were taken up by the transversely cut hippocampal slice. [3H]NA and [14C]GABA were retained as such, [3H]- (or [14C]-) adenine mainly as adenine nucleotides. There was a spontaneous overflow of all three types of compounds ranging from 0.1 (GABA) to 0.21 (NA) %/min. The rate of [3H]NA overflow increased rapidly during electrical field stimulation. The release rate was well maintained over a 15-min period. The rate of [14C]GABA release also increased rapidly but it was not maintained over a 15-min period even if uptake and/or metabolism was inhibited by nipecotic acid (1 mM) and aminooxyacetic acid (AOAA, 0.1 mM). The bulk of the purines was released after the stimulation period. For all compounds the amounts released were frequency- and calcium-dependent. At a frequency of 3 Hz a 10 V stimulation was sufficient to cause a maximal [3H]NA release and 20 V to cause maximal [14C]GABA release, but 14C-purine release was increased further by increasing the voltage to 40 V. The evoked purine release was inhibited by a nucleoside uptake inhibitor (dipyridamole). On stimulation of [3H]NA-labelled slices the released radioactivity was composed of greater than 95% unchanged NA. The specific activities of NA in the slice and in the superfusate were practically identical. In [3H]adenine-labelled slices the released radioactivity was composed of adenosine, inosine, and hypoxanthine, but the activity in the slice of ATP, ADP, and AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-methyl-D-aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV-1 envelope protein gp120, external pH and protein kinase C inhibition

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [(3)H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 microM or 1 mM) plus glycine (1 microM)-elicited [(3)H]NA, but not [(3)H]DA release. The NMDA (100 microM)-evoked release of [(3)H]NA and [(3)H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1-1 microM); in contrast, the HIV-1 envelope protein gp120 potently (30-100 pM) enhanced the NMDA-evoked release of [(3)H]NA, but not that of [(3)H]DA. Gp120 also potentiated quinolinate-evoked [(3)H]NA release. Ifenprodil (0.1-0.5 microM) or CP-101,606 (0.1-10 microM) inhibited the NMDA plus glycine-evoked release of both [(3)H]catecholamines. Zinc (0.1-1 microM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [(3)H]NA elicited by NMDA plus glycine, whereas the release of [(3)H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 microM) or H7 (10 microM) selectively prevented the effect of NMDA plus glycine on the release of [(3)H]NA. GF 109203X also blocked the release of [(3)H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.     

Effect of ethanol on [3H]Dopamine release in rat nucleus accumbens and striatal slices

Ethanol (10–200 mM) transiently increased tritium overflow from superfused rat nucleus accumbens slices previously incubated with [³H]dopamine (DA) and [¹⁴C]choline. The effect was greater in striatal tissue and did not appear to be a non-specific membrane effect since [¹⁴C]acetylcholine (ACh) release was not affected. Lack of antagonism by picrotoxin suggested that -aminobutyric acid (GABA) receptors were not involved. Calcium was not a requirement and the DA uptake blocker, nomifensine, was without effect. Ethanol appeared to be causing [³H]DA release into the cytoplasm. K⁺-stimulated release of [³H]DA and [¹⁴C]ACh from nucleus accumbens and striatal slices was not affected. Clonidine-mediated inhibition of the K⁺-evoked release of [³H]DA remained unaltered. Ethanol attenuated the isoproterenol-induced enhancement of [³H]DA release. Ethanol therefore appeared to interact with components of the DA terminal causing a transient increase in the release of neurotransmitter without impairing K⁺-evoked release but apparently interfering with the isoproterenol-induced effect.     

Serotonin,dopamine, noradrenaline and their metabolites: Levels in the brain of the house cricket (Acheta domesticus L.) during a 24-hour period and after administration of quipazine—a 5-HT2 receptor agonist

1. The levels of 5-HT, DA, NA and DA metabolites (NADA, DOPAC) measured by HPLC (with electrochemical detection) in the brain of the house cricket did not change over a 24-hr period. The level of 5-HIAA, a 5-HT metabolite, was below the limit of detection.2. The 5-HT and DOPAC levels decreased and NADA increased after quipazine injection but DA and NA levels did not change after it.3. [³H]Ketanserin was used to identify serotonin receptors bound to sites in the house cricket brain with a K_d of 5 nM and a concentration of B_max 180 fmol/mg protein.     

Dopamine Stimulates [3H]Phorbol 12,13-Dibutyrate Binding in Cultured Striatal Cells

The effect of dopamine (DA) on the binding of [3H]phorbol 12,13-dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 +/- 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half-maximal stimulation was observed with 10(-6) M DA. The peak response was observed at 2-3 min after addition of 10(-4) M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10(-6) M) blocked the response to NE, suggesting mediation by an alpha 1-adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH-23390 (10(-6) M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10(-6) M) inhibited the response to DA by 46 +/- 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF-38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding.     

Regional release of [3H]dopamine from rat brain in vitro: effects of opioids on release induced by potassium, nicotine, and L-glutamic acid

Previous studies have suggested that the release of dopamine (DA) in the rat brain may be sensitive to modulation by opioid agents, including the endogenous opioid peptides (enkephalins and endorphins). The present study examined the effects of morphine and the enkephalin analogue D-Ala2-Met5-enkephalinamide (DALA) on the release of radiolabeled DA from superfused slices of rat brain regions. The release of preloaded [3H]DA was evoked from slices of the caudate-putamen (CP) by application of potassium (K+), nicotine (NIC), or L-glutamic acid (L-GLU). The release of [3H]DA from slices of the nucleus accumbens (NA), olfactory tubercle (OT), and substantia nigra (SN) was evoked by L-GLU. Both K+ and NIC evoked a concentration-related release of [3H]DA from CP slices. K+-induced release was only partially dependent on calcium (Ca2+), while NIC-evoked release was completely Ca2+ independent. Neither morphine nor DALA influenced the release of [3H]DA evoked by K+ or NIC. L-GLU produced a concentration-dependent release of [3H]DA from slices of CP, NA, OT, and SN. In all four brain regions, this release was (a) Ca2+-dependent, (b) strongly inhibited by low concentrations of magnesium (Mg2+), (c) greater than the release evoked by D-GLU, (d) attenuated by the putative L-GLU receptor antagonist glutamic acid diethylester (GDEE), and (e) insensitive to tetrodotoxin (TTX) except in the SN. Morphine produced a significant inhibition of L-GLU-evoked [3H]DA release from all four regions. Naloxone, which by itself had no significant effect on the L-GLU-evoked release of [3H]DA, blocked the inhibitory effect of morphine on this release in the CP but not in the other regions. Levorphanol and dextrorphan were equipotent in reducing the glutamate-stimulated release of [3H]DA from CP slices. DALA had no effect on L-GLU-induced release in any of the brain regions examined. The results indicate that L-GLU provokes regional release of DA by acting at a Mg2+-sensitive glutamate receptor. This release is selectively modified by morphine through a mechanism which is insensitive to naloxone.     

Chronic nicotine differentially affects the function of nicotinic receptor subtypes regulating neurotransmitter release

It is known that nicotine can activate several subtypes of release-regulating presynaptic nicotinic receptors (nAChRs) including those situated on central noradrenergic, dopaminergic, cholinergic and glutamatergic axon terminals. The objective of this study was to investigate the effects of chronic administration of (-)nicotine on the function of the above autoreceptors and heteroreceptors using rat superfused synaptosomes. In hippocampal synaptosomes prelabelled with [3H]noradrenaline (NA) the nicotine-evoked overflow of [3H]NA was higher in rats treated with nicotine for 10 days (via osmotic mini-pumps) than in vehicle-treated rats. In striatal synaptosomes, prelabelled with [3H]dopamine (DA), chronic nicotine did not modify the releasing effect of nicotine. No significant change was observed in experiments with synaptosomes from nucleus accumbens prelabelled with [3H]DA. Exposure of hippocampal synaptosomes prelabelled with [3H]choline to nicotine elicited release of [3H]acetylcholine; this effect was almost abolished in synaptosomes from animals administered nicotine for 10 days, suggesting down-regulation of nicotinic autoreceptors. In hippocampal synaptosomes prelabelled with [3H]D-aspartate, the releasing effect of epibatidine following chronic nicotine treatment did not differ from that in controls. The K+-evoked exocytotic release of the neurotransmitters tested was not modified by long-term nicotine administration. The results show that chronic nicotine differentially affects the function of release-regulating nAChR subtypes.     

Pregnenolone sulfate induces NMDA receptor dependent release of dopamine from synaptic terminals in the striatum

Neuromodulators that alter the balance between lower-frequency glutamate-mediated excitatory and higher-frequency GABA-mediated inhibitory synaptic transmission are likely to participate in core mechanisms for CNS function and may contribute to the pathophysiology of neurological disorders such as schizophrenia and Alzheimer's disease. Pregnenolone sulfate (PS) modulates both ionotropic glutamate and GABA(A) receptor mediated synaptic transmission. The enzymes necessary for PS synthesis and degradation are found in brain tissue of several species including human and rat, and up to 5 nM PS has been detected in extracts of postmortem human brain. Here, we ask whether PS could modulate transmitter release from nerve terminals located in the striatum. Superfusion of a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes with brief-duration (2 min) pulses of 25 nM PS demonstrates that PS increases the release of newly accumulated [3H]dopamine ([3H]DA), but not [14C]glutamate or [3H]GABA, whereas pregnenolone is without effect. PS does not affect dopamine transporter (DAT) mediated uptake of [3H]DA, demonstrating that it specifically affects the transmitter release mechanism. The PS-induced [3H]DA release occurs via an NMDA receptor (NMDAR) dependent mechanism as it is blocked by D-2-amino-5-phosphonovaleric acid. PS modulates DA release with very high potency, significantly increasing [3H]DA release at PS concentrations as low as 25 pM. This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid.     

Ventricular Cerebrospinal Fluid Monoamine Transmitter and Metabolite Concentrations Reflect Human Brain Neurochemistry in Autopsy Cases

Concentrations of dopamine (DA), its metabolites 3-methoxytyramine and homovanillic acid (HVA), noradrenaline (NA), its metabolites normetanephrine (NM) and 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT, serotonin), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in 14 brain regions and in CSF from the third ventricle of 27 human autopsy cases. In addition, in six cases, lumbar CSF was obtained. Monoamine concentrations were determined by reversed-phase liquid chromatography with electrochemical detection. Ventricular/lumbar CSF ratios indicated persistence of rostrocaudal gradients for HVA and 5-HIAA post mortem. Ventricular CSF concentrations of DA and HVA correlated positively with striatal DA and HVA. CSF NA correlated positively with NA in hypothalamus, and CSF MHPG with levels of MHPG in hypothalamus, temporal cortex, and pons, whereas CSF NM concentration showed positive correlations with NM in striatum, pons, cingulate cortex, and olfactory tubercle. CSF 5-HT concentrations correlated positively with 5-HT in caudate nucleus, whereas the concentration of CSF 5-HIAA correlated to 5-HIAA levels in thalamus, hypothalamus, and the cortical areas. These data suggest a specific topographic origin for monoamine neurotransmitters and their metabolites in human ventricular CSF and support the contention that CSF measurements are useful indices of central monoaminergic activity in man.     

Increased dopamine D2 receptor-mediated inhibition of [14C]acetylcholine release in the dorsomedial part of the nucleus accumbens

Dopamine (DA) D2 receptor-mediated inhibition of the K⁺-stimulated release of [¹⁴C]acetylcholine (ACh) from prelabeled rat dorsomedial nucleus accumbens slices was found to be 1.7 times greater than that observed in dorsorostral and ventromedial slices. This observation is consistent with the 1.9 fold higher DA D2 receptor density found in the dorsomedial area. In contrast, there were no differences in the DA D2 receptor-mediated effects on [³H]DA release in these areas. In addition, DA D2 receptor-mediated effects on [³H]DA and [¹⁴C]ACh release could not be demonstrated in the ventrorostral part of the nucleus accumbens consistent with the fact that DA D2 receptors were barely detectable in this area. The results suggest that cholinergic terminals in the dorsomedial part of the nucleus accumbens are under greater inhibitory DA control than in other areas of the nucleus accumbens.     

Effects of the neoclerodane Hardwickiic acid on the presynaptic opioid receptors which modulate noradrenaline and dopamine release in mouse central nervous system

We have comparatively investigated the effects of Hardwickiic acid and Salvinorin A on the K⁺-evoked overflow of [³H]noradrenaline ([³H]NA) and [³H]dopamine ([³H]DA) from mouse hippocampal and striatal nerve terminals, respectively. The K⁺-evoked overflow of [³H]DA was inhibited in presence of Salvinorin A (100 nM) but not in presence of Hardwickiic acid (100 nM). Hardwickiic acid (100 nM) mimicked Salvinorin A (100 nM) in facilitating K⁺-evoked hippocampal [³H]NA overflow and the two compounds were almost equipotent. Facilitation of [³H]NA overflow caused by 100 nM Hardwickiic acid was prevented by the κ-opioid receptor (KOR) antagonist norbinaltorphimine (norBNI, 100 nM) and by the selective δ-opioid receptor (DOR) antagonist naltrindole (100 nM), but was not altered by 100 nM D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), a selective μ-opioid receptor (MOR) antagonist. We conclude that Hardwickiic acid modulates hippocampal [³H]NA overflow evoked by a mild depolarizing stimulus by acting at presynaptic opioid receptor subtypes.     

Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy

The release of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca²⁺ further increased the release rates of [³H]DA and [³H]NA induced by ischemic conditions. This finding indicated that the Na⁺/Ca²⁺ exchanger (NCX), working in reverse in the absence of extracellular Ca²⁺, fails to trigger the influx of Ca²⁺ in exchange for Na⁺ and fails to counteract ischemia by further increasing the intracellular Na⁺ concentration ([Na⁺]_i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca²⁺ was removed. Hypothermia inhibited the excessive release of [³H]DA in response to ischemia, even in the absence of Ca²⁺. These findings further indicate that the NCX plays an important role in maintaining a high [Na⁺]_i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [³H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H₂O₂), a mediator of ischemic brain injury enhanced the striatal resting release of [³H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca²⁺]_o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na⁺/Cl^?-dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

     

Modulation of Dendritic Release of Dopamine by N-Methyl-D-Aspartate Receptors in Rat Substantia Nigra

A superfusion system was used to study the effects of excitatory amino acids (EAA) on release of [3H]dopamine ([3H]DA) previously taken up by rat substantia nigra (SN) slices. The EAA tested (20-250 microM), with the exception of quisqualate and kainate, markedly evoked [3H]DA release from nigral slices when Mg2+ ions were omitted from the superfusion medium. The EAA receptor agonists exhibited the following relative potency in stimulating [3H]DA release: L-glutamate (L-Glu) greater than N-methyl-D-aspartate (NMDA) greater than NM(D,L)A greater than D-Glu much greater than quisqualate = kainate. D-2-Amino-5-phosphonovalerate (100-200 microM), an antagonist for NMDA receptors, substantially reduced [3H]DA release evoked by L-Glu or NMDA. In contrast, L-Glu diethyl ester (100-200 microM) produced a lesser blocking effect on [3H]DA release evoked by the EAA. Further experiments showed that the NMDA-mediated release of [3H]DA was totally suppressed by the omission of Ca2+ or by the addition of tetrodotoxin (0.1 microM) to the superfusion medium. In addition, strychnine, an antagonist for glycine (Gly) receptors, significantly decreased NMDA (100 microM)-evoked as well as glycine (100 microM)-evoked release of [3H]DA from nigral slices. The results shown support the idea that activation of NMDA subtype receptors in SN may trigger a Ca2+-dependent release of DA from dendrites of nigro-striatal DA-containing neurons. Furthermore, a transsynaptic mechanism that may partially involve Gly-containing interneurons is proposed to account for some of the events mediating NMDA receptor activation and DA release in SN.     

Physiological presynaptic facilitation of dopamine release in the cat caudate nucleus

Using halothane-anaesthetized cats implanted with push-pull cannulae, we investigated the effects of GABA application into the VM/VL on the release of [3H] DA continuously synthetized from [3H] tyrosine in the ipsilateral CN and SN and on single unit activity of nigral DA cells. GABA was applied (30 min) at a concentration of 10(-3) or 10(-5) M since the higher concentration reduces the local multi-unit activity in the VM/VL while the opposite response is observed with the lower one. The application of GABA into the VM/VL at a concentration of 10(-3) M resulted in an increase in nigral [3H] DA release, an inhibition of DA cell firing and a decrease in [3H] DA release in the CN. The latter effect is likely due to the inhibition of DA neuron activity which is triggered through DA autoreceptors by DA released from dendrites. In contrast, when applied at a concentration of 10(-5) M into the VM/VL, GABA stimulated [3H] DA release in the CN despite its inhibitory effect on single unit activity of DA cells. Furthermore, the nigral release of [3H] DA was no longer affected. These results indicated that DA release from nerve terminals was not dependent on nerve activity and they favour the existence of a potent facilitatory presynaptic regulation of DA release. The intervention of a presynaptic mechanism was further established by examining the effect of GABA (10(-5) M) application into the VM/VL on [3H] DA release in the CN shortly after a complete ipsilateral hemisection of the brain made at the meso-diencephalic level.(ABSTRACT TRUNCATED AT 250 WORDS)