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

European Journal of Pharmacology, 106 (1985) 27-37

27

Elsevier

E F F E C T O F N E U R O L E P T I C D R U G S O N S T R I A T A L D O P A M I N E RELEASE AND M E T A B O L I S M IN T H E AWAKE RAT S T U D I E D BY I N T R A C E R E B R A L DIALYSIS T Y R A Z E T T E R S T R O M *, T R E V O R S H A R P and U R B A N U N G E R S T E D T

Department of Pharmacology, Karolinska Institute, P.O. Box 604 00, S- 104 O0 Stockholm, Sweden Received 11 May 1984, revised MS received 10 July 1984, accepted 23 July 1984

T. ZETTERSTROM, T. SHARP and U. UNGERSTEDT, Effect of neuroleptic drugs on striatal dopamine release and metabolism in the awake rat studied by intracerebral dialysis, European J. Pharmacol. 106 (1985) 27-37. 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 homovaniilic 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. Suipiride (10, 50 and 250 mg/kg), cis-flupenthixol (0.5 and 2 mg/kg) and haioperidol (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 extraceilular 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. Dopamine release in vivo DOPAC

Intracerebral dialysis HVA

Dopamine metabolism 5HIAA

1. Introduction

It has long been recognized that neuroleptic drugs are potent antagonists at dopamine receptors in the central nervous system (CNS). This property of neuroleptic drugs is evident from studies showing that neuroleptics specifically inhibit behavioural (see Costall and Naylor, 1979) and electrophysiological (Aghajanian and Bunney, 1977) effects induced by dopamine receptor * To whom all correspondence should be addressed: Karolinska Institute, P.O. Box 60400, S-10400 Stockholm, Sweden. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.

Neuroleptics

Catalepsy

agonists, and potently displace these agonists from brain tissue membrane preparations (see Seeman, 1980). Furthermore, it is proposed that antagonism of central dopamine receptors by neuroleptics accounts for their antipsychotic action in man (Creese et al., 1976) as well as their cataleptic effect in animals (see Costall and Naylor, 1979). Neuroleptic drugs, together with dopamine agonists, have proved to be useful tools for investigating mechanisms which regulate central dopaminergic neurotransmission. Based on the evidence that dopamine antagonists enhance while dopamine agonists inhibit the release of preloaded radiolabeUed dopamine from rat striatal slices in

28 vitro, Farnebo and Hamberger (1971) proposed the existence of presynaptic dopamine receptors modulating dopamine release from the nerve terminal. More recently, several groups of workers, using similar techniques, have presented findings in good agreement with the earlier study (Starke et al., 1978; Arbilla and Langer, 1981). Dopamine receptor blockade by neuroleptic drugs has also been shown in biochemical studies on rat brain tissue to in.crease dopamine turnover (And6n, 1972; Westerink and Korf, 1976). This effect on turnover has been explained by an increase in dopamine synthesis and release following an action of neuroleptics on postsynaptic dopamine receptors, mediated via a neuronal feedback loop (Carlsson and Lindqvist, 1963; Bunney et al., 1973), and also by an action on presynaptic dopamine receptors (Carlsson, 1975; Di Chiara et al., 1977; Garcia-Munoz et al., 1977; Nowycky and Roth, 1978). In vivo techniques have been used as an alternative approach to study the effects of dopamine agonists and antagonists on dopamine release and metabolism in the brain. These in vivo experiments have been carried out using methods such as push-pull perfusion (Bartholini et al., 1976; Nieoullon et al., 1979) to measure radiolabelled dopamine, or ventricular perfusion (Nielsen and Moore, 1982) and in vivo voltammetry (Gonon et al., 1981; Sharp et al., 1984) for the metabolites. However, direct in vivo measurement of endogenous rather than radiolabelled dopamine release has been difficult due to the low extracellular levels of this neurotransmitter. Recently, using an intracerebral dialysis method (Ungerstedt et al., 1982; Zetterstrt~m et al., 1983) developed from earlier ideas (Delgado et al., 1972; Ungerstedt and Pycock, 1974), we have been able to monitor decreases in extracellular levels of endogenous dopamine and its metabolites in the striatum of the intact rat following dopamine agonist administration (ZetterstriSm and Ungerstedt, 1984). To further investigate the regulation of dopaminergic neurotransmission the present experiments were carried out to examine the effect of certain neuroleptic drugs on striatal dopamine release and metabolism in vivo. Three neuroleptics were chosen for the study: sulpiride, which is a

selective antagonist at dopamine D-2 receptors and two others, haloperidol and cis-flupenthixol, which are less specific for this receptor (see Seeman, 1980). The study was carried out using intracerebral dialysis with awake, freely moving rats so that neurochemical changes could be compared directly with the cataleptic effects of the drugs.

2. Materials and methods

2.1. Preparation of dialysis loops Dialysis loops were made using flexible Dow 50 cellulose dialysis tubing (Dow Company 300 # m diameter), with a molecular weight cut-off of 5000. The loops were prepared as previously described (Zetterstr~m et al., 1983). Briefly, a short length of dialysis tubing was glued inside two steel cannulae (23G) to expose a 12 mm length of tubing between the cannulae. The exposed tubing was folded into a tight loop and the cannulae were clamped in a stereotaxic holder. One cannula was connected to a microinfusion pump using polyethylene tubing whilst the other served as an outlet to a collecting tube.

2.2. In vitro recovery experiments In vitro experiments were carried out to test the recovery of monoamines through the dialysis membrane. In these experiments dialysis loops were perfused (2 / d / m i n ) with Ringer's solution (147 mmol Na +, 2.3 mmol Ca 2÷, 4 mmol K +, 155.6 m m o l / C l - , pH 6.0) and placed in this solution containing the monoamines at concentrations 10-7 to 10-5 M. Perfusate samples were collected at 20 min intervals from each monoamine solution, utilizing at least three different loops.

2.3. lntracerebral dialysis method Male Sprague Dawley rats (275-325 g) were anaesthetized with halothane and placed in a stereotaxic frame (David Kopf Instruments). The skull was exposed and a hole drilled to allow implantation of the dialysis loop into the caudate nucleus (A, + 1.5 L, + 2.3 V, - 7.0 mm relative to bregma).

29 The coordinates were chosen according to the stereotaxic atlas of K~Snig and Klippel (1963). With the dialysis loop held in its implantation holder a fine tungsten wire was temporarily positioned in the tube lumen. The wire was used to extend the loop to a point during implantation. The supporting steel cannulae of the implanted dialysis loop were secured to the cranium by dental cement. Following surgery, the animals were allowed to recover from halothane anaesthesia for at least 1 h, then placed in a hemispherical bowl (55 cm diameter). The dialysis loop was perfused with physiological Ringer solution (2/~l/min) using polyethylene tubing connected to a microinfusion pump (Microject) via a liquid swivel held above the bowl. Each animal was secured in a harness which was attached to the swivel by a length of wire. This arrangement made it possible for the animal to move freely within the confines of the bowl. The perfusates were collected in a small (150 /xl) replaceable test tube which was secured to the head of the animal. The collecting tube contained 10/~1 1 M perchloric acid to prevent oxidation of monoamines. Samples were collected at 20 rain intervals and at least four control samples were taken before drug administration.

2.5. Measurement of cataleptic behaviour following neuroleptic administration The cataleptic behaviour of the animals was tested every 20 min following drug administration. Following the collection of each perfusate sample the rat was placed with its front paws on the edge (10 mm) of the hemispherical bowl. The time that the rat remained in this position was recorded and used as an index of catalepsy.

2.6. Drugs The following drugs were used at the doses indicated: haloperidol (Leo, Sweden), 0.05-2.0 mg/kg; cis-flupenthixol (Lundbeck, Denmark), 0.05-2.0 mg/kg; and racemic sulpiride (Delagrange, France), 10-250 mg/kg. Haloperidol and sulpiride were dissolved in saline containing 1% lactic acid and 1% acetic acid respectively. Cisflupenthixol was dissolved in saline alone. All doses of neuroleptic drugs were injected s.c. in a volume of 0.1 ml/100 g body weight. Brain perfusate samples were taken for at least 180 min after neuroleptic or saline treatment.

2. 7. Statistical analysis 2.4. Assay of monoamines The perfusates were assayed for dopamine, DOPAC, HVA and 5HIAA by high performance liquid chromatography (HPLC) with electrochemical detection. Dopamine was separated by cation exchange using Nucleosil SA resin (10 /xm) with 0.05 M acetate citrate buffer pH 5.2. The monoamine metabolites were separated by reverse phase chromatography using spherisorb ODS resin (5 #m) and 0.1 M acetate citrate buffer, pH 4.2, containing 10% methanol. Electrochemical measurements were made using a carbon paste working electrode set at +0.65 V. Using 2 /tl/min perfusion speed each 20 min period yielded 40/~1 dialysis perfusate, of which 30/~1 was analysed for dopamine and the remaining 10 #1 was assayed for the monoamine metabolites. Perfusate samples were injected directly onto the HPLC column without any need for sample preparation.

Statistical analysis of the data was carried out using the Mann-Whitney U-test (two-tailed).

3. Results

3.1. Recovery of monoamines through the dialysis membrane in vitro In vitro experiments showed that the amount of monoamine recovered through the dialysis membrane was directly proportional to the amount of monoamine in the external medium (fig. 1). This relationship was constant for 10 -7 to 10 -5 M monoamine in the external medium, a concentration range which was comparable to the amounts of monoamine recovered from the brain in vivo. There was a similar percentage recovery (20-25%) of individual monoamines across the dialysis membrane over this concentration range.

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Fig. 4. Effect of haloperidol on levels of dopamine (A), DOPAC (B) and HVA (C) in striatal perfusates from the awake rat. Perfusates were collected for 200 rain after drug treatment. The data are expressed as % (mean + S.E.M., n - 4) of the mean of 3 control values obtained immediately before drug injection. Statistically significant changes are as indicated; * P < 0.05, • * P < 0.02 (Mann-Whitney U-test).

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a n d 50 m g / k g ) also elevated striatal d o p a m i n e levels b u t these doses were less effective t h a n the higher dose. In comparison, 2.0 a n d 0.5 m g / k g cis-flupenthixol caused a small (41-51%) b u t significant rise in d o p a m i n e over the 3 h post drug period (fig. 3). However, 0.05 m g / k g cisflupenthixol had n o effect on striatal d o p a m i n e levels. Haloperidol at the highest dose, 2 m g / k g , caused a p r o n o u n c e d increase in d o p a m i n e release

(fig. 4). This increase was m a x i m a l (190%) 20 m i n post-injection a n d d o p a m i n e levels did n o t return to baseline for 3 h after the drug. I n contrast, lower doses of haloperidol, 0.5 a n d 0.05 m g / k g , had n o effect o n d o p a m i n e release for 3 h following drug a d m i n i s t r a t i o n . The effect of the neuroleptics on d o p a m i n e release is s u m m a r i z e d in fig. 5.

33

3.4. Effect of neuroleptic drugs on extracellular levels of DOPAC, HVA and 5HIAA in rat striatum in vivo

levels of the d o p a m i n e metabolites, D O P A C a n d H V A (figs. 2, 3, 4 a n d 5). However, n o n e o f these neuroleptics caused a strictly d o s e - d e p e n d e n t increase in either D O P A C or H V A . Figs. 2, 3 a n d 4 show that changes in D O P A C after n e u r o l e p t i c a d m i n i s t r a t i o n were closely followed over the time course b y H V A . The increase in d o p a m i n e m e t a b o l i t e s after n e u r o l e p t i c a d m i n i s t r a t i o n showed a similar pattern. T h e increase in m e t a b o l i t e s started after 20-40 rain a n d was m a x i m a l (100-

In a d d i t i o n to d o p a m i n e , m o n o a m i n e m e t a b o lites were m e a s u r e d in striatal perfusates following n e u r o l e p t i c d r u g a d m i n i s t r a t i o n . Sulpiride (10, 50 a n d 250 m g / k g ) , cis-flupenthixol (0.05, 0.5 a n d 2.0 m g / k g ) a n d h a l o p e r i d o l (0.05, 0.5 a n d 2.0 m g / k g ) each caused a m a r k e d elevation in extracellular

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Inj. Fig. 6. The degree of catalepsy induced following neuroleptic drug administration. Catalepsy is expressed as the length of time (s) the animal remained in position when placed with its front paws on the edge of a bowl (see text for details). Each point represents the mean ( :t: S.E.M.) of n = 4 experiments. Neither haloperidol nor cis-flupenthixol at 0.05 mg/kg caused catalepsy. Sulpiride (10-250 m g / k g ) w a s a l s o inactive.

34

180% above baseline values) 80-120 min post-drug; the metabolite levels then remained higher than the control values over the rest of the 3 h experimental period. The total amount of each monoamine recovered in the perfusates over the 180 rain period after drug administration was also calculated (fig. 5). Fig. 5 shows clearly that lower doses of haloperidol (0.05 and 0.5 mg/kg) and cis-flupenthixol (0.05 mg/kg) caused pronounced increases in DOPAC and HVA but had no effect on dopamine itself. Additionally, the highest dose of haloperidol (2 mg/kg) had a marked effect on dopamine release while causing only a small increase in dopamine metabolite levels. The level of 5HIAA in striatal perfusates was also determined following neuroleptic administration (fig. 5). Neither sulpiride (10, 50 and 250 mg/kg) nor cis-flupenthixol (0.05, 0.5 and 2 mg/kg) changed the concentration of 5HIAA in striatal perfusates. However, following the highest dose of haloperidol (2 mg/kg) there was a statistically significant decrease in 5HIAA levels in the striatum.

3.5. Effect of haloperidol on dopamine release and metabolism 24 h following loop implantation In order to test whether the acute experimental conditions might affect drug-induced changes in dopamine release and metabolism, a group of animals were implanted with a dialysis loop 24 h before drug treatment. Basal monoamine levels in striatal perfusates 24 h after loop implantation (expressed as % of 1 h post-implantation values) were dopamine (72 -t- 20%, n = 5), DOPAC (96 + 16%, n = 5), HVA (86:1: 13%, n = 5) and 5HIAA (93 + 22%, n = 5). The effect of haloperidol (0.5 mg/kg) on levels of dopamine, DOPAC, and HVA in striatal perfusates was tested and no significant changes in dopamine levels were observed. However, as in the case of the acute experiments, DOPAC and HVA increased after haloperidol, with a maximal increase in DOPAC and HVA of 100% and 98% respectively. These data are in close agreement with results obtained in acutely operated animals.

3.6. Comparison of the cataleptic activity of haloperidol, cis-flupenthixol or sulpiride Following the administration of haloperidol (0.05, 0.5 and 2.0 mg/kg), cis-flupenthixol (0.05, 0.5 and 2.0 mg/kg) or sulpiride (10, 50 and 250 mg/kg) rats were tested for the degree of catalepsy (see Materials and methods). These measurements were made in the same animals in which extracellular levels of the monoamines in striatum had been determined. Both haloperidol (0.5 and 2.0 mg/kg) and cis-flupenthixol (0.5 and 2.0 mg/kg) produced cataleptic behaviour with the highest dose of each drug causing the most pronounced effect (fig. 6). The lowest doses (0.05 mg/kg) of haloperidol and cis-flupenthixol did not produce catalepsy. In comparison, no cataleptic behaviour was observed after sulpiride (10, 50 and 250 mg/kg).

4. Discussion

Intracerebral dialysis (Zetterstrt~m et al., 1983) was used in this study to measure changes in extracellular levels of endogenous dopamine and its metabolites in the striatum of awake rats following neuroleptic drug administration. The results demonstrate that each of the three neuroleptics, sulpiride, cis-flupenthixol and haloperidol, increased extracellular levels of striatal dopamine and its metabolites DOPAC and HVA. This finding follows our previous study showing that the dopamine agonist apomorphine reduced dopamine release and metabolism in vivo (Zetterstr0m and Ungerstedt, 1984). The data obtained with the dialysis technique in both of these investigations agree well with the idea of inhibitory dopamine autoreceptors regulating dopaminergic neurotransmission in the striatum, as originally proposed by Farnebo and Hamberger (1971). However, following measurement of the 5HT metabolite 5HIAA, these studies have provided little evidence for an influence of dopamine on 5HT neurons in this region. Our results are consistent with experiments carried out using other in vivo methods: it has been shown previously that haloperidol administration

35 caused an increase in efflux of radiolabelled d o p a m i n e in striatal push-pull perfusates (Bartholini et al., 1976; Nieoullon et al., 1979) and a rise in DOPAC in both striatum, measured by in vivo voltammetry (Gonon et al., 1981), and the ventricles, monitored by ventricular perfusion (Nielsen and Moore, 1982). Furthermore, push-pull perfusion studies have shown that the neuroleptic chlorpromazine enhanced the release of endogenous dopamine in the striatum (Lloyd and Bartholini, 1975). However, to our knowledge this is the first report of an in vivo study in which changes in endogenous dopamine release have been monitored simultaneously with measurements of its metabolism in the striatum following dopamine antagonist administration. An important finding in this study was that the rise in dopamine metabolites in the striatal perfusates after neuroleptic treatment did not correlate directly with changes in the amount of dopamine released. This is evident from four main observations: a) the change in time course of dopamine release after sulpiride was quite different from that of the dopamine metabolites (fig. 2); b) sulpiride had a greater effect on dopamine release than did cis-flupenthixol, but cis-flupenthixol caused a larger increase in DOPAC and HVA than did sulpiride (fig. 5); c) low doses of both haloperidol (0.05-0.5 m g / k g ) and cisflupenthixol (0.05 mg/kg) markedly enhanced dopamine metabolism while having no effect on dopamine release and (fig. 5); d) in all cases except the highest dose of haloperidol, the dopamine metabolism response was notably longer lasting than the dopamine release response (figs. 2-4). Further, in our previous study on apomorphine (Zetterstr~Sm and Ungerstedt, 1984), changes in dopamine release did not appear to correspond directly with changes in its metabolites. The finding of a poor correlation between changes in dopamine levels in the perfusates and the levels of its metabolites raises a number of points. First, it may be argued that small increases in dopamine release are not detected by the dialysis method due to rapid re-uptake of released dopamine into the nerve terminal. However, this seems unlikely in view of the fact that sulpiride in low doses induced a marked release of dopamine

(fig. 2). Furthermore, we assume that the basal levels of dopamine recovered in the perfusate represents a spillover from the synapse which reflects changes in release especially as we were able to inhibit the release with low doses of apomorphine (ZetterstriSm and Ungerstedt, 1984). Secondly, this result indicates that in vivo measurements of extracellular DOPAC or HVA may not provide a true representation of dopamine release. Similarly, other workers have reported that measurement of these metabolites in post mortem brain tissue is not a good index of dopamine release (Ponzio et al., 1981; Westerink and Spaan, 1982). Thirdly, the poor correlation indicates that the amount of extracellular DOPAC and HVA arising from recently released dopamine, which is then taken up and metabolized in the nerve terminal, is small. Further, our data suggest that the increase in extracellular DOPAC and HVA following the neuroleptics does not reflect dopamine release but probably an increase in dopamine synthesis. There is an overproduction of newly synthesized dopamine in the nerve terminal which is rapidly metabolized and does not reach the storage granules. Finally, the data suggest that the increase in dopamine release following the neuroleptics was mediated by a mechanism different from that causing an increase in dopamine metabolism. This idea is supported by the finding that while the individual neuroleptics had markedly different effects on dopamine release (sulpiride > flupenthixol > haloperidol) they were notably similar in their effects on the dopamine metabolites. It is possible that the apparently potent effect of sulpiride on dopamine reflects its high selectivity for the dopamine D2-receptor compared to cis-flupenthixol and haloperidol (Seeman, 1980). However, if this is the case it is not clear why all three neuroleptics appeared similar in their effects on dopamine metabolism. The present study showed that while DOPAC and HVA increased after neuroleptic administration this effect was not strictly dose-dependent (fig. 5), although the lowest doses of sulpiride and cis-flupenthixol were the least effective. In vitro experiments using the dialysis probe demonstrated that the amount of monoamine recovered through the membrane was strictly dependent on the -

36 monoamine concentration outside the tube. It is therefore unlikely that the poor dose-response was due to a non-linear recovery of the monoamines across the dialysis membrane from the extracellular medium. In addition, we are confident of the accuracy of the D O P A C data since D O P A C measurements obtained in a recent comparative study using the dialysis probe paralleled those obtained by in vivo voltammetry (Sharp et al., 1984). The higher doses of the neuroleptic drugs might be beyond the maximal effective dose for their actions on dopamine metabolism and this may contribute to the poor dose-responses. In this respect it has been reported that the dose-response curve for the effect of haloperidol on tissue levels of D O P A C and HVA reaches a plateau at about 1 m g / k g (Nicolau, 1980). The present experiments were performed using freely moving rats and it was therefore possible to directly relate neurochemical changes to behavioural observations. There was no correlation between changes in dopamine release or metabolism in the striatum and the occurrence of catalepsy following neuroleptic drug treatment. This point is emphasized by the fact that there were doses of sulpiride, haloperidol and cis-flupenthixol which increased D O P A C and HVA but did not cause catalepsy. Additionally, haloperidol and cisflupenthixol caused catalepsy at doses which increased dopamine release while this was not the case with sulpiride. It has been hypothesized that catalepsy induced by certain neuroleptic drugs is due to blockade of postsynaptic dopamine receptors in the striatum (see Costall and Naylor, 1979). Additionally, antagonism of these receptors is suggested to cause an increase in dopamine synthesis and its subsequent metabolism via neuronal feedback loops (Carlsson and Lindqvist, 1973; Bunney et al., 1973). However, lesioning of the striatonigral pathway does not prevent the D O P A C and HVA increase in rat striatum after haloperidol (Di Chiara et al., 1977; Garcia-Munoz et al., 1977), which indicates a presynaptic action of the neuroleptic. The results presented here directly demonstrate a clear dissociation of catalepsy and the neuroleptic-induced increase in dopamine metabolism and release, suggesting that the two effects are not mediated via the same mechanism. Fur-

ther, these data strongly support the idea that neuroleptic-induced catalepsy is not related to dopaminergic presynaptic events. In summary, sulpiride, haloperidol and cisflupenthixol were demonstrated to increase dopamine release and metabolism in the rat striatum while these drugs had little apparent effect on 5HT metabolism in this region. The effect of these neuroleptics on dopamine metabolism appeared to be separate from the effect on dopamine release and neither the increase in dopamine metabolism nor the increase in dopamine release were related to catalepsy. Finally, the present findings obtained with the dialysis technique demonstrate that this method should be a useful tool for future investigations of central dopamine function in vivo.

Acknowledgements We thank Ase HallstriSm for her skilled technical assistance and Kjerstin Cason for typing the manuscript. This work was supported by the Swedish Medical Research Council (B 8203574, A1-5/253). T.S. is sponsored by a Karolinska Institute Postdoctoral Fellowship.

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