Effect of fluoxetine on serotonin and dopamine concentration in microdialysis fluid from rat striatum

Life Sciences, Vol. Printed in the U S A

50, pp.

1683-1690

Pergamon

Press

EFFECT OF FLUOXETINE ON S E R O T O N I N AND D O P A M I N E C O N C E N T R A T I O N IN M I C R O D I A L Y S I S FLUID FROM RAT S T R I A T U M

Kenneth W. Perry and Ray W. Fuller Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285 (Received

in final

form March 23,

1992)

Summarv

Fluoxetine injected i.p. into rats at a dose of 10 mg/kg rapidly increased serotonin concentration in microdialysis fluid from the striatum by at least 4-fold, an increase that was maintained throughout the 3 hr observation period. Dopamine concentration in the microdialysis fluid did not change. The concentration of the two dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, was not changed in the microdialysis fluid, whereas the concentration of the serotonin metabolite, 5-hydroxyindoleacetic acid, was significantly decreased after fluoxetine injection. The increased extracellular concentration of serotonin no doubt resulted from inhibition of the serotonin uptake carrier by fluoxetine, and the lack of change in dopamine is evidence for the specificity of action of this uptake inhibitor. Fluoxetine is a selective inhibitor of serotonin uptake that is now widely used in the treatment of mental depression. The inhibition of the uptake carrier on serotonin nerve terminals is expected to amplify serotonergic signals by increasing the concentration of serotonin in the synaptic cleft. Although there is extensive evidence from animal studies that serotonergic function is enhanced after administration of fluoxetine or other serotonin uptake inhibitors, based on neurochemical, behavioral, neuroendocrine and other functional changes which are generally subtle (1,2), there has been limited ability to get direct evidence for increased amounts of serotonin in the synaptic cleft. Geyer et al (3) used a cytofluorimetric technique called "fading" to show that fluoxetine increased extracellul~r concentrations of serotonin in raphe nuclei of rat brain region, whereas monoamine oxidase inhibitors mainly increased intracellular serotonin. Marsden et al (4) used the then-new technique of in vivo voltammetry to show that fluoxetine increased extracellular levels of serotonin in rat striatum. They provided evidence that the voltammetric signal they were measuring was actually serotonin by showing that p-chloroamphetamine caused an initial large increase in the peak, the increase being blocked by fluoxetine pretreatment which prevents the carrier-dependent release of serotonin by p-chloroamphetamine. Guan and McBride (5) used a push-pull cannula in the rat nucleus accumbens to show that fluoxetine caused a several-fold increase in serotonin concentration (measured by liquid chromatography with electrochemical detection) in extracellular fluid. Most recently, Auerbach et al (6) have reported that addition of fluoxetine via the microdialysis probe increases serotonin concentration in the microdialysis fluid collected from rat hypothalamus. Our purpose here is to report that systemic administration of fluoxetine to rats at the dose most commonly used to inhibit serotonin uptake results in a rapid and sustained Copyright

0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights

reserved.

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several-fold increase in serotonin concentration in microdialysis fluid from rat striatum. In contrast, dopamine concentration in the microdialysis fluid was unchanged.

Materials and Methods Surgical. Surgery was performed on male Sprague-Dawley rats (260-300 g) (Charles River Laboratories, Portage, MI) under chloral hydrate/pentobarbital anesthesia (170 and 36 mg/kg i.p. in 30% propylene glycol, 14% ethanol). Each rat was placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA), then the skull was exposed and a small hole was drilled to insert a miniature dialysis probe into the anterior lateral striatum. The coordinates used were 2 mm anterior to bregma, 3 mm from the mid-sagittal suture and 6 mm ventral from dura (7). The probe was slowly lowered into position and fixed in place with cranioplastic cement (Plastics One, Roanoke, VA). The ends of the plastic tubing were plugged with fine wire and the rat was allowed to recover for two days before being used in microdialysis experiments. Rats were given free access to food and water during the recovery period. Probe. A loop style probe was made with dialysis tubing (C-D Medical, Miami, FL) heat shrunk into PE-10 polyethylene tubing which was fused to PE-20 tubing (Clay Adams, Parsippany, NJ) (see diagram below): PE 20 tubing

Melted seal PE 10 tul Enamel coated Fine wire

Heat shrunk (no ! Dialysis tubing

~m 0.6 mm

This probe is similar in design to that described previously (8); however, it has two advantages over previous types of probes: 1) the plastic tubing is flexible enough to move with the brain if the rat shakes its head vigorously, an important consideration when using freely moving rats, and 2) there is no metal or glue used in the probe, which eliminates two possible sources of contamination for the HPLC-EC assay. The in vitro recoveries for this probe at 1.5 I~l/min were: 3,4-dihydroxyphenylacetic acid (DOPAC) = 26%, homovanillic acid (HVA) = 24%, 5-hydroxyindoleacetic acid (5-HIAA) = 28%, dopamine = 21%, serotonin = 24%. J~.~V.~.. Two days after surgery the rat was placed in a plastic bowl and connected to a liquid swivel (CMN120 system for freely moving animals, BioAnalytical Systems, West Lafayette, IN). The input of the dialysis probe was connected to a syringe pump (Harvard Instruments, Model 22, South Natick, MA) which delivered an artificial corebrospinal fluid (CSF) to the probe at a rate of 1.5 I~l/min. The artificial CSF was composed of 150 mM NaCI, 3 mM KCI, 1.7 mM CaCI2 and 0.9 mM MgCI2 and was sterilized by filtration through a 0.2 micron filter. The output from the swivel was attached to an electrically actuated switching valve (Valco Instruments, Houston, TX) which was part of an on-line analysis system that assayed the dialysis output from two rats in parallel. This system allowed a control rat to be studied at the same time as a drug-treated rat. The switching valve alternated between the output of the two rats every 15 min and directed the output from one of the rats into the sample loop of an electrically actuated

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ten-port HPLC injection valve (Valco) while the output from the other rat went to a waste line. The HPLC valve rotation was synchronized with the switching valve by use of an electronic timer (Chrontrol, Lindburg Enterprises, Inc., San Diego, CA) so that an injection onto the HPLC column was made every 15 min. Assay. A ten-port HPLC valve with a 20 I~1 sample loop was used in a configuration with a small sample cleanup column (Spherisorb 31~ ODS2, 2x10 mm, Keystone Scientific, Bellefonte, PA) which trapped a late-eluting peak contained in the striatal dialysate samples. When the valve was in the inject position, dialysate sample from the sample loop was injected onto the sample cleanup column then onto the analytical column (Spherisorb 31~ ODS2, 2x150 mm, Keystone Scientific). When the valve was in the load position and dialysate sample was being collected in the sample loop, the sample cleanup column was being backflushed with mobile phase. The valve was in the inject position for 3 min and in the load position for 12 min (see diagram): SAMPLE IN

R~

INJECT POSITION (3 MIH.)

SAMPLE IN (FROM

LOAD POSITION (12 MIN.)

The mobile phase for both columns was the same and consisted of 75 mM sodium acetate, 75 mg/I sodium octanesulfonic acid, 10% methanol, 0.5 mM EDTA at pH 4.4 (adjusted with acetic acid). The flow rate for both columns was 0.22 ml/min and the analytical column was maintained at 40°C while the cleanup column was mounted close to the valve at room temperature. An electrochemical detector (EG&G PARC, Princeton, NJ) with a dual glassy carbon electrode was used to detect dopamine and serotonin and their metabolites. Dopamine and serotonin were assayed at a potential of 600 mV, sensitivity setting of 0.2 nAN, and DOPAC, 5-HIAA and HVA at a potential of 750 mV, sensitivity setting of 20 nAN. These assay conditions allowed the measurement of serotonin without any interference from 3-methoxytyramine (3-MT) which is a metabolite of dopamine that can sometimes co-elute with serotonin. The 3-MT peak eluted slightly later than serotonin and was not detectable at the low potential used to measure serotonin and dopamine. The output of both channels was sent to a HewlettPackard HP1000 chromatography data system which calculated peak heights and sample concentrations. The sensitivity for serotonin and dopamine was approximately 0.1 pmol/ml dialysate or 2 fmol injected onto the column. Values for all samples are expressed in pmol/ml for a 20 I~1 sample. Results

Fig. 1 compares the effect of saline injection and the injection of fluoxetine hydrochloride at a dose of 10 mg/kg i.p. During alternate 15 min sampling periods, serotonin concentration was stable during 150 min before injection. After saline injection, there was no change in serotonin concentration. But after the injection of fluoxetine, serotonin concentration increased quickly and reached a new plateau within 90 min, slightly more than 4 times the mean basal level. The increase in serotonin concentration was maintained for more than 3 hr after fluoxetine injection.

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.

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Minutes FIG. 1 Serotonin concentration in extracellular microdialysate fluid from rat striatum. Six samples were taken before the injection; the arrow marks the time of i.p. injection of saline or of fluoxetine hydrochloride (10 mg/kg). Mean values + standard errors for 4 (saline) or 6 (fluoxetine) rats per group are shown• The * indicates P<.05 relative to saline controls.

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FIG. 2 Dopamine concentration in extracellular microdialysate fluid from rat striatum. All conditions as in FIG• 1•

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Effect of Fluoxetine

(a)5-HIAA

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FIG. 3 Concentrations of serotonin and dopamine metabolites in extracellular dialysis fluid from rat striatum (a) 5-HIAA, (b) DOPAC, (c) HVA. All conditions as in FIG. 1. FIG. 2 shows dopamine concentration in the microdialysis fluid. Dopamine concentration was approximately 30 times the concentration of serotonin during the preinjection period. Administration of either saline or fluoxetine did not change dopamine concentration in any pronounced manner, although both lines tended to drift upward during the total 6 hr observation period. No significant difference between the fluoxetine-treated and saline-treated groups was found at any time. The concentrations of DOPAC and HVA and of the serotonin metabolite 5-HIAA are shown in FIG. 3. There was a significant decrease in the concentration of 5-HIAA but no significant change in DOPAC or HVA after fluoxetine injection.

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Serotonin

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FIG. 4 Serotonin and dopamine concentrations in rat striatal dialysate after injection of fluoxetine and infusion of tetrodotoxin. Fluoxetine was injected i.p. at 10 mg/kg as in FIG. 1. Tetrodotoxin was infused through the dialysis probe at a concentration of 3 IIM for a period of 1 hr (TTX rectangle). In FIG. 4 fluoxetine injection caused an increase in serotonin with no change in dopamine, as seen previously in FIG. 1. Then infusion of tetrodotoxin through the dialysis probe led to an abrupt drop in both serotonin and dopamine concentrations to less than 5% of pre-infusion levels. Serotonin concentration recovered to the same level as before tetrodotoxin infusion at about 90 min after the end of infusion. However, dopamine concentration did not return to normal levels during this time period.

Discussion After fluoxetine injection into rats, there is a rapid and sustained inhibition of the serotonin uptake carrier in brain (9,10,11). The 10 mg/kg i.p. dose of fluoxetine is the dose typically used to inhibit serotonin uptake in rats. It is the lowest dose causing essentially maximum (a) protection against p-chloroamphetamine-induced depletion of brain serotonin (10), (b) lowering of brain concentration of 5-HIAA (11 ), and (c) potentiation of L-5-hydroxytrytophan-induced elevation of serum corticosterone concentration (12), for example. The rapid increase in extracellular serotonin concentration which we have observed after fluoxetine injection is an expected consequence of inhibition of the uptake carrier. Serotonin released into the synaptic cleft would remain there longer when the membrane uptake carrier is inhibited, because the function of the carrier is to inactivate serotonin by transporting it back inside the nerve terminal. Sharp et al (13) used citalopram in the perfusate to inhibit serotonin uptake and obtain measurable serotonin levels in rat hippocampal microdialysates. Auerbach et al (6) and Schwartz et al (14) have shown that direct administration of fluoxetine into rat hypothalamus via the microdialysis probe increases serotonin concentration in the dialysate approximately six-fold. The present study shows that systemic administration of fluoxetine, at a dose previously found to inhibit the serotonin uptake carrier selectively (9,10), also increases serotonin concentration in striatal microdialysate. The

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decrease in 5-HIAA concentration in the extracellular fluid agrees with the decrease in tissue concentrations of 5-HIAA previously seen after fluoxetine, which resulted from a compensatory reduction in serotonin turnover (11). However, tissue levels of serotonin were not changed after fluoxetine. Tetrodotoxin ('I-I'X) was first utilized by Westerink et al (15) to demonstrate that basal dopamine in striatal dialysate was action potential dependent and thus was released from neurons. Carboni and Di Chiara (16) showed that basal serotonin in transcortical dialysate was TTX sensitive and the increase in serotonin caused by systemic injection of chlorimipramine was also TTX sensitive. Our results are consistent with these previous findings in that both the dopamine and serotonin in striatal dialysate are TTX sensitive and the increase in serotonin caused by fluoxetine is r l X sensitive. A new observation from our results in striatal dialysates is that serotonin neurons recovered from TTX infusion faster than dopamine neurons. Since the introduction of fluoxetine and other selective inhibitors of serotonin uptake into clinical use, it has been speculated that these agents might cause extrapyramidal disorders by reducing dopaminergic function through enhancement of inhibitory serotonergic input to dopamine neurons important in motor function (17-21). Baldessarini and Marsh (17) have reported that fluoxetine administration to rats causes a small but significant reduction in dopa accumulation after decarboxylase inhibition in striatum and some other brain regions. In striatum the accumulation of dopa is a measure of dopamine synthesis rate, so the inference was that fluoxetine ~ecreases dopamine turnover in striatum. In contrast, Waldmeier and Delini-Stula (22) have shown that fluoxetine and other selective inhibitors of serotonin uptake increased dopamine turnover (measured by increased DOPAC and HVA concentrations) in brain of rats treated with neuroleptic drugs, but did not change DOPAC or HVA by themselves. Our data on DOPAC and HVA concentrations in the microdialysis fluid show no changes, just as tissue concentrations of DOPAC and HVA are reported not to change after fluoxetine injection. In fact, there is an extensive literature on interactions between dopamine and serotonin neuronal systems in rat brain, and there is neuroanatomic evidence that serotonergic afferents make input to the substantia nigra and to the striatum (23, 24). De Simoni et al (23) showed that electrical stimulation of the dorsal raphe, the source of serotonergic projections to the substantia nigra and to the striatum, increased dopamine metabolism as measured by DOPAC levels in striatum but did not affect dopamine release as measured by 3-methoxytyramine levels or accumulation after monoamine oxidase inhibition. Although dorsal raphe stimulation to increase release might be expected to have the same effect as inhibition of serotonin uptake by fluoxetine, because both would increase serotonergic input to dopamine neurons, De Simoni et al (23) observed an increase in striatal dopamine turnover after raphe stimulation and Baldessarini and Marsh (17) reported a decrease in striatal dopamine turnover after fluoxetine. Our inability to measure any effect on dopamine release by striatal microdialysis after a dose of fluoxetine reported to decrease striatal dopamine synthesis (16) is consistent with the suggestion of De Simoni et al (23) that dopamine metabolism can be affected without dopamine release being affected. Kelland et al (24) have argued that serotonergic input exerts only minor influences on nigrostriatal dopamine neurons. Thus caution should be used in suggesting a change in striatal dopaminergic function as a result of serotonin uptake inhibition. In summary, the present findings show that systemic administration of fluoxetine at a dose that inhibits the serotonin uptake carrier selectively causes a rapid and sustained increase in extracellular concentration of serotonin in rat striatum. No change in extracellular dopamine concentration was observed. Showing that at the 10 mg/kg dose

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commonly used, fluoxetine does not inhibit the dopamine uptake carrier and suggesting that enhancement of serotonergic input to nigrostriatal dopamine neurons does not alter dopamine release.

Acknowledamen|~ We thank Dr. Lee A. Phebus for the design of the dialysis probe and his helpful instruction and advice on dialysis techniques. We also thank Sue Luecke for her help with the diagrams and figures.

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