As Horn

Scope and limitations of in vivo brain dialysis: A comparison of its application to various neurotransmitter systems

Brain dialysis is rapidly becoming a routine research method with a wide range of applications. Since 1982 this sampling technique is frequently used as a method to study the in vivo release of endogenous neurotransmitters such as dopamine, noradrenaline, serotonin, acetylcholine and certain amino acids. In this review most of the studies that have appeared in this field, are evaluated. Special attention was given to the question whether the neurotransmitter content in the dialysate is related to neurotransmission. Criteria such as the presence of a high tissue/dialysate concentration ratio, the sensitivity of the transmitters to membrane active compounds and the occurrence of receptor-mediated effects, are discussed. It is concluded that dopamine, noradrenaline and acetylcholine found in the dialysate are directly derived from neurotransmission, whereas the overflow of excitatory amino acid neurotransmitters is related to neurogenic as well as to metabolic events.

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.

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.

Basal acetylcholine release in freely moving rats detected by on-line trans-striatal dialysis: Pharmacological aspects

The basal release of acetylcholine (without the use of an esterase inhibitor) from brain tissue was quantified by means of transversal striatal dialysis in freely moving rats, coupled on-line to an HPLC analysis system. Basal release of acetylcholine was shown to be fully calcium dependent and tetrodotoxin sensitive. A comparison between a U-shaped and a transversally localized dialysis probe was made and some important differences were noticed. The use of a transversal probe resulted in a 20 times higher recovery when compared with the U-shaped cannula. The effect of the cholinomimetic oxotremorine and the anticholinergic atropine on the basal acetylcholine output was determined. Atropine increased the output of acetylcholine, whereas oxotremorine induced a decrease in the output of the transmitter. Application of various degrees of esterase inhibition (by addition of neostigmine to the perfusion fluid) did not qualitatively interfere with the effects of oxotremorine or atropine on the release of acetylcholine.

N,N-disubstituted 2-aminotetralins are potent D-2 dopamine receptor agonists

Mono- and di-N-substituted 2-amino-5-hydroxytetralins stimulate the D-2 dopamine receptor, 2-(N-n-propyl-N-phenylethylamino)-5-hydroxytetralin being the most potent D-2 agonist encountered to date. In contrast, 2-amino-5-hydroxytetralins only marginally stimulate the D-1 receptor; however, 2-(di-N-n-propylamino)-5, 6-dihydroxytetralin is equipotent with dopamine as a D-1 agonist. The results are discussed within the context of the two dopamine receptor hypothesis.

Stereoselective reversal of MPTP-induced parkinsonism in the marmoset after dermal application of N-0437

The selective dopamine D-2 receptor agonist N-0437 produced a rapid and dose-dependent reversal of motor deficits lasting 90-120 min following i.p. or oral administration of the racemate to MPTP-treated common marmosets. In contrast, topical application of (+/-)-, (+)- or (-)-N-0437 to the skin of MPTP-treated animals did not alter locomotor activity in the initial 4 h although other motor disabilities were reduced. However, 24 h following application of the racemate or the (-) enantiomer both locomotor activity and the other motor deficits induced by MPTP were improved. The increase in locomotor activity returned to basal values by 48-52 h following application of the racemate to the skin and by 72-76 h following administration of (-)-N-0437; the other motor deficits induced by MPTP were reduced for up to 72-76 h by both (+/-)- and (-)-N-0437. Application to skin of the (+) enantiomer produced no behavioural improvement or stimulation of locomotor activity. Transdermal administration of the active enantiomer of N-0437 may be of value in producing a prolonged reversal of parkinsonian motor deficits in man.

Synthesis and radioreceptor binding activity of N-0437, a new, extremely potent and selective D2 dopamine receptor agonist

The synthesis of a new, potent and selective D2 dopamine receptor agonist, N-0437, of the 2-aminotetralin group is described. The results of a radioreceptor binding assay using a homogenate of porcine anterior pituitary as a tissue source for D2 dopamine receptors and3H-spiperone as radioligand demonstrate that this compound is one of the most potent compounds so far evaluated in this test system.

Is TL-99 a selective presynaptic dopamine receptor agonist?

The claim that the 2-aminotetralin analogue TL-99 is a selective presynaptic dopamine (DA) receptor agonist has been investigated both in vivo and in vitro in the rat. The pharmacological specificity of the hypomotility caused by TL-99 has been examined using various selective antagonists. In addition its effects on DA metabolism and noradrenaline (NA) and DA turnover (alpha-MT method) as well as its distribution in the brain have been studied. These in vivo studies provide evidence that although TL-99 is able to activate presynaptic DA receptors it is also a potent agonist of NA receptors as shown by the fact that the hypomotility could be partly reversed by the selective alpha 2-adrenoceptor antagonists yohimbine and piperoxan. Further supporting evidence for these findings was provided by in vitro studies on the inhibition of K+-induced [3H]dopamine, [14C]acetylcholine and [3H]noradrenaline release from striatal and cortical slices where it was shown that TL-99 is not only active at both pre- and postsynaptic DA receptors but also at alpha 2-NA receptors. For the latter receptor it had a potency comparable to that of the potent alpha 2-agonist clonidine and this may explain, to some extent, the hypomotility caused by TL-99. Thus, ascribing this hypomotility solely to an interaction with presynaptic DA receptors may be an oversimplification. It is therefore concluded that TL-99 should not be considered as a selective presynaptic DA receptor agonist.

A comparison of the potencies of various dopamine receptor agonists in models for pre- and postsynaptic receptor activity.

SummarySeveral dopamine (DA) receptor agonists, notably N,N-dipropyl-2-aminotetralin analogues differing in the number and position of phenolic hydroxyl groups, were evaluated in model systems for pre- and postsynaptic dopaminergic activity. Apomorphine, piribedil and pergolide were included for comparison. All compounds inhibited the γ-butyrolactone (GBL)-induced increase in DA concentrations in the rat striatum and olfactory tubercle, although a dosedependency could not be demonstrated for one of the compounds, i.e. N,N-dipropyl-2-amino-5,6-dihydroxytetralin. In addition to the reversal of the DA-increase all compounds decreased the HVA and DOPAC levels in a dose-dependent manner, in much the same way as in normal, non GBL-pretreated rats.The potencies of the drugs to decrease HVA in normal rats and to inhibit the DA-increase and to decrease HVA in GBL-pretreated rats, both in the striatum and the olfactory tubercle were compared with each other and with the potencies to induce stereotyped behaviour. It may be concluded that (1) N,N-dipropyl-2-amino-7-hydroxytetralin shows the largest difference in activity in the biochemical and the behavioural models, suggesting a selective presynaptic activity. This was corroborated by the appearance of a marked hypomotility after low doses of this compound; (2) The potencies to decrease striatal HVA concentrations are generally somewhat different from the potencies to inhibit GBL-induced DA-increases, but appear to be comparable to the potencies to inhibit GBL-induced dihydroxyphenylalanine (DOPA)-increases; (3) There is no indication that the DA agonists in general are more potent at presynaptic receptors in the tubercle than in the striatum.

The effectiveness of yohimbine in blocking rat central dopamine autoreceptors in vivo

SummaryThe influence of various α-adrenoceptor antagonists (10 mg/kg i.p.) upon the rate of turnover of dopamine (DA) in the rat brain was investigated. Taking the levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as a measure of the rate of DA turnover, it was found that prazosin and phenoxybenzamine decreased, whereas piperoxane and yohimbine increased the turnover rate both in the corpus striatum and in the tuberculum olfactorium. Azapetine, phentolamine and tolazoline as well as the β-adrenoceptor antagonist propranolol were without a significant effect, whereas the DA antagonist haloperidol increased DOPAC and HVA levels and decreased the levels of DA itself.The possibility that the yohimbine-induced increase in the DA turnover rate was produced by a direct blockade of DA autoreceptors, was investigated under conditions where influences other than those elicited via DA autoreceptors are thought to be eliminated, i.e. in rats treated with reserpine or γ-butyrolactone (GBL). In rats that were pretreated with reserpine, yohimbine (10 mg/kg i.p.) was found to be ineffective in antagonizing the reduction of the accumulation of 3,4-dihydroxyphenylalanine (DOPA) following decarboxylase inhibition, that was produced by the DA agonist apomorphine (2.0 mg/kg i.p.). In rats pretreated with reserpine, yohimbine (10 mg/kg i.p.) was also ineffective in antagonizing the reduction of the DOPAC and HVA levels produced by apomorphine (2.0 mg/kg i.p.), but it was effective in antagonizing the reduction of the HVA level that was produced by the selective DA autoreceptor agonist N,N-di-n-propyl-7-hydroxy-2-aminotetralin (DP-7-AT, 1.0 mg/kg i.p.). In rats treated with GBL and a decarboxylase inhibitor, apomorphine (2.0 mg/kg i.p.) and DP-7-AT (1.0 mg/kg i.p.) induced a maximal suppression of the GBL-induced increase in the accumulation of DOPA. The effects of both apomorphine and DP-7-AT were partially inhibited by yohimbine (10 mg/kg i.p.). In inhibiting the effect of DP-7-AT, yohimbine appeared to be 100–200 times less effective than the DA antagonist haloperidol when both antagonists were administered at a fixed pretreatment time (1 h). It is concluded that yohimbine does indeed possess direct central DA autoreceptor blocking properties in vivo, and that this has to be taken into consideration if yohimbine is used as a pharmacological tool in order to achieve a selective blockade of α2-adrenoceptors.