Endogenous GABA potentiates the potassium-induced release of dopamine in striatum of the freely moving rat: a microdialysis study

Brain Research Bulletin, Vol. 50, No. 3, pp. 209 –214, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter

PII S0361-9230(99)00199-9

Endogenous GABA potentiates the potassium-induced release of dopamine in striatum of the freely moving rat: A microdialysis study A. Galindo, A. Del Arco and F. Mora Department of Physiology, Faculty of Medicine, Universidad Complutense, Ciudad Universitaria, Madrid, Spain [Received 4 February 1999; Revised 23 July 1999; Accepted 27 July 1999] [2,12,21,22] or reduce [2,11,13] stimulated-[3H]dopamine release. Therefore, it is still unclear how striatal GABA modulates the dopaminergic neurotransmission in the striatum. All the above studies were performed using pharmacological agonists of GABA receptors or exogenous GABA. The present study investigated the effects of endogenous GABA on basal and potassium-stimulated extracellular concentration of dopamine in the striatum of the rat. For this purpose, increases in the extracellular concentrations of endogenous GABA were produced by the GABA uptake inhibitor nipecotic acid (NIP) perfused through the microdialysis probe into the striatum.

ABSTRACT: Using microdialysis, a study was made of the effects of an increase of endogenous GABA on basal and potassium-stimulated release of dopamine in striatum of the awake rat. The dopamine metabolites DOPAC and HVA were also measured. Extracellular concentrations of GABA were increased by inhibiting its uptake with nipecotic acid. TTX (10 ␮M) reduced basal extracellular concentrations of dopamine, and dopamine metabolites, but not GABA. Nipecotic acid (200, 500, and 1000 ␮M) produced a dose-related increase in basal extracellular concentrations of GABA, but did not change basal extracellular concentrations of dopamine and dopamine metabolites. However, nipecotic acid significantly enhanced the dopamine release produced by perfusion of potassium (50 mM) and also enhanced the extracellular increase of GABA produced by high potassium. These results suggest that an increase of endogenous GABA is facilitating the stimulated release, but not the basal release, of dopamine in the striatum of the awake rat. © 1999 Elsevier Science Inc.

MATERIALS AND METHODS Animals and Surgery Male Wistar rats (2–3 months, 250 –350 g weight) were housed in individual wire mesh cages, provided with food and water ad libitum, and maintained in a temperature-controlled room under a light/dark cycle (lights on/off at 2000 p.m./0800 h). All in vivo experiments, performed at the Universidad Complutense of Madrid, were conducted during the dark period of the light/dark cycle and followed the guidelines of the International Council for Laboratory Animal Science (ICLAS). Under Equithesin (2 ml/kg intraperitoneal [i.p.]) anaesthesia, bilateral guide-cannulae were stereotaxically implanted in the brain to accommodate microdialysis probes in striatum of the rats. A guide-cannulae assembly [18] was then fixed to the skull by means of two anchorage screws and the application of dental cement. When inserted, the tip of the microdialysis probe was placed into the striatum (oral bar set at ⫺4 mm): 0.6 mm rostral, 2.5 mm lateral from bregma and 2.8 mm ventral from dura mater (Fig. 1) [8].

KEY WORDS: GABA, Dopamine, DOPAC, HVA, Nipecotic acid, Potassium stimulation, Striatum, Microdialysis, Rat.

INTRODUCTION The interaction between GABA and dopamine is an important focus of research in striatum because of the role of both these neurotransmitters in the physiology as well as in the pathology of the basal ganglia. In striatum, GABA is located in interneurons and projection neurons to the substantia nigra [19]. In striatum GABA neurons receive dopamine terminals from substantia nigra through the nigrostriatal dopaminergic pathway [19]. Several studies have suggested that GABA is modulating dopaminergic activity in striatum [20,27] but there are very few in vivo studies regarding GABA-dopamine interaction, and the results are controversial. Recent work has reported the effects of GABAergic drugs on dopamine release in striatum. Microdialysis studies in vivo have shown that GABA agonists decrease [20], increase [27] or do not modify [6,14] basal extracellular concentrations of dopamine while in vitro studies have shown that GABA agonists can increase [1,12] or not modify [21] the basal release of [3H]dopamine. These in vitro studies have also shown that GABA agonists can enhance

In Vivo Microdialysis Three to four days after surgery, microdialysis probes were inserted and experiments were performed in the freely moving rat. Probes of concentric design were constructed in our own workshop, of an active dialyzing length of 4 mm. The dialysis membrane had a molecular-weight cut-off of 5,000 da (Hospal). The

* Address for correspondence: Prof. Francisco Mora, Department of Physiology, Faculty of Medicine, Universidad Complutense, Ciudad Universitaria, s/n, 28040 Madrid, Spain. Fax: ⫹34-1-394-16-28. E-mail: [email protected]

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FIG. 1. Schematic view of the stereotaxic location of the microdialysis probe.

probes were perfused with artificial cerebrospinal fluid (CSF) (composition in mM: NaCl, 137; CaCl2, 3.4; KCl, 3; MgSO4, 1; NaH2PO4, 0.5; Na2HPO4, 2; glucose, 3; pH 7.3) at a flow rate of 1.5 ␮l/min. NaCl 137 mM was replaced by NaCl 90 mM when KCl 50 mM was used in the artificial CSF. The average relative in vitro recovery obtained was (room temperature): GABA ⫽ 14.6 ⫾ 1%; dopamine ⫽ 12.1 ⫾ 1.8%; HVA ⫽ 13.2 ⫾ 0.8; DOPAC ⫽ 10.4 ⫾ 0.7% (mean ⫾ SEM, n ⫽ 8). Once basal concentrations of amino acids and catecholamines were established, 20-min samples were collected for 240 min and immediately stored at ⫺80°C until analyzed. The change in the perfusion medium during experiments was made by a liquid switch (Harvard Apparatus, South Natick, MA, USA). Nipecotic acid (Tocris Cookson Ltd, Bristol, UK) (200, 500, and 1000 ␮M) was perfused for 60 min TTX (RBI, Natick, MA, USA) (10 ␮M) was perfused for 60 min. The drugs were dissolved in artificial CSF before infusion through the microdialysis probe. The effective extracellular concentration of the drug, estimated from the relative recovery of the microdialysis probes, is ⬍15% of the artificial CSF concentration. The microdialysis system (tubing and swivel) was filled with a diluted solution of benzolkonium chloride (Armil威) between experiments to prevent bacterial growth. At the end of the experiments the animals were anaesthetized with Equithesin and perfused intracardially with 0.9% saline followed by 10% formalin. The brain was removed and the placement of the microdialysis probe was verified with a cryostat microtome and viewing lens.

Amino Acid and Catecholamine Analysis The amino acid content of samples was analyzed by reversephase high-performance liquid chromatography (HPLC) and fluorometric detection by means of precolumn derivatization of 5-␮l samples with an O-phthalaldehyde solution as previously described [17,18]. Samples were injected in a Rheodine injector (20-␮l loop) running first through a precolumn C18 (Spherisorb威, Waters, Milford, MA, USA) and then through a 4.6 ⫻ 150 mm C18 column (Spherisorb威 ODS-2, Waters). A gradient program of two mobile phases at a flow rate of 1 ml/min was used. Solution A was a 95:5 (vol./vol.) mixture of 50 mM sodium acetate buffer (pH 5.67) and methanol, to which 12.5 ml/l isopropyl alcohol was added; solution B was a 70:30 (vol./vol.) methanol/water mixture. These conditions allowed GABA to be detected in 15 min. The detection limit in our 5-␮l samples was 0.05 ␮M for GABA. The catecholamine content of samples was analyzed by reverse-phase HPLC and electrochemical detection [17]. Samples were injected in a Rheodyne injector (25-␮l loop) running first in a C18 precolumn (Nova-Pack威, Waters) and then in a C18 column of 4-␮m particles, and 3.9 ⫻ 150 mm (Nova-Pack威, Waters). The mobile phase consisted of 0.1 M acetate-citrate buffer (pH 3.5 adjusted with HCl 1 equ/l), 1 mM EDTA, 2.9 mM sodium octyl sulphonate, and 18% methanol. The flow rate was maintained at 1 ml/min. These conditions allowed dopamine to be detected within 4 min. The compounds were measured by a coulometric detector (Coulochem II model 5200A, ESA, Chelmsford, MA, USA). A

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FIG. 2. Effects of intracerebral perfusion of nipecotic acid (NIP) for 60 min on dialysate concentrations of GABA, dopamine, DOPAC, and HVA in the striatum of the awake rat. Data (mean ⫾ SEM) are presented as percentages of control values (n ⫽ 5–9; *p ⬍ 0.001).

conditioning cell (ESA 5021) was set at ⫹50 mV and the analytical cells (ESA 5011) were set at ⫹340 mV (cell 1) and at ⫺250 mV (cell 2). The detection limit in our 25-␮l samples was 0.2 nM for dopamine. The amino acids and catecholamines chromatograms were processed using the MAXIMA 820 (Waters) software. Statistical Analysis Data are reported as absolute concentrations and percentages of baseline, not corrected for in vitro recovery. The effects of high potassium and nipecotic acid on dialysate GABA, dopamine, DOPAC, and HVA were calculated as the difference between the average of the samples in which these drugs were perfused and the average of three control samples. The effects of TTX on dialysate GABA, dopamine, DOPAC, and HVA were calculated as the difference between the average of the last six samples (140 min– 240 min) and the average of three control samples. A two factorial test (time-dose; time-treatment) with repeated measures followed by Dunnett’s t-test was performed for multiple comparisons. A regression analysis (Pearson’s coefficient) was performed for the study of correlation between GABA and nipecotic acid. RESULTS Effects of TTX on Basal Dialysate Concentrations of GABA, Dopamine and Dopamine Metabolites The basal dialysate concentrations of amino acids and catecholamines were: 0.185 ⫾ 0.04 ␮M for GABA; 1.78 ⫾ 0.35 nM

for dopamine; 699.8 ⫾ 123 nM for DOPAC; and 481.6 ⫾ 38 nM for HVA (means ⫾ SEM, n ⫽ 7). TTX (10 ␮M) perfused through the microdialysis probe reduced significantly ( p ⬍ 0.01, n ⫽ 5) basal dialysate concentrations of dopamine by 65% [F(3,81) ⫽ 26.3], DOPAC by 70% [F(3,81) ⫽ 78.3], and HVA by 50% [F(3,81) ⫽ 62.41] (as a maximal effect after 60-min perfusion of TTX), but did not change basal dialysate concentrations of GABA. Effects of NIP on Basal Dialysate Concentrations of GABA, Dopamine, and Dopamine Metabolites NIP (200, 500, and 1000 ␮M) produced a dose-related (r ⫽ 0.54, p ⬍ 0.02) increase in dialysate concentrations of GABA by 0.18 ⫾ 0.02 [F(6,135) ⫽ 7.07], 0.25 ⫾ 0.07 [F(6,135) ⫽ 15.68], and 0.39 ⫾ 0.05 ␮M [F(6,135) ⫽ 65.28], respectively (n ⫽ 5–9, p ⬍ 0.001). NIP did not change basal dialysate concentrations of dopamine, DOPAC, and HVA at any dose used (Fig. 2). Effects of NIP on Potassium-Stimulated Dialysate Concentrations of GABA, Dopamine and Dopamine Metabolites The perfusion of high potassium (50 mM) for 40 min produced a maximal increase in dialysate concentrations of GABA by 190% [F(8,99) ⫽ 14.4], and dopamine by 230% [F(8,99) ⫽ 27.98], and reduced dialysate concentrations of DOPAC by 40%, and HVA by 45% (n ⫽ 7, p ⬍ 0.01) (Fig. 3). NIP (1000 ␮M) significantly enhanced increases in dialysate

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FIG. 4. Effects of intracerebral perfusion nipecotic acid (NIP) on the increases on dialysate concentrations of GABA produced by 50 mM potassium in the striatum of the awake rat. Data (mean ⫾ SEM) are presented as absolute values (as the average of two samples with potassium) (n ⫽ 5; **p ⬍ 0.01).

FIG. 3. Effects of intracerebral perfusion of 50 mM potassium (40 min) and 50 mM potasium plus nipecotic acid (NIP) on dialysate concentrations of dopamine, DOPAC, and HVA in the striatum of the awake rat. Data (mean ⫾ SEM) are presented as absolute values (n ⫽ 5; *p ⬍ 0.05).

concentrations of dopamine [F(1,99) ⫽ 5.65, p ⬍ 0.05] (Fig. 3) and GABA [F(1,99) ⫽ 29.05, p ⬍ 0.01] (Fig. 4) produced by potassium 50 mM, but did not alter the decreases in dialysate concentrations of DOPAC and HVA produced by potassium at the same concentration. DISCUSSION As shown in this study, an increase in the extracellular concentrations of endogenous GABA (produced by the GABA uptake inhibitor NIP) did not change basal extracellular concentrations of dopamine, DOPAC or HVA, but enhanced dopamine release stimulated by potassium. NIP also enhanced GABA release stimulated by potassium. As already reported [15,26], the perfusion of TTX reduced basal extracellular concentrations of dopamine and dopamine me-

tabolites in striatum. This experimental approach using TTX was of relevance to our study because it allowed us to investigate the possible inhibition of basal dopamine release produced by endogenous GABA under our experimental conditions. The increase in the extracellular concentration of the endogenous GABA did not, however, change basal extracellular concentrations of dopamine, DOPAC, and HVA. These results are in agreement with other in vivo and in vitro studies using GABA agonists in striatum [6,14, 21], which suggests that GABA is not modulating spontaneous dopamine release in this structure of the brain. In contrast, other in vivo or in vitro studies have reported that GABA agonists decrease or increase basal extracellular dopamine in striatum [1,20,27]. It might be that different experimental conditions may account for the different effects of GABA agonists in striatum (i.e., in vivo vs. in vitro; awake vs. anaestethized; chronic vs. acute probe implantation). As shown in the Results section, potassium-stimulated dopamine release was enhanced by an increase of endogenous GABA. Other in vitro studies have shown similar results with GABA agonists after electrical- or potassium-stimulation of dopamine release [12,21,22]. This suggests that GABA is facilitating the stimulated dopaminergic activity in striatum of the rat. This facilitatory effect of endogenous GABA could be produced by a direct action on GABA receptors or GABA transporters located in the dopaminergic terminal. In fact, in vitro studies have suggested that GABA can enhance dopamine release activating GABA transporters located on dopaminergic terminals (heterotransporters) [12]. However, since GABA transporters are inhibited by NIP in the present study, the possibility that GABA acts on GABA heterotransporters to enhance dopamine release seems unlikely. Alternatively, endogenous GABA can act on GABA receptors, presumably located on dopaminergic terminals [13,20], to enhance dopamine release. However, there is not in vivo experimental support indicating that GABA can increase striatal dopamine release acting directly on GABA receptors located on dopamine terminals. Thus, the effects of endogenous GABA-enhancing dopamine release in striatum of the awake rat could be mediated through GABA receptors involving a striatum-substantia nigra-striatum loop. This possibility has been suggested in another in vivo study using intrastriatal infusion of GABAA agonists [27]. Thus, an increase in endogenous GABA in striatum could be reducing the activity of GABA projection neurons to substantia nigra [7] and facilitating the release of striatal dopamine through the nigrostriatal pathway. This would be supported by experiments in which intranigral infusions of GABA antagonists increase

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FIG. 5. Schematic representation of the posible mechanisms underlying the potentiation of the stimulated-dopamine release by increased extracellular GABA in striatum. (1) Indirect mechanism, through a striatum-substantia nigra-striatum loop. (2) Local mechanism, involving a GABAergic action on heteroreceptors or heterotransporters located on dopaminergic terminals. For further explanation see text.

striatal dopamine [16], whereas GABA agonists decrease it [10,16]. A schematic diagram depicting all these possibilities is shown in Fig. 5. This research is the first evidence that, in the awake animal, an increase of endogenous GABA, produced by blocking its high affinity transporter system, potentiates the release of stimulated dopamine. In view of the controversial results reporting the effects of pharmacological GABA agonists or exogenous GABA on dopamine release both in vitro and in vivo [2,11,12,14,20,28] it provides further and direct evidence of a role of endogenous GABA in promoting the release of dopamine in the striatum of the rat. As shown, TTX did not modify basal concentrations of GABA in striatum. This result is in agreement with other microdialysis studies and suggests that the basal extracellular GABA could have an important glial origin [24]. NIP produced a dose-related increase of extracellular GABA, which confirms that NIP is a very potent and selective GABA uptake inhibitor in in vivo preparations [4,9]. NIP also enhanced the increase of GABA stimulated by potassium. This is in agreement with other studies [3,23,25], and suggests that the high specific transporters for GABA are important in removing GABA released by depolarization with potassium [3,23]. The same conclusion has been reported in other studies using similar experimental conditions for other amino acid neurotransmitters, such as glutamate [5].

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10. ACKNOWLEDGEMENTS

This research was supported by DGICYT PM96-0046. We also thank Gregorio Segovia for his criticism of the manuscript. 11.

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