Modulation of striatal dopamine release in cerebral ischemia byL-arginine

The effect ofL-arginine, the precursor of nitric oxide, on ischemic dopamine release from the striatum was investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (2 h). Dopamine and its metabolites were measured in the striatal extracellular space dialysate after continuous perfusion (2 l/min) of artificial extracellular fluid in the presence or absence of 15 mmol/literL- orD-arginine or 1 mmol/liter nitro-L-arginine.L-Arginine but notD-arginine increased the striatal content of dopamine in pre- and postischemia whereas it lowered the levels of dopamine and 3-methoxytyramine induced by ischemia. In contrast, nitro-L-arginine reduced the preischemic levels of dopamine and 3,4-dihydroxyphenyl-acetic acid, and had no effect on the ischemic release of dopamine. These findings indicate thatL-arginine stereospecifically modified the ischemic release and metabolism of dopamine. The data also suggest that the basal level of nitric oxide is not involved in dopamine release during ischemia but may participate in regulating dopamine release under physiological conditions.Presented in part at the 19th International Joint Conference on Stroke and Cerebral Circulation, San Diego, California, February 17–19, 1994.     

Effects of long-term recovery from transient cerebral ischemia in rat brain: Tissue levels of acetylcholine,monoamines, and their metabolites

Concentrations of acetylcholine and the monoaminergic neurotransmitters dopamine, serotonin and their respective metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenylacetic acid (HVA), 5-hydroxyindolacetic acid (5-HIAA) and choline were simultaneously determined in the corpus striatum of rats after 15 min. complete cerebral ischemia (CCI) and in different intervals (1, 24, 48, 72, 96 hours) of postischemic cerebral reperfusion. Results were compared to respective sham-operated control animals. After 15 min. CCI acetylcholine concentration decreased to 15%, and dopamine concentration to 56% of the control values. The metabolite levels of DOPAC decreased to 40% and HVA to 64% of the control values. Acetylcholine, dopamine, serotonin and choline concentrations were not changed significantly after reperfusion. The metabolites HVA and 5-HIAA showed their maximum increases after 1 and 24 hours of reperfusion, additionally HVA was decreased both, after 72 and 96 hours of reperfusion. The data indicate that surprisingly little permanent damage could be caused by a 15 min. ischemia in the striatum. Tissue levels of the neurotransmitters appeared differentially altered but similarly regulated during ischemia and subsequent recirculation. Acetylcholine and dopamin levels decreased profoundly during ischemia. However, acetylcholine levels could be compensated rapidly during reperfusion, whereas the dopaminergic system showed a long-lasting change in its turnover rate. Although serotonin levels were unaffected by CCI, there was an increase of its presumed turnover rate during reperfusion.     

Corelease of dopamine and serotonin from striatal dopamine terminals

The striatum receives rich dopaminergic and more moderate serotonergic innervation. After vesicular release, dopamine and serotonin (5-hydroxytryptamine, 5-HT) signaling is controlled by transporter-mediated reuptake. Dopamine is taken up by dopamine transporters (DATs), which are expressed at the highest density in the striatum. Although DATs also display a low affinity for 5-HT, that neurotransmitter is normally efficiently taken up by the 5-HT transporters. We found that when extracellular 5-HT is elevated by exogenous application or by using antidepressants (e.g., fluoxetine) to inhibit the 5-HT transporters, the extremely dense striatal DATs uptake 5-HT into dopamine terminals. Immunohistochemical results and measurements using fast cyclic voltammetry showed that elevated 5-HT is taken up by DATs into striatal dopamine terminals that subsequently release 5-HT and dopamine together. These results suggest that antidepressants that block serotonin transporters or other factors that elevate extracellular 5-HT alter the temporal and spatial relationship between dopamine and 5-HT signaling in the striatum.     

Neuroactive metabolites of L-tryptophan, serotonin and quinolinic acid, in striatal extracellular fluid. Effect of tryptophan loading

Extracellular fluid levels of the neurotoxin quinolinic acid in the corpus striatum of rats, measured by in vivo microdialysis, were increased in a dose-dependent manner following the intraperitoneal administration of tryptophan. The lowest dose of tryptophan (12.5 mg/kg), equivalent to about 5% of the normal daily intake, increased peak quinolinic acid levels nearly 3-fold. At higher doses of tryptophan (up to 250 mg/kg), concentrations of quinolinic acid increased over 200-fold and exceeded potentially neurotoxic levels (10 microM). In contrast, the increase in extracellular serotonin following even the highest tryptophan dose was small (less than 2-fold). These data indicate that quinolinic acid is present in the extracellular fluid where it may function as a neuromodulator and that it is very responsive to physiological changes in precursor availability.     

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.     

Oxalic acid stabilizes dopamine, serotonin, and their metabolites in automated liquid chromatography with electrochemical detection

Use of antioxidative agents is required in automated LC assay of microdialysis samples, due to rapid degradation of the monoamine neurotransmitters and their metabolites. Addition of oxalic acid prevented degradation of dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid efficiently: after a 24-h incubation at room temperature the decreases in peak heights were less than 10%. The long-term stability of the analytes, however, was still enhanced when acetic acid and -cysteine were included in the solution. Using this antioxidative solution, the monoamine neurotransmitters and their metabolites could be determined with an automated LC assay even at room temperature.     

Dose-related effects of cysteamine treatment on hypothalamic and striatal dopamine, noradrenaline and serotonin neurotransmission

The dose-related effects of cysteamine treatment on hypothalamic and striatal neurotransmission were investigated. Cysteamine pretreatment with a dose of 150 mg/kg slightly increased the dopamine, and markedly decreased the noradrenaline, content of the hypothalamus in a dose-related manner. The serotonin levels of the hypothalamus and striatum were not affected. Cysteamine pretreatment with a higher dose (300 mg/kg sc) slightly increased the uptake of noradrenaline into hypothalamic slices. The drug did not influence dopamine and serotonin uptake into hypothalamus and striatal slices. These results suggest that cysteamine decreases rather selectively the noradrenaline content of the hypothalamus.     

Effects of K-stimulation and precursor loading on the in vivo release of dopamine, serotonin and their metabolites in the nucleus accumbens of the rat

The efflux of endogenous 3,4-dihydroxyphenylethylamine (DA) 5-hydroxytryptamine (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the nucleus accumbens of the anesthetized rat was studied using a push-pull cannula. Local perfusion for 10 minutes with 35 mM K⁺ significantly (P<0.01) increased the release of DA and 5-HT, but not their metabolites, from their respective control levels of 0.95 and 0.04 pmol/15 min to 2.5 and 0.23 pmol/15 min. Exposure to 35 mM K⁺ a second and third time resulted in a decrement in the amount of stimulated release for both DA and 5-HT. This decrease was prevented by local perfusion for 10 minutes with 50 uM L-tyrosine and -tryptophan starting 30 minutes before each episode of depolarization. The baseline amounts of DOPAC, HVA and 5-HIAA observed in the perfusates were several fold higher than the basal levels found for 5-HT and Da. In the absence of precursors, the efflux of DOPAC, HVA and 5-HIAA decreased approximately 60, 40 and 25%, respectively, from the first to the last baseline fraction collected. Addition of precursors prevented the decrease for DOPAC and 5-HIAA but not for HVA. The data indicated that (a) the release of DA and 5-HT, along with their metabolites, could be simultaneously measured with the present procedure, and (b) when using the push-pull cannula, local perfusion with precursors may be necessary following periods of sustained and/or repeated stimulation in order to replenish the monoamine transmitter pools.     

Electro-cortical signs of early neuronal damage following transient global cerebral ischemia in rat

During recovery after a transient global cerebral ischemia (TGCI), rat electrocorticogram (ECoG) shows epochs of synchronized activity (SA) alternating with epochs of low amplitude background activity (BA). The aim of this study was to compare the changes in these electrical activities during a 30-min recovery period that followed either a noninjuring (3 minutes, N=10) or an injuring (10 minutes, N=10) TGCI. During TGCI there was a 3 fold reduction in amplitudes of both SA and BA but no changes in frequency. During reperfusion following a 3 minutes TGCI, the amplitudes of both SA and BA recovered to about 70%. During the reperfusion that followed a 10 minutes TGCI, BA showed no recovery, whereas SA recovered to about 40%. During the 30 min reperfusion, there was a timedependent decrease in the frequency of SA, but independent on the duration of TGCI. In contrast, the frequency of the BA did not change during reperfusion. Our data indicate that following cerebral ischemia the recovery of SA can take place independently of BA. The lack of recovery in BA may indicate early subcortical neuronal damage.     

Morphological manifestations of local functional activation of astrocytes induced by transient global cerebral ischemia

The goal of the work was to study changes of structural and cytochemical organization of activated hippocampal astrocytes in the rat exposed to transient global ischemia of the brain. Intermediate filament proteins immunocytochemistry revealed functional activation of astrocytes of dorsal hippocampus 7 days following the ischemia, which was manifested as changes of size and shape of the cells and processes and accumulation of intermediate filament proteins GFAP and nestin. This is accompanied by formation of two populations of activated astrocytes: GFAP-positive astrocytes, which are more abundant and nestin-positive astrocytes distributed predominantly in the area of massive loss of neural cells. The obtained data suggest that astrocytes activated post-ischemically obtain properties typical for immature cells of nervous tissue, but lack of morphological signs of dedifferentiation do not support their contribution to reparative neurogenesis in the hippocampus.     

Protective effect of resveratrol against neuronal damage following transient global cerebral ischemia in mice

Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenol which is rich in grape seeds and skin. Several studies have revealed that resveratrol possesses neuroprotective effects. In the case of global brain ischemia, there are few reports regarding the protective effect of resveratrol. Therefore, the influence of resveratrol on neuronal damage after transient global brain ischemia remains to be clarified. In the current study, C57BL/6 black mice were subjected to 20 min of transient global brain ischemia and followed by 72 h of reperfusion. Resveratrol (20 or 40 mg/kg, once daily, dissolved in 0.5% carboxymethylcellulose) was administered orally for 7 days before ischemia and daily until the mice were euthanized. The effect of lower or higher dose of resveratrol on neuronal damage, matrix metalloproteinase (MMP) activity and in situ DNA fragmentation (TUNEL) assay in the hippocampus after global ischemia was examined. Neuronal damages were remarkable in CA1 and CA2 pyramidal cell layers after global ischemia. In resveratrol-treated mice (40 mg/kg), neuronal damage was significantly reduced compared with vehicle-treated mice. Mice treated with resveratrol showed reduced MMP-9 activity. Resveratrol also inhibited TUNEL staining. These data suggest that resveratrol, a natural polyphenol, reduces hippocampal neuronal cell damage following transient global ischemia by reducing MMP-9 activity.     

In vivo electrochemical studies of rat striatal dopamine and serotonin release after morphine

The effect of the reference opiate, morphine (d-morphine-sulfate), on endogenously released striatal dopamine and serotonin was studied in male, adult, anesthetized Sprague-Dawley rats. The intraperitoneal administration of morphine produced a biphasic effect on striatal dopamine release. A significant increase in the dopamine signal was seen in the first hour after drug administration; a significant decrease in the dopamine signal was seen in the second and third hour after drug administration. On the other hand, the effect of morphine on striatal serotonin release was monophasic. Morphine significantly increased serotonin release from rat striatum. The effect lasted three hours after morphine administration, i.e., the effect persisted significantly throughout the study. These data show a simultaneous opiate-dopaminergic and opiate-serotonergic interaction in rat striatum. These data further extend studies which have suggested that the pharmacological mechanism of action of morphine may have its etiology in the concurrent modulation of more than one neurotransmitter.     

Animal models of focal and global cerebral ischemia

The use of appropriate animal models is essential to predict the value and effect of therapeutic approaches in human subjects. Focal (stroke) and global (cardiac arrest) cerebral ischemia represents diseases that are common in the human population. Stroke and cardiac arrest, which are major causes of death and disability, affect millions of individuals around the world and are responsible for the leading health care costs of all diseases. Understanding the mechanisms of injury and neuroprotection in these diseases is critical if we are ever to learn new target sites to treat ischemia. There are many animal models available to investigate injury mechanisms and neuroprotective strategies. This review summarizes many (but not all) small and large animal models of focal and global cerebral ischemia and discusses their advantages and disadvantages.     

Chronic nicotine modifies the effects of morphine on extracellular striatal dopamine and ventral tegmental GABA

Previously, we have shown that 7-week oral nicotine treatment enhances morphine-induced behaviors and dopaminergic activity in the mouse brain. In this study, we further characterized the nicotine-morphine interaction in the mesolimbic and nigrostriatal dopaminergic systems, as well as in the GABAergic control of these systems. In nicotine-pretreated mice, morphine-induced dopamine release in the caudate putamen and nucleus accumbens was significantly augmented, as measured by microdialysis. Chronic nicotine treatment did not change basal extracellular concentrations of dopamine and its metabolites in the caudate putamen and nucleus accumbens, nor did it affect the rate of dopamine synthesis, as assessed by 3-hydroxybenzylhydrazine dihydrochloride-induced DOPA accumulation. GABAergic control of dopaminergic activity was studied by measuring extracellular GABA in the presence of nipecotic acid, an inhibitor of GABA uptake. Acute (0.3 mg/kg or 0.5 mg/kg i.p.) and chronic nicotine, as well as morphine (15 mg/kg s.c.) in control mice decreased nipecotic acid-induced increase in extracellular GABA in the ventral tegmental area/substantia nigra (VTA/SN). In contrast, in nicotine-treated mice, morphine increased GABA levels in the presence of nipecotic acid. We did not find any alterations in GABA(B)-receptor function after chronic nicotine treatment. Thus, our data show that chronic nicotine treatment sensitizes dopaminergic systems to morphine and affects GABAergic systems in the VTA/SN.     

Protective effect of olive leaf extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in Mongolian gerbils

The beneficial effects of antioxidant nutrients, as well as complex plant extracts, in cerebral ischemia/reperfusion brain injury are well known. Mediterranean diet, rich in olive products, is associated with lower incidence of cardiovascular disease, cancer, inflammation and stroke. In this study, the possible neuroprotective effect of standardized dry olive leaf extract (OLE) is investigated for the first time. Transient global cerebral ischemia in Mongolian gerbils was used to investigate the OLE effects on different parameters of oxidative stress and neuronal damage in hippocampus. The biochemical measurements took place at different time points (80 min, 2, 4 and 24 h) after reperfusion. The effects of applied OLE were compared with effects of quercetin, a known neuroprotective plant flavonoid. Pretreatment with OLE (100 mg/kg, per os) significantly inhibited production of superoxide and nitric oxide, decreased lipid peroxidation, and increased superoxide dismutase activity in all time points examined. Furthermore, OLE offered histological improvement as seen by decreasing neuronal damage in CA1 region of hippocampus. The effects of applied OLE were significantly higher than effects of quercetin (100 mg/kg, per os). Our results indicate that OLE exerts a potent neuroprotective activity against neuronal damage in hippocampus after transient global cerebral ischemia, which could be attributed to its antioxidative properties.     

Reverse-phase high-performance liquid chromatographic separation and electrochemical detection of norepinephrine,dopamine, serotonin,and related major metabolites

A convenient method for the determination of biogenic amines and their metabolites in small samples of brain tissue weighing from 0.5 to 5 mg, based on reverse-phase chromatography, is described. The biogenic amines, norepinephrine, dopamine, and serotonin, and the metabolites normetanephrine and 3-methoxytyramine were separated by ion-pair chromatography on a μBondapak phenyl column with an aqueous eluant, while the metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid were separated on a μBondapak C18 column with a methanol aqueous mobile phase. The sensitivity of the method is in the picogram range, from 20 to 100 pg, depending on the substances analyzed.     

Effects of haloperidol on extracellular contents of dopamine and its metabolites in the septum of rats during interspecies aggression]

The extracellular levels of dopamine (DA) and its metabolites in the septum of freely moving rats were studied using "push-pull" method during muricidal aggression before and after haloperidol (2 mg/kg, i. p.) treatment. Mouse-killing activity of the rats was accompanied by significant reduction of the output of DA and metabolites in this brain area. Haloperidol suppressed muricidal activity of the rats and increased the extracellular levels of DA metabolites. In contrast, the decrease of DA release monitoring during killing activity was not affected by haloperidol administration. Possible role of the septal dopaminergic mechanisms underlying muricidal behaviour and the drug effects is discussed.     

多巴胺D1和D2受体激动剂和拮抗剂对脑缺血/再灌注损伤的影响

实验应用开阔法、组织病理学方法、原位末端标记(in situ terminal deoxynucleotidyl transferase-metliated de-oxy-UTP mick end labeling,TUNEL)法及免疫组织化学等方法,探讨多巴胺D1、D2受体激动剂和拮抗剂对沙土鼠前脑缺血／再灌注损伤海马CA1区神经元凋亡及凋亡相关基因bcl-2、bax表达的影响。结果显示：前脑缺血5min可引起沙土鼠探索活动增加;再灌注3d,海马CA1区约95％的锥体细胞凋亡;再灌注7d,海马CA1区仅残存约2％—7％的存活锥体细胞;前脑缺血5min可抑制bcl-2的表达并诱导bax表达增高;预先应用D2受体激动剂培高利特可减轻缺血后沙土鼠行为学异常、抑制海马CA1区锥体细胞凋亡、提高锥体细胞存活数、显著诱导bcl-2的表达并抑制bax的表达。预先应用SKF38393、SCH23390及螺哌隆对以上结果无明显影响。实验结果提示,培高利特具有确切的脑保护作用,诱导bcl-2并抑制bax的表达可能是其脑保护作用机制之一。     

New antioxidant mixture for long term stability of serotonin, dopamine and their metabolites in automated microbore liquid chromatography with dual electrochemical detection

An automated microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin, dopamine and their metabolites, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Due to the chemical instability of the compounds, the addition of an antioxidant is required for automated analysis over a long period of time (e.g., 20 h). Therefore, the time stability of these substances was tested with different antioxidants. The stability for serotonin and 5-hydroxyindoleacetic acid was poor in acidic medium containing Na₂EDTA but could greatly be improved by the addition of

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-cysteine and ascorbic acid. Using this assay, the neurotransmitters and their metabolites could easily be determined in microdialysates obtained from different rat brain areas.