Bhc Westerink

THE USE OF TETRODOTOXIN FOR THE CHARACTERIZATION OF DRUG-ENHANCED DOPAMINE RELEASE IN CONSCIOUS RATS STUDIED BY BRAIN DIALYSIS

SummaryThe effect of TTX (infused during brain dialysis of the striatum and nucleus accumbens) on the in vivo release of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid, was investigated. In addition it was studied whether the increase in the release of dopamine, induced by various pharmacological treatments, was sensitive to TTX infusion. The following drugs were studied: haloperidol, amphetamine, haloperidol co-administered with GBR 12909, morphine and MPP+. Dialysis was carried out in the striatum, except for morphine, which was studied in the nucleus accumbens. The infusion of TTX revealed three different types of pharmacologically enhanced dopamine release in conscious rats. First, action potential dependent dopamine release (exocytosis), which was observed in untreated animals as well as in animals treated with haloperidol, haloperidol + GBR 12909, and morphine. Second, action potential independent release (carrier-mediated) was established in the case of amphetamine. Third, action potential independent DA release, probably caused by neurotoxic reactions was observed during MPP+ infusion.

MEASUREMENT OF ACETYLCHOLINE-RELEASE IN FREELY MOVING RATS BY MEANS OF AUTOMATED INTRACEREBRAL DIALYSIS

Abstract: The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC‐assay system that comprises a cation exchanger, a post‐column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 μM) of neostigmineresultedingraduallyincreasingamounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.

The significance of extracellular calcium for the release of dopamine, acetylcholine and amino acids in conscious rats, evaluated by brain microdialysis

SummaryThe influence of the calcium concentration of the perfusion fluid on the release of striatal dopamine recorded by brain dialysis was investigated. The release of dopamine appeared very sensitive to the calcium concentration of the Ringer. Next we studied whether three different methods known to antagonize the effects of calcium entry, were able to affect the release of dopamine. The conditions investigated were: the use of calcium-free Ringer, infusion of magnesium, and infusion of the calcium-antagonist verapamil. Calcium-antagonism was studied on the day of implantation of the cannula as well as on several days thereafter. It appeared that magnesium infusion was the most effective condition to antagonize the effects of calcium on the release of dopamine. Magnesium infusion was also most effective in preventing drug-evoked voltage-dependent dopamine release (induced by coadministration of haloperidol and GBR 12909). In addition magnesium infusion appeared a potent antagonist of acetylcholine release. In contrast, the dialysate content of aminoacids was not influenced by magnesium infusion.

Characterization of in vivo dopamine release as determined by brain microdialysis after acute and subchronic implantations: methodological aspects.

Abstract: Infusion of tetrodotoxin (TTX) through the dialysis membrane and perfusion with calcium‐free Ringer solution (calcium depletion) were used to evaluate the dopamine release determined by in vivo brain dialysis. Several hours after implantation, the dopamine release recorded by the U‐shaped cannula did not respond to calcium depletion and was only partly (˜50%) TTX dependent. The half‐life of the TTX‐independent dopamine overflow was determined to be 2 h. In contrast, when a transstriatal cannula was used, the dopamine output displayed calcium and TTX dependency. Differences in the dimensions of the two types of probes are a likely explanation for the observed effects. Twenty‐four hours after implantation, both types of cannula fulfilled the criteria of calcium and TTX dependency. The results indicate that infusion of TTX‐contain‐ing or calcium‐free Ringer solution can be used to estimate the functional damage caused by the implantation of the cannula.

Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes

SummaryMicrodialysis of dopamine was performed in the striatum with four different microdialysis cannulas: a trans-cerebral probe, a U-shaped probe, a I-shaped probe and a commercially available I-shaped probe (Carnegie). The commercial cannula was studied with as well as without a guide cannula. The effect of infusion of tetrodotoxin (TTX) or calcium free-Ringer solution on the dialysate content of dopamine released was determined 2 h after implantation (day 1) as well as 24 h after implantation (day 2). Two hours after implantation, all cannulas displayed a certain amount of TTX-independent overflow of dopamine. The best results were obtained with the I-shaped cannula: already 2 h after implantation of the probe, about 85% of the release of dopamine was TTX-dependent. In case of the trans-cerebral cannula and the Carnegie cannula (implanted without guide in anesthetized rats), 70–75% of the output of dopamine was TTX-dependent. When the Carnegie cannula was implanted with a guide cannula, only 45% of the dopamine output was TTX-dependent. The responses to calcium-free perfusion were much more variable. Dopamine output in I-shaped and the U-shaped probes was virtually insensitive to calcium-free perfusion in acute experiments. Twenty-four hours after implantation of the probes, the calcium- and TTX-dependency was much more pronounced. All types of cannulas studied now sampled dopamine that was completely TTX-dependent. Calcium-free perfusion caused a reproducible disappearance of dopamine from the dialysates to 30% of controls, in all cannulas studied. When the removable Carnegie cannula was re-implanted 24 h after its first implantation, 91% of the dopamine overflow was TTX-sensitive. These results confirm earlier conclusions that microdialysis experiments should be carried out at least 24 h after implantation of the probe.

Chronic Use of Intracerebral Dialysis for the In Vivo Measurement of 3,4-Dihydroxyphenylethylamine and Its Metabolite 3,4-Dihydroxyphenylacetic Acid

Abstract: The intracerebral dialysis technique was studied with a method in which the rat was directly connected to the HPLC equipment. The effect of three pharmacological treatments [perfusion of 60 mmol K+ or 5 × 10−5M (+)‐amphetamine or subcutaneous injection of 2 mg/kg (+)‐amphetamine] on the release of 3,4‐dihydroxy‐phenylethylamine (dopamine) and 3,4‐dihydroxyphenylacetic acid was followed over a period of 7 days. The marked rise of dopamine output seen after infusion of K+ had almost disappeared on day 3. Tissue reactions around the membrane presumably formed a barrier preventing K+ from reaching dopaminergic terminals. In contrast, the pronounced rise in dopamine level after amphetamine (infused as well as systemically administered) was still present (although diminished) 8 days after implantation. It is concluded that, with certain restrictions, brain dialysis of dopamine is still useful several days after implantation of the membrane.

On the Significance of Endogenous 3-Methoxytyramine for the Effects of Centrally Acting Drugs on Dopamine Release in the Rat Brain

Abstract: 3‐Methoxytyramine (3‐MT) was measured in the striata of rats killed by microwave radiation. Apomorphine, γ‐butyrolactone (GBL), and reserpine decreased the 3‐MT content. A slight but transient increase in 3‐MT was observed after haloperidol. The turnover rate of 3‐MT was unchanged 60 min after haloperidol treatment. (+)‐Amphetamine induced a pronounced rise in the 3‐MT content, which was potentiated after combined treatment with haloperidol. The increased 3‐MT turnover rate that was observed after amphetamine treatment suggests that monoamine oxidase (MAO) inhibition is no explanation for the mechanism of interaction of this drug with dopamine (DA) metabolism. The central stimulants amphonelic acid and nomifensine in‐creased 3‐MT levels; no substantial change was seen after benztropine, morphine, or oxotremorine. It is concluded that a decreased release of DA is closely and rapidly reflected by decreased formation of 3‐MT. 3‐MT seems to be a much better indicator for decreased DA release than 3,4‐dihydroxyphenylacetic acid or homovanillic acid.

DETERMINATION OF ACETYLCHOLINE AND CHOLINE IN THE FEMTOMOLE RANGE BY MEANS OF HPLC, A POST-COLUMN ENZYME REACTOR, AND ELECTROCHEMICAL DETECTION

SummaryThe measurement of choline and acetylcholine by means of HPLC, a post-column enzyme reactor, and electrochemical detection has been simplified and optimised. The use of a cation exchanger and enzyme reactor fitted in a cartridge holder appeared to result in reproducible, sensitive, and selective measurement of endogenous choline and acetylcholine with a lower detection limit of 50 fmole.

THE RELEASE OF DOPAMINE FROM NERVE-TERMINALS AND DENDRITES OF NIGROSTRIATAL NEURONS INDUCED BY EXCITATORY AMINO-ACIDS IN THE CONSCIOUS RAT

SummaryThe possible localization of excitatory amino acid (EAA) receptors on dopaminergic neurons was studied by microdialysis in conscious male rats. Varying concentrations of 3 specific EAA agonists, N-methyl-D-aspartate (NMDA), kainate and amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), were infused into the striatum or into the substantia nigra, and the extracellular dopamine (DA) was recorded by the same probe. All 3 compounds induced a dose-dependent increase in both striatal and nigral extracellular DA. Kainate and AMPA were more potent than NMDA. Nigral DA release was stimulated by lower concentrations of kainate and AMPA than striatal DA release.The effects of two concentrations of NMDA and kainate on the release of DA were analyzed in terms of tetrodotoxin (TTX) dependency and sensitivity to ibotenic acid-induced striatal lesion. It appeared that NMDA and kainate stimulated DA release by 3 different mechanisms. The first mechanism is seen at low concentrations of kainate, it fulfills the criteria for a functional receptor-interaction: it is TTX-sensitive and independent of the ibotenic acid lesion. The second mechanism was observed when relatively low concentrations of NMDA stimulate the release of DA; in this effect postsynaptic structures are involved. The third mechanism lacks specificity as it is seen after high concentrations of kainate as well as of NMDA. The latter mechanism is TTX-independent and is probably of a toxic nature. Finally NMDA and kainate were infused into the nigra, whereas DA was recorded with a second probe implanted into the striatum. Kainate and NMDA induced an increase of striatal DA, but kainate was about 100 times more potent in this model than NMDA.The present data therefore support localization of kainate and (probably) AMPA-receptors on nigrostriatal dopaminergic neurons. The receptors on the somatoden-dritic sites were observed to be more sensitive than those on the nerve terminals.

ON THE MECHANISM OF NEUROLEPTIC INDUCED INCREASE IN STRIATAL DOPAMINE RELEASE - BRAIN DIALYSIS PROVIDES DIRECT EVIDENCE FOR MEDIATION BY AUTORECEPTORS LOCALIZED ON NERVE-TERMINALS

The influence of sulpiride on the in vivo release of dopamine and DOPAC from the rat striatum was investigated by microdialysis. Racemic sulpiride was administered systemically (i.p.) to control rats and to rats in which a striatum was pretreated by kainic acid. In addition various concentrations (10(-8) to 10(-5) M) of the two enantiomers of sulpiride were infused into the striatum and the effects on the release of dopamine were recorded. Infusion as well as systemic administration of sulpiride caused a maximal increase in the release of dopamine of about 180-190% of basal values. A similar increase was seen in kainic acid-pretreated rats. The rises in dopamine seen after systemic administration or infusion of sulpiride were not additive, suggesting that similar mechanisms were involved. DOPAC dialysate levels also increased during infusion of the neuroleptic but the rise was significantly less when sulpiride was administered i.p. to kainic acid pretreated rats. It is concluded that the rise in dopamine release seen after neuroleptics is mediated by autoreceptors localized on nerve terminals. This implies that the well-known increase in electrical activity of dopaminergic neurons during neuroleptic treatment, is not responsible for the increased release of the transmitter.