T. Zetterström

In Vivo Measurement of Dopamine and Its Metabolites by Intracerebral Dialysis: Changes After d-Amphetamine

Abstract: By using a new technique, intracerebral dialysis, in combination with high performance liquid chromatography and electrochemical detection, it was possible to recover and measure endogenous extracellular dopamine, together with its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) from the striatum and nucleus accumbens of anaesthetized or freely moving rats. In addition, measurements of extracellular 5‐hydroxyindoleacetic acid, ascorbic acid, and uric acid were made. Basal extracellular concentrations of dopamine and DOPAC in the striatum were estimated to be 5 × 10−8M and 5 × 10−6M, respectively. d‐Amphetamine (2 mg/kg s.c.) increased dopamine levels in the striatum perfusates by 14‐fold, whereas levels of DOPAC and HVA decreased by 77% and 66%, respectively.

In vivo measurement of extracellular dopamine and DOPAC in rat striatum after various dopamine-releasing drugs; implications for the origin of extracellular DOPAC.

In order to further examine the likely origin of the dopamine (DA) metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), certain drugs known to release DA from different intraneuronal pools were tested for their effects on extracellular striatal DA and DOPAC levels by means of brain microdialysis in the halothane-anaesthetized rat. Amphetamine (10(-6) and 10(-5) M), nomifensine (10(-5) M), potassium chloride (30 and 60 mM), methylphenidate (10(-5) and 10(-4) M) and tyramine (10(-5) M), when added to the perfusion medium and administered locally into the striatum via the dialysis membrane, increased the level of DA in striatal perfusates during the 20 min of application. In comparison, the level of DOPAC in the perfusates was decreased by both amphetamine (10(-5) M) and potassium chloride (60 mM), but was not significantly changed by nomifensine, methylphenidate or tyramine. The effect of amphetamine (10(-6) M) and nomifensine (10(-5) M) on DA and DOPAC levels was further studied by administering the drugs over a longer period of time (3 X 20 min). Although both of these treatments produced a similar increase of DA, only amphetamine reduced the levels of DOPAC. DA (10(-4) but not (10(-5) M) increased the levels of DOPAC but this effect was also seen in DA-denervated animals. These data indicate that when the DA nerve terminal is exposed to drugs which release newly synthesized DA, DOPAC declines possibly because intraneuronal monoamine oxidase is deprived of its main substrate. We suggest that these findings support the hypothesis that a major portion of the DA metabolite, DOPAC, is derived from an intraneuronal pool of newly synthesized DA.

A direct comparison of amphetamine-induced behaviours and regional brain dopamine release in the rat using intracerebral dialysis

An intracerebral microdialysis method was used in awake rats to directly compare the effect of amphetamine on dopamine (DA) release in the striatum and nucleus (n.) accumbens with alterations in behaviour. Amphetamine (0.5-5.0 mg/kg, s.c.) caused a dose-dependent release of DA in both brain regions; however the n. accumbens appeared for the most part more sensitive to amphetamine than the striatum. At each individual dose of the drug, 0.5, 2.0 and 5.0 mg/kg s.c., DA release was closely followed over the time course by the overall behavioural syndrome. Certain components of behaviour showed a regional-specific association with DA release. The intensity of stereotyped head and forepaw movements was closely correlated over the dose range with the amount of DA released in striatum but not n. accumbens. Over the time course, however, the occurrence of this behaviour was delayed compared to increased striatal DA release. In contrast, increased locomotor activity was correlated with the time course change in, and amount of, DA released in n. accumbens by low doses of amphetamine, but not at any dose with DA released in striatum. Repetitive sniffing was better correlated with DA released in n. accumbens than striatum. These in vivo measurements of DA release add further support to the hypothesis that amphetamine-induced stereotypy and locomotion are mediated via DA released in striatum and n. accumbens, respectively. Our data suggest that the occurrence of intense stereotypy rather than locomotor activity at high doses of amphetamine is not due to a selection action in striatum but probably competition between the two behaviours.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of neuroleptic drugs on striatal dopamine release and metabolism in the awake rat studied by intracerebral dialysis

This study investigated the effect of three neuroleptic drugs, (+/-)-sulpiride, haloperidol and cis-flupenthixol, on dopamine release and metabolism in the striatum of the awake rat. Endogenous extracellular dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as the 5-hydroxytryptamine (5HT) metabolite 5-hydroxyindoleacetic acid (5HIAA), were determined in striatal perfusates in awake rats by using intracerebral dialysis together with high performance liquid chromatography with electrochemical detection. Sulpiride (10, 50 and 250 mg/kg), cis-flupenthixol (0.5 and 2 mg/kg) and haloperidol (2 mg/kg) all increased the levels of dopamine in striatal perfusates. However, the time course and magnitude of these effects differed markedly depending upon the neuroleptic used. Sulpiride (10, 50 and 250 mg/kg), cis-flupenthixol (0.05, 0.5 and 2 mg/kg) and haloperidol (0.05, 0.5 and 2 mg/kg) increased extracellular levels of DOPAC and HVA while having little effect on 5HIAA. In contrast to the effect on dopamine levels the changes in DOPAC and HVA followed similar time courses and were of similar magnitude independent of the neuroleptic used. The response of the dopamine metabolites seemed to occur at lower doses of the neuroleptics than the response of dopamine release itself. Furthermore, there was no close relationship between changes in dopamine as compared to changes in DOPAC and HVA. Finally, there was no correlation between any of the neurochemical changes measured and the occurrence of catalepsy. These data suggest that neuroleptic drugs have two separate actions on the dopamine neuron in vivo, one causing an increase in dopamine release and another producing an increase in dopamine metabolism, which is probably a consequence of increased dopamine synthesis. Furthermore neither of these effects are related to catalepsy.

An in vivo study of dopamine release and metabolism in rat brain regions using intracerebral dialysis

Intracerebral dialysis was used with a specifically designed HPLC with electrochemical detection assay to monitor extracellular levels of endogenous 3,4‐dihydroxyphenylethylamine (dopamine, DA) and its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in brain regions of the halo‐thane‐anesthetized rat. Significant amounts of DA, DOPAC, and HVA were detected in control perfusates collected from striatum and n. accumbens whereas the medial prefrontal cortex showed lower monoamine levels. The ratio of DA in perfusate to DA in whole tissue suggests that in f. cortex, compared to n. accumbens and striatum, there is a greater amount of DA in the extracellular space relative to the intraneuronal DA content. The DOPAC/HVA ratio in control perfusates varied between regions in accordance with whole tissue measurements. This ratio was highest in n. accumbens and lowest in f. cortex. The monoamine oxidase inhibitor pargyline (100 mg/kg i.p.) caused an exponential decline in DOPAC, but not of HVA, in regional perfusates, an effect that was associated with an increase in DA. The data indicated a higher turnover of extracellular DOPAC in n. accumbens than in striatum and the lowest DOPAC turnover in f. cortex. The rate of decline in extracellular DA metabolite levels was slow compared to whole tissue measurements. In the perfusates there was no statistical correlation between basal amounts of DA in the perfusates and DOPAC and HVA levels or DOPAC turnover for any of the areas, indicating that measurement of DA metabolism in the brain under basal conditions does not provide a good index of DA release. In summary, this study shows clear regional differences in basal DA release and metabolite levels, metabolite patterns, and DOPAC turnover rates in rat brain in vivo.

Effects of apomorphine on the in vivo release of dopamine and its metabolites, studied by brain dialysis

The effect of apomorphine (0.05-0.5 mg/kg s.c.) on the release of endogenous dopamine and extracellular levels of the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5HIAA) were examined in vivo by intracerebral dialysis. A dialysis tube was implanted stereotaxically through both caudate nuclei of rats and perfused with Ringer solution at a rate of 2 microliters/min. The amount of dopamine, DOPAC, HVA and 5HIAA in the perfusates was measured by HPLC with electrochemical detection. With the dialysis tube implanted into the striatum of anaesthetized rats it was possible to measure basal levels of dopamine and the metabolites in the perfusates; dopamine, 0.27 +/- 0.05 pmol/20 min (n = 15), DOPAC 43.3 +/- 2.57 pmol/20 min (n = 15), HVA 24.5 +/- 1.89 pmol/20 min (n = 15) and 5HIAA 13.9 +/- 1.77 pmol/20 min (n = 15). The % recoveries of the monoamines through the membrane were estimated to be 12% (dopamine), 21% (DOPAC), 23% (HVA) and 25% (5HIAA). Apomorphine 0.05-0.2 mg/kg decreased the spontaneous release of dopamine by a maximum of approximately 50%. When the dose of apomorphine was raised up to 0.5 mg/kg there was a 100% inhibition of dopamine release. Also, the extracellular levels of the metabolites DOPAC and HVA decreased following apomorphine administration; however there was no consistent change in 5HIAA. These findings indicate that the dopamine autoreceptors decrease dopamine release in vivo by 0-50% while larger decreases probably involve postsynaptic neurons engaging short- as well as long-loop reflexes.

Dopamine Synaptic Mechanisms Reflected in Studies Combining Behavioural Recordings and Brain Dialysis

By using a new technique of brain dialysis, it has been possible to recover endogenously released dopamine and aminoacids in awake as well as anaesthetized rats. By combining dialysis experiments with behavioural recordings we have studied the relationship between neurotransmission and behaviour. The results indicate that changes in receptor sensitivity may take place in direct response to changes in transmitter release. Studies with rotational behaviour strongly suggest the existence of dopamine receptor sites preferentially stimulated by apomorphine or pergolide and preferentially inhibited by cis-flupenthixol and sulpiride respectively. These receptor sites seem to relate differently to cholinergic and GABA-ergic mechanisms. Finally we describe the surprising finding that methylxanthines induce rotational behaviour in a way very similar to known dopamine agonists.

Simultaneous measurement of dopamine release and rotational behaviour in 6-hydroxydopamine denervated rats using intracerebral dialysis

Amphetamine- and L-DOPA-induced changes in extracellular levels of striatal dopamine (DA) were monitored during rotational behaviour in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal DA pathway. In vivo DA release measurements were obtained using intracerebral dialysis combined with high-performance liquid chromatography with electrochemical detection (HPLC-ED). In unilaterally denervated rats no DA was detected in dialysates collected from the striatum ipsilateral to the lesion while the contralateral side showed a compensatory increase in basal DA levels when compared to non-lesioned controls. Amphetamine (0.5-5.0 mg/kg s.c.) caused a dose-dependent release of DA in the striatum of normal rats and in the intact striatum of DA denervated rats but did not significantly release DA in the lesioned striatum. In response to amphetamine (0.5-5.0 mg/kg s.c.) lesioned animals showed dose-related ipsilateral rotational behaviour which in general correlated well with the time-course change in, and the total amount of, DA release in striatum contralateral to the lesion. In contrast to amphetamine, in lesioned rats, the DA precursor L-DOPA (20 mg/kg s.c.) induced readily detectable amounts of DA in dialysates collected from the DA denervated striatum. The L-DOPA-induced appearance of DA, which approached basal DA levels seen on the intact side, was concomitant with contralateral rotational behaviour. However, the time-course change in DA levels did not closely correlate with the apparently two-peak pattern of contralateral rotation induced by L-DOPA.

Further evaluation of the mechanism by which amphetamine reduces striatal dopamine metabolism: a brain dialysis study

An intracerebral dialysis method was used in the halothane-anaesthetized rat to further clarify the site which mediates the amphetamine-induced decrease of the striatal dopamine (DA) metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Following subcutaneous injection of amphetamine (0.1-5.0 mg/kg), DOPAC and HVA in striatal perfusates decreased over the 2 h time course, with 0.5 mg/kg of the drug having maximal effect. In comparison, amphetamine (0.1-5.0 mg/kg) caused a strictly dose-dependent increase of DA in striatal perfusates. Following low (0.1-5.0 mg/kg) but not high (2.0-5.0 mg/kg) doses of amphetamine there was a negative correlation between the increase of DA and decrease of DOPAC in the striatum. Amphetamine (0.5 mg/kg) caused a reduction in DA metabolism in the ibotenic acid-lesioned striatum. Intranigral injection of 10 micrograms, but not of 1 microgram amphetamine, decreased DOPAC (-27%) in the striatal perfusates. However, injection of 1 microgram amphetamine into the striatum caused a strong decrease in striatal DOPAC (-46%) and HVA (-22%). The possible mechanisms of action of amphetamine are discussed in the light of these data.

Effect of dopamine D-1 and D-2 receptor selective drugs on dopamine release and metabolism in rat striatum in vivo.

SummaryThe effect of the dopamine (DA) D-1 agonist SKF 38393, the D-2 agonist LY 171555 and the mixed D-1/D-2 agonist apomorphine on striatal DA release and metabolism was tested in vivo using an intracerebral dialysis method in halothane-anaesthetized rats. The specificity of responses to these agonists was tested using the selective DA antagonists SCH 23390 (D-1) and sulpiride (D-2).Both LY 171555, 0.01 mg/kg, and SKF 38393, 10 mg/kg, reduced levels of DA in striatal perfusates. Neither SCH 23390, 0.5 and 5 mg/kg, nor sulpiride, 10 mg/kg, affected levels of DA in striatal perfusates, but 250 mg/kg sulpiride caused a DA increase. The decrease of DA levels induced by LY 171555 (0.01 mg/kg) was prevented by pretreatment with sulpiride (10 mg/kg) but not SCH 23390 (0.5 mg/kg). In comparison, pretreatment with SCH 23390 (0.5 mg/kg) completely inhibited the reduction of DA induced by SKF 38393 (10 mg/kg) while sulpiride (10 mg/kg) was without effect. Apomorphine (0.05 mg/kg) also decreased DA in striatal perfusates and this action was partially inhibited by both SCH 23390 (0.5 mg/kg) and sulpiride (10 mg/kg).Levels of the DA metabolite DOPAC in striatal perfusates also significantly decreased following LY 171555 (0.01 mg/kg) and apomorphine (0.05 mg/kg) but not SKF 38393 (10 mg/kg). The antagonist SCH 23390, in a dose, 0.5 mg/kg, that alone did not increase levels of DOPAC, inhibited the reduction of DOPAC induced by both LY 171555 and apomorphine. Sulpiride, 10 mg/kg, caused a marked increase in striatal DOPAC and this was not affected by a subsequent injection of LY 171555, SKF 38393 or apomorphine.We conclude from these data that DA release in rat striatum is autoregulated by independent D-1 and D-2 receptor-linked mechanisms. In contrast, the level of DA metabolism is controlled by a D-2 receptor-coupled mechanism which can be influenced by the D-1 receptor. This study provides further evidence that DA release and DA synthesis/metabolism are able to change independent of each other.