European Journal of Pharmacology, 219 (1992) 175-181
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52574
The role of GABA receptors in the control of nigrostriatal dopaminergic neurons: dual-probe microdialysis study in awake rats M. Santiago
a
and B.H.C. Westerink b
a Department of Biochemistry, Faculty of Pharmacy, University of Seville, Seville, Spain and b University CentreforPharmacy, Department of Medicinal Chemistry, University of Groningen, Groningen, Netherlands
Received 12 March 1992, revised MS received6 May 1992, accepted 26 May 1992
A microdialysis probe implanted into the substantia nigra was used to infuse y-aminobutyric acid-ergic (GABAergic) compounds onto cell bodies/dendrites of dopaminergic neurons, while a second microdialysis probe was used to record the extraceilular concentrations of dopamine and 3,4-dihydroxy-phenylacetic acid (DOPAC) in the ipsilateral striatum. The GABA k receptor agonist muscimol (10 /zmol/l) increased the release of dopamine in the ipsilateral striatum to 120% of the control values. The GABA a receptor agonist, (Z)-3[(aminoiminomethyl)-thiol]-prop-2-enoic acid (500 /~mol/l), was without effect. Infusion of the G A l A A receptor antagonists, bicuculline (50 ~mol/l) and picrotoxin (50 /xmol/1), stimulated the release of dopamine in the ipsilateral striatum to 160 and 130% of the controls, respectively. The GABA B receptor agonist, baclofen (10 and 50 /xmol/I), strongly inhibited the release of striatal dopamine, whereas infusion of the GABA B receptor antagonist, 2-hydroxy-saclofen (100 p.moi/l), was without effect. The results indicate that, in the substantia nigra, GABA A as well as GABA B receptors participate in controlling the activity of dopaminergic neurons. Striatum; Substantia nigra; Dopamine release; Microdialysis; GABA A receptors; GABA a receptors
I. Introduction The nigrostriatal dopaminergic pathway is believed to be under inhibitory control of y-aminobutyric acidergic (GABAergic) neurons. This hypothesis is based on both electrophysiological (Aghajanian and Bunney, 1975) and biochemical evidence (And6n and Stock, 1973). The nature of this inhibitory GABAergic pathway is not yet fully understood. Some authors suggest a direct GABAergic striatonigral or pallidonigral projection, but other speculate about the involvement of a second inhibitory interneuron located in the substantia nigra pars reticulata (Groves et al., 1975; Leviel et al., 1979; Grace and Bunney, 1979). The effect of intranigral application of GABA receptor agonists on the post-mortem tissue content of dopamine and its metabolites in the ipsilateral striatum has been much studied. Some authors concluded that the GABA receptor agonists induced an increase in the release of striatal dopamine (Martin and Haubrich, 1978; Sperber et al., 1989), but others postulated a
Correspondence to: B.H.C. Westerink, UniversityCentre for Pharmacy, Antonius Deusinglaan 2, 9713 AW Groningen, Netherlands. Tel. 31.50.633307, fax 31.50.633311.
decrease in the release of the transmitter (Waddington, 1980; Wood, 1982). The controversy is partly explained by the fact that different dopamine metabolites were considered to reflect dopamine release. Various workers have used in vivo methods to investigate the biochemical nature of the G A B A / d o p a m i n e interaction. GABAergic drugs were administered to the nigra via direct injections or push-pull cannulas, whereas the release of radiolabelled or endogenous dopamine was recorded with push-pull cannulas (Ch6ramy et al., 1977, 1978, 1979; Leviel et al., 1979) or microdialysis probes (Reid et al., 1988, 1990b) from the ipsilateral striatum. Although all authors concluded that GABA receptors play an important role in the regulation of nigrostriatal dopaminergic cells, some of the reported data are controversial. For example, intranigrally applied GABA caused stimulation of [3H]dopamine in the ipsilateral striatum of the cat (Ch6ramy et al., 1978), but suppressed striatal endogenous dopamine in the rat (Reid et al., 1988). Moreover GABA receptor agonists as well as antagonist were reported to increase the activity of dopaminergic cells (Ch6ramy et al., 1978; Leviel et al., 1979). Methodological differences, such as the use of different species, the use of anesthesia and artificial respiration, the use of push-pull cannulas vs. microdialysis
176
probes, are likely to be responsible for these discrepancies. That the dose used is also important was shown by Reid et al. (1990b), who demonstrated that a low dose of bicuculline increased dopamine release whereas higher doses of the GABA' receptor antagonist inhibited the ipsilateral release of dopamine. An alternative method for delivering drugs to brain areas is by continuous infusion via a microdialysis probe. An important advantage of this method is that the brain concentration of the infused drugs does not reach extreme levels. A second advantage is that the infused drug will probably reach an equilibrium concentration gradient in brain tissue. In the present study we have used the dual-probe microdialysis technique to study the GABA-dopamine interactions in the substantia nigra. Two microdialysis probes were implanted simultaneously. The first probe was used to infuse GABA receptor-specific drugs into the substantia nigra, whereas a second probe was located in the ipsilateral striatum and collected extracellular dopamine. All experiments were performed in conscious rats. (Z)3[(aminoiminomethyl)-thiol]-prop-2-enoic acid (ZAPA), and the GABA A receptor antagonists, bicuculline and picrotoxin, were studied, In addition the GABA a receptor agonist, baclofen, and the GABA B receptor antagonist, 2-hydroxy-saclofen, were infused into the nigra. Evidence was provided that, in the substantia nigra, GABA a as well as GABA B receptors participate in the control of activity of dopaminergic neurons.
2. Materials and methods
2.1. Animals Male albino rats of a Wistar-derived strain (275-320 g) (C.D.L., Groningen, The Netherlands) were used for the experiments. The rats were housed in plastic cages (35 × 35 × 40 cm) and allowed free access to food and water. 2.2. Drug treatment The following drugs were dissolved in the perfusion fluid and infused via a microdialysis membrane into the substantia nigra: muscimol, picrotoxin, (Z)-3[(aminoiminomethyl)-thiol]-prop-2-enoic acid (ZAPA), ( - ) - b i cuculline (Tocris Neuramin, Essex, UK), d,l-baclofen and 2-hydroxy-saclofen (Research Biochemicals Inc., Natick, USA). 2.3. Surgery and brain dialysis Microdialysis recordings and infusions were performed using a 1-shaped cannula (Santiago and West-
erink, 1990). The exposed tip of the dialysis membrane was 2 mm (substantia nigra) or 4 mm (striatum). The dialysis tube (inner diameter: 0.22 mm; outer diameter: 0.31 mm)was prepared from polyacrylonitrile/sodium methalyl sulfonate copolymer (AN 69, Hospal, Bologna, Italy). The in vitro efficiency of the probe (4 mm exposed) for dopamine was 23.2 + 1.9% (mean + S.E.M.; N = 4). Coordinates of the nigral cannula were: A / P 3.8, L / M 3.8, V / D 8.7, from the interaural line, at an angle of 12°; and for the striatal cannula: A / P 0.7, L / M 2.5, V / D 6.0, from the bregma point and dura (Paxinos and Watson, 1982). The probes were implanted under general chloral hydrate (400 mg/kg i.p.) and local lidocaine (6%) anesthesia. The perfusion experiments were carried out 24-48 h after implantation of the probe. Brain dialysis of dopamine was performed with a fully automated on-line system as described elsewhere (Westerink et al., 1987). In brief, two polyethylene tubes (length: 45 cm; inner diameter = 0.28 mm) were connected to the outlets of the dialysis tube. One tube was connected to the perfusion pump, and the other to the injection valve of the HPLC apparatus. The connection with the HPLC equipment introduced a lag time of about 30 min, for which the data presented are corrected. An electronic timer was used for holding the injection valve in the load position for 15 min during which time the sample loop (40 /xl) was filled with dialysate. The valve then switched automatically to the injection position for 15 s. This procedure was repeated every 15 min, which was the time needed to record a complete chromatogram. The substantia nigra and the striatum were perfused with a Ringer solution at a flow rate of 2.8-3.0 /zl/min (perfusor VI, B. Braun, Melsungen, F.R.G.). The composition of the Ringer solution was (in mmol/1): NaC1 140.0; KC1 4.0; CaC12 1.2; and MgC12 1.0; the solution was unbuffered (pH: 6-6.5). When the experiment was terminated the rat was given an overdose of chloral hydrate and the brain was fixed with 4% paraformaldehyde via intracardiac perfusion. Coronal sections (40 ~m thick) were made, and dialysis probe placement verified according to the atlas of Paxinos and Watson (1982). 2.4. Chem&al assays Dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were quantitated by HPLC with electrochemical detection. A Perkin-Elmer series 10 HPLC pump was used in conjunction with a glassy carbon working electrode set at - 7 8 0 mV (with respect to an Ag/AgCI reference electrode) (ANTEC, The Netherlands). An Altech-RSL cartridge (150 × 4.6 mm) column filled with reverse-phase Cls 5 izm material was used. The mobile phase consisted of a mixture of 0.1 mol/1 of sodium acetate adjusted to pH 4.1 with acetic
177 acid, 1.8 mmol/1 of 1-heptanesulfonic acid, 0.3.mmol/1 of NAEEDTA, and 120 ml methanol/l; at a flow of 0.7 ml/min. The detection limit of the assay was about 10-15 fmol per sample.
2.5. Expression of results and statistics
200
percentage of controls 150
The average of the last four stable samples before drug treatment was considered as the control and was defined as 100%. All values given are expressed as percentages of the controls. Absolute values of the control data for the individual experiments are given in Results. Saline experiments were carried out, but these results are not presented as no time-dependent tendency in the output of dopamine and DOPAC was noticed. Differences between the average dialysate concentrations of the control and drugs treatment were compared by Kruskal-Wallis analysis of variance by ranks. Comparison of the means (when the H value greater than the 95% confidence level) was carried out using the Wilcoxon matched-pairs signed-ranks twosided test.
3. Results
3.1. Effect of infusion of muscimol and ZAPA into the substantia nigra on the dialysate content of dopamine and DOPAC recorded from the ipsilateral striatum The GABA A receptor agonist, muscimol, was infused for 60 min into the substantia nigra in a concentration of 10 /xmol/l (fig. 1). Basal values were ( + S.E.M.; n = 5), dopamine: 12.4 + 1.5 fmol/min and
!
t
100
50
I
0
I
1
I
2
I
3
h--Fig. 2. Effect of intranigral infusion of bicuculline (50 p,m o l / l ) (black bar) on the dialysate content of dopamine (open circles) and DOPAC (filled circles) in the ipsilateral striatum. The data are given as means + S.E.M. (N = 4) expressed as percentages of controls. Significantly different values are indicated: * P < 0.05, compared with the control value (Kruskal-Wallis followed by Wilcoxon test).
DOPAC: 0.64 5:0.04 pmol/min. Muscimol had a slight, statistically significant, stimulatory effect (to about 120% of controls) on the release of dopamine in the ipsilateral striatum. DOPAC levels in the striatum increased to about 170% of the controls. Infusion of 50 /~mol/1 muscimol induced strong behavioral effects and these experiments were not pursued further. The GABA A receptor agonist, ZAPA, infused in a concentration of 500 ~ m o l / l for 60 min did not affect the release of dopamine or DOPAC in the ipsilateral striatum (results not shown).
3.2. Effect of infusion of bicuculline or picrotoxin into the substantia nigra on the dialysate content of dopamine and DOPAC recorded from the ipsilateral striatum
200
percentage of controls 150
~
S
100
50 I
I
I
I
0
1
2
3 h'"
Fig. 1. Effect of the intranigral infusion of muscimol (10 /zmol/l) (black bar) on the dialysate content of dopamine (open circles) and DOPAC (filled circles) in the ipsilateral striatum. The data are given as means + S.E.M. (N = 5) expressed as percentages of the controls. Significantly different values are indicated: * P < 0.05, compared with the control value (Kruskal-Wallis followed by Wilcoxon test).
The GABA A receptor antagonists, bicuculline and picrotoxin, both induced an increase in the release of dopamine and DOPAC in the ipsilateral striatum. After 60 min infusion of 50 /xmol/1 bicuculline, striatal dialysate levels of dopamine as well as of DOPAC increased to 160% of controls (fig. 2). After 60 min infusion of 50 ~ m o l / l picrotoxin, dopamine in the dialysates of the ipsilateral striatum increased to 130% of controls, whereas DOPAC levels rose to 190% of the controls (fig. 3). Basal values for the bicuculline experiments were (_S.E.M.; n = 5), dopamine: 9.7 5:1.5 fmol/min and DOPAC; 0.74 5:0.08 pmol/min. Basal values for the picrotoxin experiments were (5:S.E.M.; n = 5 ) , dopamine: 10.7 5:1.0 fmol/min and DOPAC; 0.61 5: 0.08 pmol/min.
178
200
I percentage of controls
150
100
50 I
I
I
I
0
1
2
3
h---
Fig. 3. Effect of intranigral infusion of picrotoxin(50 lzmol/l) (black bar) on the dialysatecontent of dopamine (open circles) and DOPAC (filled circles) in the ipsilateral striatum. The data are given as means + S.E.M. (bars) (N = 6) expressed as percentages of controls. Significantlydifferent values: * P < 0.05, compared with the control value (Kruskal-Wallis followedby Wilcoxontest).
3.3. Effect of infusion of baclofen and 2-hydroxy-saclofen into the substantia nigra on the dialysate content of dopamine and DOPAC recorded from the ipsilateral striatum When the G A B A B receptor agonist, baclofen, was infused for 60 min in a dose of 10 /~mol/1 into the substantia nigra, a pronounced decrease of dopamine to about 50% of control values was recorded in the
200
150
) percentage of controls 100
50
0
1
2
3 h--Fig. 4. Effect of intranigral infusion of (+)-baclofen (10 or 50 /zmol/l) (black bar) on the dialysate content of dopamine (open symbols) and DOPAC (filled symbols) in the ipsilateral striatum. Circles represent 10 /xmol/l and squares 50/.~mol/l. The data are given as means+S.E.M. (N=4) expressed as percentages of the controls. Significantlydifferent values are indicated: * P < 0.05, compared with the control value (Kruskal-Wallis followed by Wilcoxon test).
dialysates of the ipsilateral striatum (fig. 4). The decrease of dopamine lasted for 2 h. In contrast, D O P A C levels in the striatum increased to about 160% of the controls. When baclofen was infused in a concentration of 50 /xmol/1, dopamine was further decreased to about 15% of the controls, whereas E, OPAC increased to about 200% of the controls. Basal values were ( + S.E.M.; n = 9), dopamine: 10.4 + 1.5 f m o l / m i n and DOPAC: 0.69 + 0.06 p m o l / m i n . The G A B A a receptor antagonist, 2-hydroxy-saclofen, was infused in a concentration of 100 izmol/l for 60 min into the substantia nigra. No effect on the dialysate content of dopamine and D O P A C was observed in the ipsilateral striatum (results not shown).
4. Discussion
A GABAergic neuronal pathway that controls the activity of nigrostriatal dopaminergic neurons has been the object of numerous studies but remains controversial. Methodological differences between the various studies may be responsible for the conflicting results. One methodological aspect is the administration of drugs to selected brain areas.
4.1. The dual-probe protocol Administration of drugs to small brain areas via injection or push-pull cannulas has certain disadvantages. The use of injection cannulas may cause locally high concentrations of the injected drugs and damage to the surrounding neuronal tissue has been demonstrated (Reid et al., 1990a). When push-pull cannulas are used damage is also likely because of the intimate mechanical contact between perfusion fluid and nervous tissue. In the present study we administered drugs via a microdialysis probe. Drugs were infused into the substantia nigra whereas dopamine was recorded from the ipsilateral striatum with a second probe (the 'dualprobe protocol'). An advantage of a microdialysis probe is that mechanical contact between the infused solution and brain tissue is prevented and damage is minimized (Benveniste et al., 1987; Quan and Blatteis, 1989). Moreover, a regular concentration gradient of the drug - which does not exceed the concentration in the infusion fluid - is built up in the surrounding tissue. A disadvantage of all types of intracerebral administrations - including the present use of microdialysis probes - is the uncertainty about the actual concentration of the drug at the site of action. The development of quantitative microdialysis (Morrison et al., 1991) is a most promising approach in this respect.
4.2. Nigral GABA A receptors The two G A B A A receptor agonists (muscimol and ZAPA), infused into the substantia nigra, had rela-
179
tively few effects on the release of striatal dopamine. In the case of muscimol, a moderate but statistically significant increase in the release of dopamine was detected in the ipsilateral striatum. Such an increase is at variance with the supposed inhibitory effect of GABAergic neurons. Similar observations have been reported in the literature. Various authors, using different methods, have observed that intranigral administration of GABA receptor agonists stimulates nigral dopaminergic neurons (Martin and Haubrich, 1978; Chdramy et al., 1978; Grace and Bunney, 1979; Leviel et al., 1979). Several authors have speculated that a second inhibitory interneuron located in the substantia nigra pars reticulata may participate in the GABAergic striatonigral inhibitory pathway (Leviel et al., 1979; Grace and Bunney, 1979). The finding of stimulation after application of GABA A receptor agonists was explained by the fact that this interneuron is 20 times more sensitive to GABA than the nigrostriatal neuron (Grace and Bunney, 1979)~ However, other workers have reported opposite effects of GABA A receptor agonists. A decrease in the release of striatal dopamine after intranigral application of GABA A receptor agonists was described by Waddington (1980) and Wood (1982). These observations were based on the interpretation of changes in striatal dopamine metabolites. However, recent microdialysis studies - including the present one - have demonstrated that dopamine metabolites do not always reflect dopamine release (Zetterstr6m et al., 1988). In a microdialysis study Reid et al. (1988) injected different doses of GABA into the nigra. The injections were without effect on dopamine release, except for the highest concentration applied (300 nmol in 0.2 p,l), which induced a short-lasting decrease in the release of the transmitter in the ipsilateral striatum. Interpretations of latter observations are complicated by the fact that these injections may have caused aspecific effects because of the high concentrations of GABA involved (1.5 mol/l). The GABA A receptor antagonists, bicuculline and picrotoxin, clearly stimulated the release of striatal dopamine. If we accept the possibility of a second GABAergic interneuron (Leviel et al., 1979; Grace and Bunney, 1979) the present observations then imply that this neuron is relatively insensitive to GABA A receptor antagonists. The present results for picrotoxin are in line with those of the cat perfusion study (Ch6ramy et al., 1977), but why bicuculline was inactive in the latter model remains unexplained (Leviel et al., 1979). Our results are somewhat at variance with those of Reid et al. (1990b). These authors observed an increase in ipsilateral striatal extracellular dopamine after intranigral injections of low doses of bicuculline and a decrease after high doses of bicuculline in anesthetized animals. Reid et al. (1990b) suggest that a second, indirectly acting, G A B A A mechanism, probably lo-
cated in the pars reticulata, is of relevance. This second mechanism is hypothesized to be activated at relatively high concentrations or with more penetrating infusion methods such as push-pull perfusion. Reproduction of the findings of Reid et al. (1990b) would have required higher doses of bicuculline, which could not be tested because of the strong behavioral effects of bicuculline in conscious animals. It remains to be established whether the second GABAergic neuron hypothesized by Reid et al. (1990b) is similar to the earlier supposed GABAergic interneuron (Leviel et al., 1979; Grace and Bunney, 1979). 4.3. Nigral GABA B receptors
The present finding that intranigral infusion of baclofen potently inhibited the release of dopamine in the ipsilateral striatum, supports the role of GABA a receptors in controlling the activity of nigrostriatal dopaminergic neurons. The results would be explainable by the existence of GABA B receptors located directly on the somata of the dopaminergic neurons. A second possibility is that baclofen acts on GABA B receptors that are located presynaptically to dopaminergic neurons (Giralt et al., 1990). It is emphasized that the present data are in strong contrast with results obtained with push-pull perfusions in cats, when nigral application of baclofen stimulated the release of ipsilateral striatal radiolabelled dopamine (Chdramy et al., 1978, 1979). Intranigral infusion of the GABA B receptor antagonist, 2-hydroxy-saclofen, was without effect on the release of dopamine in the ipsilateral striatum. This would indicate that the tonus of the GABA B inhibition is relatively weak. However, a more likely explanation for the absence of effects of 2-hydroxy-saclofen is its apparent low potency as a GABA B receptor antagonist. It has been known for many years that systemically administered baclofen causes inhibition of the impulse flow of nigrostriatal dopaminergic neurons, resulting in a sharp increase of the dopamine content of the striatum (Da Prada and Keller, 1976; Anddn and Wachtel, 1977). The present results for baclofen are in good agreement with these observations and suggest that GABA B receptors play a crucial role in this effect. It is speculated that compounds that are pharmacologically closely related to baclofen might also interact with GABA B receptors, y-Hydroxybutyric acid is such a compound and its pharmacological effect is the basis for the well-known presynaptic dopamine autoreceptor model (Roth et al., 1973; Walters and Roth, 1976). 4.4. Changes in DOPAC
Finally, we would like to comment on the drug-induced changes in extracellular DOPAC. It is striking
180
that the extracellular levels of DOPAC were increased during both an increase of extracellular dopamine (during infusion of muscimol, bicuculline or picrotoxin) and during a decrease of extracellular dopamine (during infusion of baclofen). A similar tendency was found in the microdialysis studies of Reid et al. (1988, 1990a,b). There is increasing evidence that dopamine does not need to be released first before it is metabolized to DOPAC (Zetterstr6m et al., 1988). The mechanism behind the increase in ipsilateral DOPAC is presently under investigation. 4.5. In conclusion
The present study showed that microdialysis probes can be used to deliver drugs to restricted brain areas and provided an example of the experimental conditions for 'remote control' of dopamine release in the striatum. The observations on GABA A receptor antagonists support the hypothesis that GABA A receptors are located on dopaminergic cell bodies a n d / o r dendrites in the nigra. The present results are best explained when a second projection of GABAergic interneurons in the substantia nigra is hypothesized (Leviel et al., 1979; Grace and Bunney, 1979). This second neuron is apparently much more sensitive to GABA A receptor agonists that to GABA A receptor antagonists. The observed effects on baclofen strongly suggest that GABA B receptors also participate in the regulation of the activity of dopaminergic neurons.
Acknowledgement This work was supported by the Ministerio de Educacion y Ciencia, Spain.
References Aghajanian, G.K. and B.S. Bunney, 1975, Dopaminergic and nondopaminergic neurons of the substantia nigra: differential response to putative transmitters, in: Neuropsychopharmacology, eds. J.R. Boissier, H. Hippius and P. Pocht (Excerpta Medica, Amsterdam and American Elsevier New York) p. 444. And6n, N.E. and G. Stock, 1973, Inhibitory effect of gamma-hydroxybutyric acid and gamma-aminobutyric acid on the dopamine cells in the substantia nigra, Naunyn-Schmiedeb. Arch. Exp. Pathol. Pharmakol. 279, 89. And6n, N.E. and H. Wachtel, 1977, Biochemical effects of baciofen (p-chlorophenyl-GABA) on the dopamine and the noradrenaline in the rat brain, Acta Pharmacol. Toxicol. 40, 310. Benveniste, H., J. Drejer, A. Shousboe and N.H. Diemer, 1987, Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat, J. Neurochem. 49, 729. Ch6ramy, A., A. Nieoullon and J. Glowinski, 1977, Effects of periph-
eral and local administration of picrotoxin on the release of newly synthesized 3H-dopamine in the caudate nucleus of the cat, Arch. Pharmacol. 297, 31. Ch6ramy, A., A . Nieoullon and J. Glowinski, 1978, GABAergic process involved in the control of dopamine release from nigrostriatal dopaminergic neurons in the cat, Eur. J. Pharmacol. 48, 281. Ch6ramy, A., A. Nieoullon and J. Glowinski, 1979, Effects of unilateral nigral application of baclofen on dopamine release in the two caudate nuclei of the cat, Eur. J. Pharmacol. 58, 133. Da Prada, M. and H.H. Keller, 1976, Baclofen and gamma-hydroxybutyrate: similar effects on cerebral dopamine neurons, Life Sci. 19, 1253. Giralt, M.T., G. Bonanno and M. Raiteri, 1990, GABA terminals autoreceptors in the pars compacta and pars reticulata of the rat substantia nigra are GABAB, Eur. J. Pharmacol. 175, 137. Grace, A.A. and B.S. Bunney, 1979, Paradoxical GABA excitation of nigral dopaminergic cells: indirect mediation through reticulata inhibitory neurons, Eur. J. Pharmacol. 59, 211. Groves, P.M., C.J. Wilson, S.J. Young and G. Rebec, 1975, Self-inhibition by dopaminergic neurons, Science 190, 522. Leviel, V., A. Ch6ramy, A. Nieoullon and J. Glowinski, 1979, Symmetric bilateral changes in dopamine release from the caudate nuclei of the cat induced by unilateral nigral application of glycine and GABA related compounds, Brain. Res. 174, 259. Martin, G.E. and D.R. Haubrich, 1978, Striatal dopamine release and contraversive rotation elicited by intranigrally applied muscimol, Nature 275, 230. Morrison, P.F., P.M. Bungay, J.K. Hsiao, B.A. Ball, I.N. Mefford and R.L. Dedrick, 1991, Quantitative microdialysis: Analysis of transients and application of pharmacokinetics in brain, J. Neurochem. 57, 103. Paxinos, G. and C. Watson, 1982, The Rat Brain in Stereotaxic Coordinates (Academic Press, New York). Quan, N. and C.M. Blatteis, 1989, Microdialysis: a system for localized drug delivery into the brain, Brain Res. Bull. 22, 621. Reid, M.S., M. Herrera-Marschitz, T. H6kfelt, L. Terenius and U. Ungerstedt, 1988, Differential modulation of striatal dopamine release by intranigral injection of gamma-aminobutyric acid (GABA), dynorphin A and Substance P, Eur. J. Pharmacol. 147, 411. Reid, M.S., M. Herrera-Marschitz, T. H/Skfelt, M. Ohlin, K.L. Valentino and U. Ungerstedt, 1990a, Effects of intranigral substance P and neurokinin A on striatal dopamine release. I. Interactions with substance P antagonists, Neuroscience 36, 643. Reid, M.S., M. Herrera-Marschitz and U. Ungerstedt, 1990b, Effects of intranigral substance P and neurokinin A on striatal dopamine release. II. Interactions with bicuculline and nalaxone, Neuroscience 36, 659. Roth, R.H., J.R. Waiters and G.K. Aghajanian, 1973, Effect of impulse flow on the release and synthesis of dopamine in the rat striatum, in: Frontiers in Catecholamines Research, eds. E. Usdin and S.H. Snyder (Pergamon Press, New York) p. 567. Santiago, M. and B.H.C. Westerink, 1990, Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes, Naunyn-Schmiedeb. Arch. Pharmacol. 342, 407. Sperber, E.F., J.N.D. Wurpel, N.S., Sharpless and S.L Mosh6, 1989, Intranigral GABAergic drug effects on striatal dopamine activity, Pharmacol. Biochem. Behav. 32, 1067. Waddington, J.L., 1980, GABAergic mechanisms in the substantia nigra, Nature 283, 696. Walters, J.R. and R.H. Roth, 1976, Dopaminergic neurons: An in vivo system for measuring drug interactions with presynaptic receptors, Neunyn-Schmiedeb. Arch. Pharmacol. 296, 5. Westerink, B.H.C., G. Damsma, H. Rollema, J.B. De Vries and A.S.
181 Horn, 1987, Scope and limitations of in vivo brain dialysis: a comparison of its application to various neurotransmitter systems, Life Sci. 41, 1763. Wood, P.L., 1982, Actions of GABAergic agents on dopamine metabolism in the nigrostriatal pathway of the rat, J. Pharmacol. Exp. Ther. 222, 674.
Zetterstr6m, T., T. Sharp, A.K. Collin and U. Ungerstedt, 1988, In vivo measurement of extracellular dopamine and DOPAC in rat striatum after various dopamine-releasing drugs; implications for the origin of extracellular DOPAC, Eur. J. Pharmacol. 148, 327.
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52574
The role of GABA receptors in the control of nigrostriatal dopaminergic neurons: dual-probe microdialysis study in awake rats M. Santiago
a
and B.H.C. Westerink b
a Department of Biochemistry, Faculty of Pharmacy, University of Seville, Seville, Spain and b University CentreforPharmacy, Department of Medicinal Chemistry, University of Groningen, Groningen, Netherlands
Received 12 March 1992, revised MS received6 May 1992, accepted 26 May 1992
A microdialysis probe implanted into the substantia nigra was used to infuse y-aminobutyric acid-ergic (GABAergic) compounds onto cell bodies/dendrites of dopaminergic neurons, while a second microdialysis probe was used to record the extraceilular concentrations of dopamine and 3,4-dihydroxy-phenylacetic acid (DOPAC) in the ipsilateral striatum. The GABA k receptor agonist muscimol (10 /zmol/l) increased the release of dopamine in the ipsilateral striatum to 120% of the control values. The GABA a receptor agonist, (Z)-3[(aminoiminomethyl)-thiol]-prop-2-enoic acid (500 /~mol/l), was without effect. Infusion of the G A l A A receptor antagonists, bicuculline (50 ~mol/l) and picrotoxin (50 /xmol/1), stimulated the release of dopamine in the ipsilateral striatum to 160 and 130% of the controls, respectively. The GABA B receptor agonist, baclofen (10 and 50 /xmol/I), strongly inhibited the release of striatal dopamine, whereas infusion of the GABA B receptor antagonist, 2-hydroxy-saclofen (100 p.moi/l), was without effect. The results indicate that, in the substantia nigra, GABA A as well as GABA B receptors participate in controlling the activity of dopaminergic neurons. Striatum; Substantia nigra; Dopamine release; Microdialysis; GABA A receptors; GABA a receptors
I. Introduction The nigrostriatal dopaminergic pathway is believed to be under inhibitory control of y-aminobutyric acidergic (GABAergic) neurons. This hypothesis is based on both electrophysiological (Aghajanian and Bunney, 1975) and biochemical evidence (And6n and Stock, 1973). The nature of this inhibitory GABAergic pathway is not yet fully understood. Some authors suggest a direct GABAergic striatonigral or pallidonigral projection, but other speculate about the involvement of a second inhibitory interneuron located in the substantia nigra pars reticulata (Groves et al., 1975; Leviel et al., 1979; Grace and Bunney, 1979). The effect of intranigral application of GABA receptor agonists on the post-mortem tissue content of dopamine and its metabolites in the ipsilateral striatum has been much studied. Some authors concluded that the GABA receptor agonists induced an increase in the release of striatal dopamine (Martin and Haubrich, 1978; Sperber et al., 1989), but others postulated a
Correspondence to: B.H.C. Westerink, UniversityCentre for Pharmacy, Antonius Deusinglaan 2, 9713 AW Groningen, Netherlands. Tel. 31.50.633307, fax 31.50.633311.
decrease in the release of the transmitter (Waddington, 1980; Wood, 1982). The controversy is partly explained by the fact that different dopamine metabolites were considered to reflect dopamine release. Various workers have used in vivo methods to investigate the biochemical nature of the G A B A / d o p a m i n e interaction. GABAergic drugs were administered to the nigra via direct injections or push-pull cannulas, whereas the release of radiolabelled or endogenous dopamine was recorded with push-pull cannulas (Ch6ramy et al., 1977, 1978, 1979; Leviel et al., 1979) or microdialysis probes (Reid et al., 1988, 1990b) from the ipsilateral striatum. Although all authors concluded that GABA receptors play an important role in the regulation of nigrostriatal dopaminergic cells, some of the reported data are controversial. For example, intranigrally applied GABA caused stimulation of [3H]dopamine in the ipsilateral striatum of the cat (Ch6ramy et al., 1978), but suppressed striatal endogenous dopamine in the rat (Reid et al., 1988). Moreover GABA receptor agonists as well as antagonist were reported to increase the activity of dopaminergic cells (Ch6ramy et al., 1978; Leviel et al., 1979). Methodological differences, such as the use of different species, the use of anesthesia and artificial respiration, the use of push-pull cannulas vs. microdialysis
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probes, are likely to be responsible for these discrepancies. That the dose used is also important was shown by Reid et al. (1990b), who demonstrated that a low dose of bicuculline increased dopamine release whereas higher doses of the GABA' receptor antagonist inhibited the ipsilateral release of dopamine. An alternative method for delivering drugs to brain areas is by continuous infusion via a microdialysis probe. An important advantage of this method is that the brain concentration of the infused drugs does not reach extreme levels. A second advantage is that the infused drug will probably reach an equilibrium concentration gradient in brain tissue. In the present study we have used the dual-probe microdialysis technique to study the GABA-dopamine interactions in the substantia nigra. Two microdialysis probes were implanted simultaneously. The first probe was used to infuse GABA receptor-specific drugs into the substantia nigra, whereas a second probe was located in the ipsilateral striatum and collected extracellular dopamine. All experiments were performed in conscious rats. (Z)3[(aminoiminomethyl)-thiol]-prop-2-enoic acid (ZAPA), and the GABA A receptor antagonists, bicuculline and picrotoxin, were studied, In addition the GABA a receptor agonist, baclofen, and the GABA B receptor antagonist, 2-hydroxy-saclofen, were infused into the nigra. Evidence was provided that, in the substantia nigra, GABA a as well as GABA B receptors participate in the control of activity of dopaminergic neurons.
2. Materials and methods
2.1. Animals Male albino rats of a Wistar-derived strain (275-320 g) (C.D.L., Groningen, The Netherlands) were used for the experiments. The rats were housed in plastic cages (35 × 35 × 40 cm) and allowed free access to food and water. 2.2. Drug treatment The following drugs were dissolved in the perfusion fluid and infused via a microdialysis membrane into the substantia nigra: muscimol, picrotoxin, (Z)-3[(aminoiminomethyl)-thiol]-prop-2-enoic acid (ZAPA), ( - ) - b i cuculline (Tocris Neuramin, Essex, UK), d,l-baclofen and 2-hydroxy-saclofen (Research Biochemicals Inc., Natick, USA). 2.3. Surgery and brain dialysis Microdialysis recordings and infusions were performed using a 1-shaped cannula (Santiago and West-
erink, 1990). The exposed tip of the dialysis membrane was 2 mm (substantia nigra) or 4 mm (striatum). The dialysis tube (inner diameter: 0.22 mm; outer diameter: 0.31 mm)was prepared from polyacrylonitrile/sodium methalyl sulfonate copolymer (AN 69, Hospal, Bologna, Italy). The in vitro efficiency of the probe (4 mm exposed) for dopamine was 23.2 + 1.9% (mean + S.E.M.; N = 4). Coordinates of the nigral cannula were: A / P 3.8, L / M 3.8, V / D 8.7, from the interaural line, at an angle of 12°; and for the striatal cannula: A / P 0.7, L / M 2.5, V / D 6.0, from the bregma point and dura (Paxinos and Watson, 1982). The probes were implanted under general chloral hydrate (400 mg/kg i.p.) and local lidocaine (6%) anesthesia. The perfusion experiments were carried out 24-48 h after implantation of the probe. Brain dialysis of dopamine was performed with a fully automated on-line system as described elsewhere (Westerink et al., 1987). In brief, two polyethylene tubes (length: 45 cm; inner diameter = 0.28 mm) were connected to the outlets of the dialysis tube. One tube was connected to the perfusion pump, and the other to the injection valve of the HPLC apparatus. The connection with the HPLC equipment introduced a lag time of about 30 min, for which the data presented are corrected. An electronic timer was used for holding the injection valve in the load position for 15 min during which time the sample loop (40 /xl) was filled with dialysate. The valve then switched automatically to the injection position for 15 s. This procedure was repeated every 15 min, which was the time needed to record a complete chromatogram. The substantia nigra and the striatum were perfused with a Ringer solution at a flow rate of 2.8-3.0 /zl/min (perfusor VI, B. Braun, Melsungen, F.R.G.). The composition of the Ringer solution was (in mmol/1): NaC1 140.0; KC1 4.0; CaC12 1.2; and MgC12 1.0; the solution was unbuffered (pH: 6-6.5). When the experiment was terminated the rat was given an overdose of chloral hydrate and the brain was fixed with 4% paraformaldehyde via intracardiac perfusion. Coronal sections (40 ~m thick) were made, and dialysis probe placement verified according to the atlas of Paxinos and Watson (1982). 2.4. Chem&al assays Dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were quantitated by HPLC with electrochemical detection. A Perkin-Elmer series 10 HPLC pump was used in conjunction with a glassy carbon working electrode set at - 7 8 0 mV (with respect to an Ag/AgCI reference electrode) (ANTEC, The Netherlands). An Altech-RSL cartridge (150 × 4.6 mm) column filled with reverse-phase Cls 5 izm material was used. The mobile phase consisted of a mixture of 0.1 mol/1 of sodium acetate adjusted to pH 4.1 with acetic
177 acid, 1.8 mmol/1 of 1-heptanesulfonic acid, 0.3.mmol/1 of NAEEDTA, and 120 ml methanol/l; at a flow of 0.7 ml/min. The detection limit of the assay was about 10-15 fmol per sample.
2.5. Expression of results and statistics
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percentage of controls 150
The average of the last four stable samples before drug treatment was considered as the control and was defined as 100%. All values given are expressed as percentages of the controls. Absolute values of the control data for the individual experiments are given in Results. Saline experiments were carried out, but these results are not presented as no time-dependent tendency in the output of dopamine and DOPAC was noticed. Differences between the average dialysate concentrations of the control and drugs treatment were compared by Kruskal-Wallis analysis of variance by ranks. Comparison of the means (when the H value greater than the 95% confidence level) was carried out using the Wilcoxon matched-pairs signed-ranks twosided test.
3. Results
3.1. Effect of infusion of muscimol and ZAPA into the substantia nigra on the dialysate content of dopamine and DOPAC recorded from the ipsilateral striatum The GABA A receptor agonist, muscimol, was infused for 60 min into the substantia nigra in a concentration of 10 /xmol/l (fig. 1). Basal values were ( + S.E.M.; n = 5), dopamine: 12.4 + 1.5 fmol/min and
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t
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h--Fig. 2. Effect of intranigral infusion of bicuculline (50 p,m o l / l ) (black bar) on the dialysate content of dopamine (open circles) and DOPAC (filled circles) in the ipsilateral striatum. The data are given as means + S.E.M. (N = 4) expressed as percentages of controls. Significantly different values are indicated: * P < 0.05, compared with the control value (Kruskal-Wallis followed by Wilcoxon test).
DOPAC: 0.64 5:0.04 pmol/min. Muscimol had a slight, statistically significant, stimulatory effect (to about 120% of controls) on the release of dopamine in the ipsilateral striatum. DOPAC levels in the striatum increased to about 170% of the controls. Infusion of 50 /~mol/1 muscimol induced strong behavioral effects and these experiments were not pursued further. The GABA A receptor agonist, ZAPA, infused in a concentration of 500 ~ m o l / l for 60 min did not affect the release of dopamine or DOPAC in the ipsilateral striatum (results not shown).
3.2. Effect of infusion of bicuculline or picrotoxin into the substantia nigra on the dialysate content of dopamine and DOPAC recorded from the ipsilateral striatum
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percentage of controls 150
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Fig. 1. Effect of the intranigral infusion of muscimol (10 /zmol/l) (black bar) on the dialysate content of dopamine (open circles) and DOPAC (filled circles) in the ipsilateral striatum. The data are given as means + S.E.M. (N = 5) expressed as percentages of the controls. Significantly different values are indicated: * P < 0.05, compared with the control value (Kruskal-Wallis followed by Wilcoxon test).
The GABA A receptor antagonists, bicuculline and picrotoxin, both induced an increase in the release of dopamine and DOPAC in the ipsilateral striatum. After 60 min infusion of 50 /xmol/1 bicuculline, striatal dialysate levels of dopamine as well as of DOPAC increased to 160% of controls (fig. 2). After 60 min infusion of 50 ~ m o l / l picrotoxin, dopamine in the dialysates of the ipsilateral striatum increased to 130% of controls, whereas DOPAC levels rose to 190% of the controls (fig. 3). Basal values for the bicuculline experiments were (_S.E.M.; n = 5), dopamine: 9.7 5:1.5 fmol/min and DOPAC; 0.74 5:0.08 pmol/min. Basal values for the picrotoxin experiments were (5:S.E.M.; n = 5 ) , dopamine: 10.7 5:1.0 fmol/min and DOPAC; 0.61 5: 0.08 pmol/min.
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I percentage of controls
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h---
Fig. 3. Effect of intranigral infusion of picrotoxin(50 lzmol/l) (black bar) on the dialysatecontent of dopamine (open circles) and DOPAC (filled circles) in the ipsilateral striatum. The data are given as means + S.E.M. (bars) (N = 6) expressed as percentages of controls. Significantlydifferent values: * P < 0.05, compared with the control value (Kruskal-Wallis followedby Wilcoxontest).
3.3. Effect of infusion of baclofen and 2-hydroxy-saclofen into the substantia nigra on the dialysate content of dopamine and DOPAC recorded from the ipsilateral striatum When the G A B A B receptor agonist, baclofen, was infused for 60 min in a dose of 10 /~mol/1 into the substantia nigra, a pronounced decrease of dopamine to about 50% of control values was recorded in the
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) percentage of controls 100
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3 h--Fig. 4. Effect of intranigral infusion of (+)-baclofen (10 or 50 /zmol/l) (black bar) on the dialysate content of dopamine (open symbols) and DOPAC (filled symbols) in the ipsilateral striatum. Circles represent 10 /xmol/l and squares 50/.~mol/l. The data are given as means+S.E.M. (N=4) expressed as percentages of the controls. Significantlydifferent values are indicated: * P < 0.05, compared with the control value (Kruskal-Wallis followed by Wilcoxon test).
dialysates of the ipsilateral striatum (fig. 4). The decrease of dopamine lasted for 2 h. In contrast, D O P A C levels in the striatum increased to about 160% of the controls. When baclofen was infused in a concentration of 50 /xmol/1, dopamine was further decreased to about 15% of the controls, whereas E, OPAC increased to about 200% of the controls. Basal values were ( + S.E.M.; n = 9), dopamine: 10.4 + 1.5 f m o l / m i n and DOPAC: 0.69 + 0.06 p m o l / m i n . The G A B A a receptor antagonist, 2-hydroxy-saclofen, was infused in a concentration of 100 izmol/l for 60 min into the substantia nigra. No effect on the dialysate content of dopamine and D O P A C was observed in the ipsilateral striatum (results not shown).
4. Discussion
A GABAergic neuronal pathway that controls the activity of nigrostriatal dopaminergic neurons has been the object of numerous studies but remains controversial. Methodological differences between the various studies may be responsible for the conflicting results. One methodological aspect is the administration of drugs to selected brain areas.
4.1. The dual-probe protocol Administration of drugs to small brain areas via injection or push-pull cannulas has certain disadvantages. The use of injection cannulas may cause locally high concentrations of the injected drugs and damage to the surrounding neuronal tissue has been demonstrated (Reid et al., 1990a). When push-pull cannulas are used damage is also likely because of the intimate mechanical contact between perfusion fluid and nervous tissue. In the present study we administered drugs via a microdialysis probe. Drugs were infused into the substantia nigra whereas dopamine was recorded from the ipsilateral striatum with a second probe (the 'dualprobe protocol'). An advantage of a microdialysis probe is that mechanical contact between the infused solution and brain tissue is prevented and damage is minimized (Benveniste et al., 1987; Quan and Blatteis, 1989). Moreover, a regular concentration gradient of the drug - which does not exceed the concentration in the infusion fluid - is built up in the surrounding tissue. A disadvantage of all types of intracerebral administrations - including the present use of microdialysis probes - is the uncertainty about the actual concentration of the drug at the site of action. The development of quantitative microdialysis (Morrison et al., 1991) is a most promising approach in this respect.
4.2. Nigral GABA A receptors The two G A B A A receptor agonists (muscimol and ZAPA), infused into the substantia nigra, had rela-
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tively few effects on the release of striatal dopamine. In the case of muscimol, a moderate but statistically significant increase in the release of dopamine was detected in the ipsilateral striatum. Such an increase is at variance with the supposed inhibitory effect of GABAergic neurons. Similar observations have been reported in the literature. Various authors, using different methods, have observed that intranigral administration of GABA receptor agonists stimulates nigral dopaminergic neurons (Martin and Haubrich, 1978; Chdramy et al., 1978; Grace and Bunney, 1979; Leviel et al., 1979). Several authors have speculated that a second inhibitory interneuron located in the substantia nigra pars reticulata may participate in the GABAergic striatonigral inhibitory pathway (Leviel et al., 1979; Grace and Bunney, 1979). The finding of stimulation after application of GABA A receptor agonists was explained by the fact that this interneuron is 20 times more sensitive to GABA than the nigrostriatal neuron (Grace and Bunney, 1979)~ However, other workers have reported opposite effects of GABA A receptor agonists. A decrease in the release of striatal dopamine after intranigral application of GABA A receptor agonists was described by Waddington (1980) and Wood (1982). These observations were based on the interpretation of changes in striatal dopamine metabolites. However, recent microdialysis studies - including the present one - have demonstrated that dopamine metabolites do not always reflect dopamine release (Zetterstr6m et al., 1988). In a microdialysis study Reid et al. (1988) injected different doses of GABA into the nigra. The injections were without effect on dopamine release, except for the highest concentration applied (300 nmol in 0.2 p,l), which induced a short-lasting decrease in the release of the transmitter in the ipsilateral striatum. Interpretations of latter observations are complicated by the fact that these injections may have caused aspecific effects because of the high concentrations of GABA involved (1.5 mol/l). The GABA A receptor antagonists, bicuculline and picrotoxin, clearly stimulated the release of striatal dopamine. If we accept the possibility of a second GABAergic interneuron (Leviel et al., 1979; Grace and Bunney, 1979) the present observations then imply that this neuron is relatively insensitive to GABA A receptor antagonists. The present results for picrotoxin are in line with those of the cat perfusion study (Ch6ramy et al., 1977), but why bicuculline was inactive in the latter model remains unexplained (Leviel et al., 1979). Our results are somewhat at variance with those of Reid et al. (1990b). These authors observed an increase in ipsilateral striatal extracellular dopamine after intranigral injections of low doses of bicuculline and a decrease after high doses of bicuculline in anesthetized animals. Reid et al. (1990b) suggest that a second, indirectly acting, G A B A A mechanism, probably lo-
cated in the pars reticulata, is of relevance. This second mechanism is hypothesized to be activated at relatively high concentrations or with more penetrating infusion methods such as push-pull perfusion. Reproduction of the findings of Reid et al. (1990b) would have required higher doses of bicuculline, which could not be tested because of the strong behavioral effects of bicuculline in conscious animals. It remains to be established whether the second GABAergic neuron hypothesized by Reid et al. (1990b) is similar to the earlier supposed GABAergic interneuron (Leviel et al., 1979; Grace and Bunney, 1979). 4.3. Nigral GABA B receptors
The present finding that intranigral infusion of baclofen potently inhibited the release of dopamine in the ipsilateral striatum, supports the role of GABA a receptors in controlling the activity of nigrostriatal dopaminergic neurons. The results would be explainable by the existence of GABA B receptors located directly on the somata of the dopaminergic neurons. A second possibility is that baclofen acts on GABA B receptors that are located presynaptically to dopaminergic neurons (Giralt et al., 1990). It is emphasized that the present data are in strong contrast with results obtained with push-pull perfusions in cats, when nigral application of baclofen stimulated the release of ipsilateral striatal radiolabelled dopamine (Chdramy et al., 1978, 1979). Intranigral infusion of the GABA B receptor antagonist, 2-hydroxy-saclofen, was without effect on the release of dopamine in the ipsilateral striatum. This would indicate that the tonus of the GABA B inhibition is relatively weak. However, a more likely explanation for the absence of effects of 2-hydroxy-saclofen is its apparent low potency as a GABA B receptor antagonist. It has been known for many years that systemically administered baclofen causes inhibition of the impulse flow of nigrostriatal dopaminergic neurons, resulting in a sharp increase of the dopamine content of the striatum (Da Prada and Keller, 1976; Anddn and Wachtel, 1977). The present results for baclofen are in good agreement with these observations and suggest that GABA B receptors play a crucial role in this effect. It is speculated that compounds that are pharmacologically closely related to baclofen might also interact with GABA B receptors, y-Hydroxybutyric acid is such a compound and its pharmacological effect is the basis for the well-known presynaptic dopamine autoreceptor model (Roth et al., 1973; Walters and Roth, 1976). 4.4. Changes in DOPAC
Finally, we would like to comment on the drug-induced changes in extracellular DOPAC. It is striking
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that the extracellular levels of DOPAC were increased during both an increase of extracellular dopamine (during infusion of muscimol, bicuculline or picrotoxin) and during a decrease of extracellular dopamine (during infusion of baclofen). A similar tendency was found in the microdialysis studies of Reid et al. (1988, 1990a,b). There is increasing evidence that dopamine does not need to be released first before it is metabolized to DOPAC (Zetterstr6m et al., 1988). The mechanism behind the increase in ipsilateral DOPAC is presently under investigation. 4.5. In conclusion
The present study showed that microdialysis probes can be used to deliver drugs to restricted brain areas and provided an example of the experimental conditions for 'remote control' of dopamine release in the striatum. The observations on GABA A receptor antagonists support the hypothesis that GABA A receptors are located on dopaminergic cell bodies a n d / o r dendrites in the nigra. The present results are best explained when a second projection of GABAergic interneurons in the substantia nigra is hypothesized (Leviel et al., 1979; Grace and Bunney, 1979). This second neuron is apparently much more sensitive to GABA A receptor agonists that to GABA A receptor antagonists. The observed effects on baclofen strongly suggest that GABA B receptors also participate in the regulation of the activity of dopaminergic neurons.
Acknowledgement This work was supported by the Ministerio de Educacion y Ciencia, Spain.
References Aghajanian, G.K. and B.S. Bunney, 1975, Dopaminergic and nondopaminergic neurons of the substantia nigra: differential response to putative transmitters, in: Neuropsychopharmacology, eds. J.R. Boissier, H. Hippius and P. Pocht (Excerpta Medica, Amsterdam and American Elsevier New York) p. 444. And6n, N.E. and G. Stock, 1973, Inhibitory effect of gamma-hydroxybutyric acid and gamma-aminobutyric acid on the dopamine cells in the substantia nigra, Naunyn-Schmiedeb. Arch. Exp. Pathol. Pharmakol. 279, 89. And6n, N.E. and H. Wachtel, 1977, Biochemical effects of baciofen (p-chlorophenyl-GABA) on the dopamine and the noradrenaline in the rat brain, Acta Pharmacol. Toxicol. 40, 310. Benveniste, H., J. Drejer, A. Shousboe and N.H. Diemer, 1987, Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat, J. Neurochem. 49, 729. Ch6ramy, A., A. Nieoullon and J. Glowinski, 1977, Effects of periph-
eral and local administration of picrotoxin on the release of newly synthesized 3H-dopamine in the caudate nucleus of the cat, Arch. Pharmacol. 297, 31. Ch6ramy, A., A . Nieoullon and J. Glowinski, 1978, GABAergic process involved in the control of dopamine release from nigrostriatal dopaminergic neurons in the cat, Eur. J. Pharmacol. 48, 281. Ch6ramy, A., A. Nieoullon and J. Glowinski, 1979, Effects of unilateral nigral application of baclofen on dopamine release in the two caudate nuclei of the cat, Eur. J. Pharmacol. 58, 133. Da Prada, M. and H.H. Keller, 1976, Baclofen and gamma-hydroxybutyrate: similar effects on cerebral dopamine neurons, Life Sci. 19, 1253. Giralt, M.T., G. Bonanno and M. Raiteri, 1990, GABA terminals autoreceptors in the pars compacta and pars reticulata of the rat substantia nigra are GABAB, Eur. J. Pharmacol. 175, 137. Grace, A.A. and B.S. Bunney, 1979, Paradoxical GABA excitation of nigral dopaminergic cells: indirect mediation through reticulata inhibitory neurons, Eur. J. Pharmacol. 59, 211. Groves, P.M., C.J. Wilson, S.J. Young and G. Rebec, 1975, Self-inhibition by dopaminergic neurons, Science 190, 522. Leviel, V., A. Ch6ramy, A. Nieoullon and J. Glowinski, 1979, Symmetric bilateral changes in dopamine release from the caudate nuclei of the cat induced by unilateral nigral application of glycine and GABA related compounds, Brain. Res. 174, 259. Martin, G.E. and D.R. Haubrich, 1978, Striatal dopamine release and contraversive rotation elicited by intranigrally applied muscimol, Nature 275, 230. Morrison, P.F., P.M. Bungay, J.K. Hsiao, B.A. Ball, I.N. Mefford and R.L. Dedrick, 1991, Quantitative microdialysis: Analysis of transients and application of pharmacokinetics in brain, J. Neurochem. 57, 103. Paxinos, G. and C. Watson, 1982, The Rat Brain in Stereotaxic Coordinates (Academic Press, New York). Quan, N. and C.M. Blatteis, 1989, Microdialysis: a system for localized drug delivery into the brain, Brain Res. Bull. 22, 621. Reid, M.S., M. Herrera-Marschitz, T. H6kfelt, L. Terenius and U. Ungerstedt, 1988, Differential modulation of striatal dopamine release by intranigral injection of gamma-aminobutyric acid (GABA), dynorphin A and Substance P, Eur. J. Pharmacol. 147, 411. Reid, M.S., M. Herrera-Marschitz, T. H/Skfelt, M. Ohlin, K.L. Valentino and U. Ungerstedt, 1990a, Effects of intranigral substance P and neurokinin A on striatal dopamine release. I. Interactions with substance P antagonists, Neuroscience 36, 643. Reid, M.S., M. Herrera-Marschitz and U. Ungerstedt, 1990b, Effects of intranigral substance P and neurokinin A on striatal dopamine release. II. Interactions with bicuculline and nalaxone, Neuroscience 36, 659. Roth, R.H., J.R. Waiters and G.K. Aghajanian, 1973, Effect of impulse flow on the release and synthesis of dopamine in the rat striatum, in: Frontiers in Catecholamines Research, eds. E. Usdin and S.H. Snyder (Pergamon Press, New York) p. 567. Santiago, M. and B.H.C. Westerink, 1990, Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes, Naunyn-Schmiedeb. Arch. Pharmacol. 342, 407. Sperber, E.F., J.N.D. Wurpel, N.S., Sharpless and S.L Mosh6, 1989, Intranigral GABAergic drug effects on striatal dopamine activity, Pharmacol. Biochem. Behav. 32, 1067. Waddington, J.L., 1980, GABAergic mechanisms in the substantia nigra, Nature 283, 696. Walters, J.R. and R.H. Roth, 1976, Dopaminergic neurons: An in vivo system for measuring drug interactions with presynaptic receptors, Neunyn-Schmiedeb. Arch. Pharmacol. 296, 5. Westerink, B.H.C., G. Damsma, H. Rollema, J.B. De Vries and A.S.
181 Horn, 1987, Scope and limitations of in vivo brain dialysis: a comparison of its application to various neurotransmitter systems, Life Sci. 41, 1763. Wood, P.L., 1982, Actions of GABAergic agents on dopamine metabolism in the nigrostriatal pathway of the rat, J. Pharmacol. Exp. Ther. 222, 674.
Zetterstr6m, T., T. Sharp, A.K. Collin and U. Ungerstedt, 1988, In vivo measurement of extracellular dopamine and DOPAC in rat striatum after various dopamine-releasing drugs; implications for the origin of extracellular DOPAC, Eur. J. Pharmacol. 148, 327.
