Intrastriatal taurine increases striatal extracellular dopamine in a tetrodotoxin-sensitive manner in rats

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Neuroscience Letters 212 (1996) 175-178

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Intrastriatal taurine increases striatal extracellular dopamine in a tetrodotoxin-sensitive manner in rats Minna Ruotsalainen*, Liisa Ahtee Department of Pharmacy, Division of Pharmacology and Toxicology, P.O. Box 56 (Viikinkaari 5), FIN-O0014 University of Helsinki, Helsinki, Finland

Received 19 February 1996; revised version received 4 June 1996; accepted 10 June 1996

Abstract

In vivo effects of locally administered taurine on striatal dopamine release and metabolism were studied by microdialysis in freely moving rats. Concentrations of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in striatal dialysates were quantified by high pressure liquid chromatography (HPLC) using electrochemical detection. Infusion of 150 mM taurine into the striatum for 2 h induced a 2.5-fold increase in the extracellular dopamine concentration. Extracellular DOPAC concentration increased nearly 2-fold. Taurine infusion initially decreased HVA to 70% but afterwards increased it to 140% of the control. When taurine was infused simultaneously with 1 pM tetrodotoxin starting 60 min after tetrodotoxin, the output of dopamine did not differ from that in the presence of tetrodotoxin alone. Tetrodotoxin abolished the effects of taurine on dopamine metabolites as well. Tetrodotoxin-sensitivity of the effects of taurine on dopamine and its metabolites suggests that intrastriatal taurine elevates extracellular dopamine by releasing it from neuronal pool. Keywords: lntrastriatal taurine; Striatal dopamine release and metabolism; Tetrodotoxin-sensitivity; In vivo microdialysis; Rat

The amino acid taurine (2-aminoethanesulphonic acid) has long been known to be present in high concentrations in the central nervous system, but so far its function remains unclear. One possibility is that taurine acts as a neurotransmitter or as a neuromodulator [7]. It tends to reduce neuronal excitability, thereby mimicking the structurally related inhibitory neurotransmitters ),-aminobutyric acid ( G A B A ) and glycine [7,18]. Furthermore, there is evidence implying that taurine acts as a neurotransmitter or neuromodulator in the striatonigral pathway [3]. In our earlier studies, intraventricularly administered taurine as well as homotaurine (3-aminopropanesulphonic acid, a GABAA-receptor agonist) elevated striatal 3,4dihydroxyphenylacetic acid (DOPAC) concentration in a similar way to G A B A [1,13]. The three amino acids also clearly decreased striatal 3-methoxytyramine concentration. These results indicate that taurine inhibits the release of striatal d o p a m i n e and increases its synthesis. However, our recent microdialysis study in anaesthetised rats sug* Corresponding author. Tel.: +358 0 70859473; fax: +358 0 70859471 ; e-mail: [email protected]

gests that depending on the administration site, taurine either decreases or elevates extracellular striatal dopamine in vivo [16]. Intranigrally infused taurine decreased dopamine in ipsilateral striatum confirming the inhibitory role of taurine in the control of nigrostriatal dopaminergic neurons, whereas local infusion of taurine into the striatum increased extracellular dopamine. The present study was performed to clarify further effects of local taurine infusion on striatal dopamine and its metabolism in vivo. A microdialysis study was carried out using freely moving rats. Continuous intrastriatal infusion of the sodium channel blocker tetrodotoxin was used to demonstrate action potential dependency of taurineinduced release of striatal dopamine in dialysates [22]. Male Wistar rats (9-11 week old) weighing 2 0 0 - 3 1 0 g were maintained in groups of five to six animals at an ambient temperature 24 _+ 2°C and on a 12:12 h light/dark cycle (lights on at 0600 h and off at 1800 h). Animals had free access to tap water and standard rat diet. After surgery they were kept individually in their own cages. Microdialysis was performed using a modified Ishaped cannula [15,17]. The exposed tip of the dialysis

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membrane was 4 mm. The dialysis tube was prepared from polyacrylonitrile/sodiummethalylsulfonate copolymer (Filtral 20; o.d./i.d. 310:220/2m; Hospal, France). The probes were implanted during general pentobarbitone anaesthesia (pentobarbitone sodium 60-80 mg/kg, i.p.) and local lidocaine (5 mg/ml) anaesthesia. Coordinates were calculated relative to bregma and dura (A +1.0, L +2.7, D -6.0) according to Paxinos and Watson [14]. Perfusion experiments were carried out 42--46 h after implantation. On the day of the dialysis experiments, the rats were connected to a microperfusion pump (Harvard '22', Harvard Apparatus, MA, USA). The pump was set to a perfusion speed of 2/21/min and 40/21 samples were collected every 20 min. A 35/~1 aliquot of each sample was injected into the chromatograph. The perfusion medium was a modified Ringer solution containing in mM, NaC1 147, CaCI2 1.2, KCI 2.7, MgCI2 1.0 and ascorbic acid 0.04. Taurine and tetrodotoxin infusions were started after a stable baseline (four consecutive samples with stable dopamine levels) was obtained. Taurine (Fluka AG, Buchs, Switzerland) and tetrodotoxin (Sigma Chemical Co., St. Louis, MO, USA) were solved in the perfusion fluid and infused via a microdialysis probe into the striatum. Taurine was infused at the concentration of 150 raM, which gives a total taurine dose of 4.5 mg/rat during the 2 h infusion, because in our previous experiments i.c.v. taurine at doses of 10 and 36/2mol/rat (1.25 and 4.5 mg, respectively) altered the striatal dopamine metabolism [1,13] and when locally infused in anaesthetised rats taurine at the concentrations of 50 and 150 mM elevated tile striatal extracellular dopamine in a dose-related manner [16]. Control rats were infused with Ringer solution throughout the experiment. Moreover, the control experiments using 150 mM mannitol instead of taurine were performed to study the effect of the hyperosmolarity of the infused solution. After the experiments the rats were decapitated, their brains were rapidly taken out and immersed in phosphate buffer containing 10% formaldehyde. The correct placement of the dialysis probe was verified from sections cut with rodent brain matrix (RBM-4000C, Activational systems Inc., USA) by eye according to the atlas of Paxinos and Watson [14]. Only results derived from rats with correctly positioned dialysis probes were included in the statistical analysis. Dialysate contents of dopamine, DOPAC, and homovaniilic acid (HVA) were quantified by high pressure liquid chromatography (HPLC) with electrochemical detection. A Beckman series 110 B pump was used in conjunction with a glassy carbon working electrode set at +780 mV versus an Ag/AgCI reference electrode (ANTEC, The Netherlands). A Spherisorb ODS2 3/2 (100 x 4.6 ram) or ODS 5/2 (150 x 4.6 mm) reverse phase column was used. The mobile phase consisted of a mixture of 0.1 M NaH2PO 4 adjusted to pH 4.0 with 0.1 M citric acid, methanol (18-20%), 0 . 8 - 1 . 0 m M octanesul-

phonic acid and 0.2 mM EDTA. The flow rate was 1.0 ml/min. The average of four stable samples before drug treatment was considered as the control level (baseline), and was defined as 100%. All values given are expressed as percentages of the control level. The statistical evaluation was carried out using an analysis of variance (ANOVA) for repeated measurement with one grouping factor and one within factor (time). When significant treatment x time interaction (using Huynh-Feldt adjusted degree of freedom) was present, the analysis was continued by performing separate ANOVAs for each pair of treatments between the timepoints of interest. The absolute basal values (baselines) of the extracellular striatal concentrations of dopamine and its metabolites did not differ significantly in different experiments and are grouped together here. The means+_SEM (n = 20) were, dopamine 180 -+ 24 fmol/40/21; DOPAC 14.3 _+0.9 pmol/40/21; HVA 10.0 _ 0.7 pmol/40/~l. The concentrations of dopamine, DOPAC and HVA in dialysates from control rats infused with Ringer solution remained unchanged over the 6 h dialysis period. Furthermore, in control experiments in which rats were infused with 150 mM mannitol in Ringer solution instead of taurine to study the effect of elevated osmotic pressure, the extracellular dopamine, DOPAC or HVA were not increased. It is noted that the concentrations of drugs given represent the infused concentrations; the actual drug content reached in the tissue being smaller. The recovery of taurine in the probe used was not determined. Local administration of 150 mM taurine into the striatum for 2 h caused a maximum 2.5-fold increase in extracellular dopamine concentrations (P < 0.001 versus control rats; Fig. 1A). The extracellular DOPAC increased to a maximum of 190% (P < 0.001; Fig. 1B). Taurine infusion initially decreased HVA to about 70% (P < 0.01), but afterwards increased it to 140% (P < 0.01) of the control (Fig. 1C). Intrastriatal tetrodotoxin (TTX; 1/2M) infusion decreased dopamine concentration to a minimum of 13% of the control (P < 0.001 versus control rats, data not shown). DOPAC and HVA concentrations were not significantly altered by TTX (data not shown), q-"FX at the used concentration (1/2M) did not alter the behaviour of the animals. When taurine was infused simultaneously with TTX starting 60 min after TTX, the extracellular dopamine concentration decreased to a minimum of 9% of the control (Fig. 1A), and was similar to that in the presence of TI'X alone, q"FX abolished the effects of taurine on the dopamine metabolites DOPAC and HVA as well (Fig. 1B,C). The TTX-sensitivity of the effects of taurine suggests that taurine-induced release of dopamine is dependent on nerve action potential. Previously, taurine was found to facilitate [3H]dopamine release in striatal slices stimulated by potassium [10]. Furthermore, our present results with freely moving rats agree with our earlier findings that

M. Ruotsalainen, L. Ahtee / Neuroscience Letters 212 (1996) 175-178

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Fig. 1. Effects of TTX (1 #M) on taurine-induced (150 mM for 2 h) changes in extracellular dopamine (A), DOPAC (B), and HVA (C) concentrations in striatal dialysates, lntrastriatal infusion of TTX started 1 h before taurine was added into the perfusion fluid and continued until the end of the experiment. The data are given as means _+SEM (n = 5). Note that the scales in the ordinates differ. Statistical analyses were carried out by ANOVA for repeated measurement: (A) taurine versus control (P < 0.001), taurine versus taurine + TTX (P < 0.05); (B) taurine versus control (P < 0.001), taurine versus taurine + TTX (P < 0.001); (C) taurine versus control at 0-140 min after starting taurine (P < 0.01), taurine versus control at 140-280 min after starting taurine (P<0.01), taurine versus taurine + TTX (P < 0.001). intrastriatal taurine elevated extracellular dopamine in vivo in anaesthetised rats [16]. However, the onset of taurine-induced increase of the extracellular dopamine was delayed in freely moving rats when compared with anaesthetised rats. In part, this might be due to the differences in the experimental conditions. In freely moving rats, the lag times for administration of taurine and collecting the samples were longer because of the longer tubings needed. Moreover, d o p a m i n e baseline values are increased during halothane anaesthesia [20]. Also, in our study with anaesthetised rats the dopamine baseline val-

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ues were two to three times higher than in present study. Thus, it is possible that the pronounced effects of taurine during halothane anaesthesia is due to interaction of taurine with halothane. Other inhibitory amino acid neurotransmitters have been found to stimulate dopamine release in striatum as well. Thus, intrastriatal glycine (20 mM) increased striatal extracellular dopamine in conscious rats [23]. In striatal slice preparations both G A B A and glycine were found to stimulate spontaneous release of [3H]dopamine [5,9]. The stimulatory effects of taurine on dopamine release seem to be contradictory to earlier findings of the inhibitory role of taurine on dopaminergic neurons [1,4,13]. However, in these studies taurine was given intraventricularly and not locally as in the present study. Indeed, when we gave taurine intranigrally it reduced striatal extracellular dopamine concentration [16]. Further, effects of G A B A ergic compounds on dopaminergic neurons are somewhat contradictory. Thus, locally administered bicuculline, a G A B A A antagonist, as well as, the G A B A B antagonist phaclofen increased striatal extracellular dopamine concentration [19]. However, support to the stimulatory effect of taurine on nigrostriatal dopaminergic neurons is given by the findings that rats given taurine intranigrally showed contralateral circling behaviour which was antagonized both by bicuculline and strychnine [8]. Infusion of TTX during enhanced dopamine release enables discrimination between action potential dependent release and action potential independent release. Hence TTX-infusion during brain dialysis indicates whether drug-induced dopamine release is caused by exocytosis or by other mechanisms like carrier-mediated or neurotoxic mechanisms [22]. As found earlier by Westerink et al. [22] infusion of l / ~ M TTX significantly decreased the extracellular dopamine concentration, but did not significantly alter its metabolites, D O P A C and HVA. More interestingly, T T X blocked taurine induced elevation of extracellular dopamine, which suggests that taurine releases dopamine from neuronal pool. Intrastriatal taurine enhanced dopamine release and simultaneously elevated D O P A C concentrations. H V A was initially decreased, but afterwards increased. W e have found earlier an increase in extracellular D O P A C and a decrease in H V A during local infusion of taurine in anaesthetised rat [16]. In the present study simultaneous TTX infusion abolished taurine-induced changes in dopamine metabolites. Thus, taurine-induced changes in extracellular D O P A C and H V A seem to be related to neuronal processes as they were inhibited during simultaneous TTX infusion. At present, the pharmacological profile o f taurine is not known. Its effects on dopamine release and metabolism in the striatum appear slowly after intrastriatal administration. Also, the onset of the inhibitory action of taurine on spike discharges of Purkinje cells has been found to be slower than that of G A B A [12]. Okamoto and

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S a k a i c o n s i d e r e d it u n l i k e l y t h a t this d e l a y is solely d u e to d i f f e r i n g d i f f u s i b i l i t i e s o f t h e s e m o l e c u l e s . W h e t h e r this s l o w o n s e t o f t a u r i n e ' s e f f e c t is s u g g e s t i v e o f a n e u r o m o d u l a t o r y f u n c t i o n o f t a u r i n e , or o f a n i n d i r e c t m e c h a n i s m o f action, c a n n o t b e c o n c l u d e d at p r e s e n t . T h e effects o f t a u r i n e o n e x t r a c e l l u l a r d o p a m i n e r e s e m b l e d those of inhibitory amino acids GABA and glycine. There is e v i d e n c e t h a t t a u r i n e m i g h t act o n b o t h G A B A a n d glycine r e c e p t o r s in b r a i n [2,6,11,21]. H o w e v e r , it is n o t c l e a r w h e t h e r t a u r i n e c a u s e s its e f f e c t s b y a c t i n g o n glyc i n e r e c e p t o r s , G A B A r e c e p t o r s or a n y r e c e p t o r s . In c o n c l u s i o n , o u r r e s u l t s c o n f i r m the earlier s u g g e s t i o n s o f t h e s t i m u l a t o r y role o f intrastriatal t a u r i n e in t h e r e g u l a t i o n o f n i g r o s t r i a t a l d o p a m i n e r g i c n e u r o n s . Furthermore, present experiments showing TTX-sensitivity of taurine-induced dopamine release demonstrate that the e n h a n c e m e n t o f striatal d o p a m i n e r e l e a s e b y t a u r i n e depends on nerve action potential. W e s i n c e r e l y t h a n k Dr. J. K o r f a n d Dr. B.H.C. W e s t e r i n k f o r a d v i c e a n d i n s t r u c t i o n s in s e t t i n g u p the m i c r o d i a l y s i s t e c h n i q u e . W e w i s h to t h a n k Ph. Lic. J u h a n i T u o m i n e n , D e p a r t m e n t o f Biostatistics, U n i v e r s i t y o f T u r k u , F i n l a n d f o r v a l u a b l e a d v i c e in statistical a n a l y s e s . T h i s w o r k w a s f i n a n c i a l l y s u p p o r t e d b y g r a n t s f r o m the F i n n i s h C u l t u r a l F o u n d a t i o n , the L e i r a s R e s e a r c h F o u n d a t i o n a n d t h e R e s e a r c h C o u n c i l for H e a l t h o f t h e A c a d emy of Finland. [1] Ahtee, L. and Vahala M.-L., Taurine and its derivatives alter brain dopamine metabolism similarly to GABA in mice and rats. In S. Oja, L. Ahtee, P. Kontro, M.K. Paasonen (Eds.), Taurine: Biological Actions and Clinical Perspectives, Alan R. Liss, New York, 1985, pp. 331-341. [2] Bureau, M.H. and Olsen, R.W., Taurine acts on a subclass of GABA A receptors in mammalian brain in vitro, Eur. J. Pharmacol., 207 (1991) 9-16. [3] Della Corte, L., Bolam, J.P., Clarke, D.J., Parry, D.M. and Smith, A.D., Sites of [3H]taurine uptake in the rat substantia nigra in relation to the release of taurine from the striatonigral pathway, Eur. J. Neurosci., 2 (1990) 50-61. [4] Garcia de Yebenes Prous, J., Carlsson, A. and Mena Gomez, M.A., The effect of taurine on motor behaviour, body temperature and monoamine metabolism in the rat brain, NaunynSchmiedeberg's Arch. Pharmacol., 304 (1978) 95-99. [5] Giorguieff, M.F., Kemel, M.L., Glowinski, J. and Besson, M.J., Stimulation of dopamine release by GABA in rat striatal slices, Brain Res., 139 (1978) 115-130. [6] Horikoshi, T., Asanuma, A., Yanagisawa, K., Anzai, K. and Goto, S., Taurine and fl-alanine act on both GABA and glycine receptors in Xenopus occyte injected with mouse brain messenger RNA, Mol. Brain Res., 4 (1988) 97-105. [7] Huxtable, R.J., Taurine in the central nervous system and the mammalian actions of taurine, Prog. Neurobiol., 32 (1989) 471533.

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