gaba-dopamine receptor-receptor interactions in neostriatal membranes of the rat

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Neurochemical Research, Vol. 22, No. 8, 1997, pp. 1051-1054 GABA-Dopamine Receptor-Receptor Interactions in Neostriatal Membranes of the Rat* M. Perez de la Mora 1,3 S. Ferre, 2 and K. Fuxe 2 (Accepted January 7, 1997) Recent evidence has shown in membrane preparations that the binding of one ligand to its receptor is able to modify the binding parameters of a second receptor (receptor-receptor interactions), allowing the modulation of incoming signals onto a neuron. To further understand the y-amino- butyric acid (GABA)-dopamine (DA) interactions in the neostriatum we have carried out experi- ments to explore whether an activation of the GABA A receptor could affect the binding characteristics of the D 2 DA receptor in membrane preparations of the rat neostriatum. The results show that GAB A (30-100 nM) significantly increases the dissociation constant of the high affinity (K H ) D 2 DA binding site (labelled with the selective D 2 DA receptor antagonist [ 3 H]raclopride and that such an effect is fully counteracted by the GABA A receptor antagonist bicuculline (1 uM). It is suggested that such putative GABA A /D 2 receptor-receptor interactions may take place in the somato-dendritic membrane of the striato-pallidal GABA neurons and that it may modulate the inhibitory effects of DA on these neurons, mediated via D 2 receptors. KEY WORDS: GABA; dopamine; striatum; GABA A receptors; D 2 DA receptors; receptor-receptor interac- tions; dopamine binding. INTRODUCTION It is now generally accepted that y-aminobutyric acid (GABA) and dopamine (DA) interactions play a major role in the physiology of the neostriatum. Medium sized spiny GABA neurons are by far the most abundant neurons within the neostriatum (1,2) and DA terminals from the nigro-striatal neurons give rise to a vast inner- vation of the neostriatum making axo-somatic and axo- dendritic contacts mainly with the spiny GABA neurons 1 Departtnento de Neurociencias, Institute de Fisiologia Celular, Univ- ersidad Nacional Autonoma de Mexico, Mexico. 2 Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden. 3 Address reprint requests to: Miguel Perez de la Mora, Departmento de Neurociencias, Institute de Fisiologia Celular, Universidad Na- cional Autonoma de Mexico, Apartado postal 70-600, 04510 Mex- ico, D.F., Mexico. Phone: 525-622 5668; fax: 525-622 5607. * Special issue dedicated to Dr. Annica Dahlstrom. 1051 (1,3-5). Spiny GABA neurons receive also glutama- tergic imputs from the cerebral cortex (6). In addition, neurons of both striopallidal and strionigral GABA sys- tems, owing to a dense arborization formed by their local axonal collaterals, interact directly or indirectly (inter- neurons) either within the same GABA system or with neurons of the other GABA system (2-5). Further stud- ies have indicated that although the neurons of both sys- tems are morphologically identical, each system is regulated by DA via different types of receptors, and contains a different set of cotransmitter molecules. Thus, while the neurons of the striato-nigral system respond to DA through the activity of D 1 DA receptors and use substance P and dynorphin as cotransmitters, the striato- pallidal projection uses D 2 DA receptors and contains enkephalin (7-10). Evidence for GABA/DA interactions also arise from biochemical studies which show that DA neurotransmission either increases (D 1 action) or de- creases (D 2 action) GABA release in the striatum (11- 13) and conversely, that an increase in GABA neuro- 0364-3190/97/0800-1051$12.50/0 © 1997 Plenum Publishing Corporation

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Neurochemical Research, Vol. 22, No. 8, 1997, pp. 1051-1054

GABA-Dopamine Receptor-Receptor Interactions inNeostriatal Membranes of the Rat*

M. Perez de la Mora1,3 S. Ferre,2 and K. Fuxe2

(Accepted January 7, 1997)

Recent evidence has shown in membrane preparations that the binding of one ligand to its receptoris able to modify the binding parameters of a second receptor (receptor-receptor interactions),allowing the modulation of incoming signals onto a neuron. To further understand the y-amino-butyric acid (GABA)-dopamine (DA) interactions in the neostriatum we have carried out experi-ments to explore whether an activation of the GABAA receptor could affect the bindingcharacteristics of the D2 DA receptor in membrane preparations of the rat neostriatum. The resultsshow that GAB A (30-100 nM) significantly increases the dissociation constant of the high affinity(KH) D2 DA binding site (labelled with the selective D2 DA receptor antagonist [3H]raclopride andthat such an effect is fully counteracted by the GABAA receptor antagonist bicuculline (1 uM). Itis suggested that such putative GABAA/D2 receptor-receptor interactions may take place in thesomato-dendritic membrane of the striato-pallidal GABA neurons and that it may modulate theinhibitory effects of DA on these neurons, mediated via D2 receptors.

KEY WORDS: GABA; dopamine; striatum; GABAA receptors; D2 DA receptors; receptor-receptor interac-tions; dopamine binding.

INTRODUCTION

It is now generally accepted that y-aminobutyricacid (GABA) and dopamine (DA) interactions play amajor role in the physiology of the neostriatum. Mediumsized spiny GABA neurons are by far the most abundantneurons within the neostriatum (1,2) and DA terminalsfrom the nigro-striatal neurons give rise to a vast inner-vation of the neostriatum making axo-somatic and axo-dendritic contacts mainly with the spiny GABA neurons

1 Departtnento de Neurociencias, Institute de Fisiologia Celular, Univ-ersidad Nacional Autonoma de Mexico, Mexico.

2 Department of Neuroscience, Karolinska Institute, 171 77 Stockholm,Sweden.

3 Address reprint requests to: Miguel Perez de la Mora, Departmentode Neurociencias, Institute de Fisiologia Celular, Universidad Na-cional Autonoma de Mexico, Apartado postal 70-600, 04510 Mex-ico, D.F., Mexico. Phone: 525-622 5668; fax: 525-622 5607.

* Special issue dedicated to Dr. Annica Dahlstrom.

1051

(1,3-5). Spiny GABA neurons receive also glutama-tergic imputs from the cerebral cortex (6). In addition,neurons of both striopallidal and strionigral GABA sys-tems, owing to a dense arborization formed by their localaxonal collaterals, interact directly or indirectly (inter-neurons) either within the same GABA system or withneurons of the other GABA system (2-5). Further stud-ies have indicated that although the neurons of both sys-tems are morphologically identical, each system isregulated by DA via different types of receptors, andcontains a different set of cotransmitter molecules. Thus,while the neurons of the striato-nigral system respond toDA through the activity of D1 DA receptors and usesubstance P and dynorphin as cotransmitters, the striato-pallidal projection uses D2 DA receptors and containsenkephalin (7-10). Evidence for GABA/DA interactionsalso arise from biochemical studies which show that DAneurotransmission either increases (D1 action) or de-creases (D2 action) GABA release in the striatum (11-13) and conversely, that an increase in GABA neuro-

0364-3190/97/0800-1051$12.50/0 © 1997 Plenum Publishing Corporation

1052 Perez dela Mora, Ferre, and Fuxe

transmission leads to a reduction in DA turnover (14)and to a decrease in DA release (15).

On the other hand, during the last few years ex-perimental evidence has been obtained showing that ina number of different neuronal systems (see Zoli et al.,(16) for a review and references) the activation of oneG protein coupled receptor by its ligand induces sec-ondary changes in the binding properties of another Gprotein coupled receptor. In view of the fact that suchreceptor-receptor interactions seem to play an importantrole in the integration of the incoming signals onto themembrane of a neuron it seemed to us of interest toexplore whether the activation of the striatal GABAA

receptor could lead to any change in the binding prop-erties of the D2 DA receptor. In order to achieve thistask we studied the effects of GABA on the competitiveinhibition curves of DA vs the D2 antagonist [3H]-raclopride in membrane preparations of rat neostriarum.

EXPERIMENTAL PROCEDURE

Male Sprague-Dawley rats (200 g b.wt, B&K Universal, Stock-holm, Sweden) were maintained in a temperature-and humidity-con-trolled environment with a 12 h light/dark cycle and had free accessto water and food ad libitum. On the day of the experiment the ratswere decapitated, the brain was rapidly removed and the striata weredissected out. The competition experiments with DA vs [3H]raclopridewere performed essentially as described by Ferre et al., (17). The neo-striatal tissue was weighed, introduced in polypropylene vials and son-icated for 30 s in ice-cold Tris-HCl buffer (50 mM, pH 7.6) containing5 mM MgCl2, 0.01% L-(+) ascorbic acid and 1 mM EDTA. Thehomogenate was diluted with the same buffer and centrifuged for 10min at 45,000 g (Sorvall SS-34 rotor; RC-5B centrifuge, DuPont In-strument, Sorvall Division). The supernatant was discarded and themembrane pellet was resuspended by sonication in Tris-buffer andpreincubated for 30 min at 37°C. This preincubation was followed byanother centrifugation for 30 min at 45,000 X g. The pellet was re-

suspended in Tris-buffer (final concentration of membranes, 2.5 mgof wet weight per 0.5 ml of incubation medium). The effect of GABAon the binding competition of DA vs [3H]raclopride was followed byincubating the striatal membranes (0.5 ml) with 2 nM [3H]raclopride(ASTRA Lakemedel, Sodertalje, Sweden) and 20 single concentrations(10 pM - 1 mM) of DA in the presence and absence of GABA for30 min at room temperature. The incubation was stopped by washingthe membranes three times with 5 ml of ice-cold Tris-buffer over aWhatman GF/B filter (Millipore, Milford, MA, USA) under reducedpressure. The radioactivity content of the filters was detected by scin-tillation spectroscopy. Data was analyzed by the LIGAND program(18) for the determination of high-and low-affinity sites (KH and KL)as well as the proportion of receptors in the high affinity state (RH).Points deviating more than two residuals from the fitted curve werediscarded. Curves with more than two discarded points were not in-cluded for statistical analysis. The amount of non-specific binding(about 0.5% of the free ligand) was calculated by extrapolation of thedisplacement curves. To avoid the variability of the binding parametersassociated with the assay conditions, a repeated measures design wasapplied by using the same membrane preparation to study the effectsof different concentrations of GABA or the effect GABA in the pres-ence and absence of the GABA antagonist bicuculline. Statistical eval-uation was made by repeated measures ANOVA analysis followed bythe post-hoc Fisher protected least-square difference method. Toachieve homogeneity of variance and allow parametric statistical anal-ysis, KH and KL were logarithmically transformed (19).

RESULTS

As shown in Table I and Fig. 1, GABA at nmolarconcentrations decreased the DA-induced inhibition of[3H]raclopride binding by increasing the KH value. Theeffect did not reach statistical significance at 10 nM butwas significant at the 30 and 100 nM concentrations (Ta-ble I). The KL values also increased but due to the highvariability no significance was reached. No changes inRH were observed at any GABA concentration. On theother hand, bicuculline did not by itself affect any bind-ing parameter but fully counteracted the effect of GABAon the competitive inhibition curves of DA vs[3H]raclopride (Table II).

DISCUSSION

These results show that like glutamate (20), neu-rotensin (21) and A2a agonists (17), GABA is able toreduce the affinity of DA in neostriatal membranes forDA D2 receptors as indicated by the observed significantincrease in the KH value in the competition experimentswith DA vs [3H]raclopride. The effect on KH most prob-ably involves an activation by GABA of the GABAA

receptor, since it occurs at concentrations (nmolar range)near to the KD value for this receptor (22) and is coun-teracted by bicuculline, a well known antagonist for GA-

Table I. Studies on GABA Modulation of the Ability of DA toCompete with [3H]Raclopride for Its Binding in Neostriatal

Membranes

Treatment

Control10 nM GABA30 nM GABA100 nM GABA

KH (nM)

4.8 (2.9 - 7.7)7.7 (3.9 - 15.1)

12.2 (4.5 - 33.1)*8.9 (5.1 - 15.4)*

KL (nM)

43 (10 - 190)103 (11 - 984)155 (13 - 1500)148 (40 - 501)

RH % Total

80 ± 1978 - 1.681 ± 4.777 ± 4.6

The dissociation constants of high affinity and low affinity bindingsites (KH and KL, respectively) are presented as geometric means and,in parenthesis, the antilog of the 95% confidence limits of the geo-metric mean. The percentage of total specific binding sites in the highaffinity state (RH) is shown as means ± SEM. A repeated measuresdesign was applied by using the same membrane preparation to studythe effects of different concentrations of GABA (n = 7).* Significantly different compared with the control (p < 0.05 in allcases).

GABAA/D2 Receptor Interactions 1053

Fig. 1. Representative competitive inhibition curves illustrating theeffect of GABA (30 nM) on dopamine-induced inhibition of[3H]raclopride binding in membrane preparations from rat striatum. KH

and KL values were estimated to 1.9 and 34.2 nM under normal con-ditions and 51.2 and 48.0 nM in the presence of GABA. The RH valueswere 85.4 and 95.4%, respectively.

reduced D2 affinity demonstrated, since GABA agonistsapplied in the striatum of rats produce a reduction ratherthan an increase in DA release (15). Likewise, althoughthe cholinergic and GABA interneurons contain GABAA

and D2 DA receptors (2,24), their participation in theeffects reported in this paper would be of minor impor-tance owing to the relatively low number of interneuronsin the striatum compared with efferent neurons (2).

D1 agonists stimulate the activity of the striatonigralGABA pathway and increase GABA release in the neos-triatum and D2 agonists decrease the activity of the stria-topallidal neurons and reduce striatal GABA release(9,11,13). Therefore, the present results may suggest thatany activation of the GABAA receptors by GABA re-leased from axon collaterals on the somatodendritic sur-face of the striatopallidal neurons might antagonize,through an antagonistic GABAA/D2 interaction, the in-hibitory effects of DA on the activity of the striatopal-lidal GABA neurons. In this way excessive inhibition ofthis GABA pathway may be avoided.

In conclusion, the present results indicate thatGABA acting on striatal GABAA receptors reduces theaffinity of the D2 DA receptor via an intramembraneinteraction and opens up a possible new integrativemechanism for GABA/DA interactions in the basal gan-glia.

ACKNOWLEDGMENTS

This work has been supported by a grant from the Direccion deAsuntos del Personal Academico, Universidad Nacional Autonoma deMexico (IN208194), and by a grant (04X-715) from the Swedish Med-ical Research Council, Sweden and by a grant from the Marianne andMarcus Wallenberg Foundation. The technical assistance of Ulla-BrittFinnman and excellent secretarial help of Anne Edgren is gratefullyacknowledged.

REFERENCES

1. Gerfen, C. 1992. The neostriatal mosaic: multiple levels of com-partmental organization. Trends Neurosci. 15:133-138.

2. Kawaguchi, Y., Wilson, C., Avgood, S., and Emson, R. 1995.Striatal interneurons: chemical, physiological and morphologicalcharacterization. Trends Neurosci. 18:527-535.

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Table II. Antagonistic Effects of Bicuculline on the Ability ofGABA to Modulate the Competitive-Inhibition Curves of

[3H]Raclopride vs DA in Neostriatal Membranes

Treatment

ControlGABA (30 nM)Bicuculline (1 mM)GABA (30 nM) +Bicuculline

KH (nM)

1.8 (0.2 - 13.9)9.4 (4.9 - 17.7)*2.8 (0.3 - 22.9)

1.3 (0.2 - 7.6)

KL (nM)

79.4 (39 - 213)280 (108 - 726)127 (39 - 398)

95 (43 - 208)

RH% Total

80 ± 1.981 ± 570 ± 7

63 ± 5

Means ± SEM. For further details, see text to Table I. A repeatedmeasures design was applied by using the same membrane preparationto study the effects of GABA in the presence and absence of theGABA antagonist bicuculline (n = 4).*Significantly different compared to the control group (p < 0.05).

BAA receptors. Washed neostriatal membranepreparations were used indicating that an intramembraneinteraction must be involved. Therefore, it is possiblethat the changes in the binding characteristics of the D2

receptors occurred through an interaction of the activatedGABAA receptors with the D2 DA receptors located ad-jacent to the GABAA receptors in the same membrane.

Within the neostriatum D2 DA receptors are locatedto a large extent on the spiny striatopallidal GABAergicneurons (7,23) which through a dense arborization oftheir axons synapse inter alia among themselves andwith neurons of the striatonigral GABAergic projection(3-5) containing D1 DA receptors. Therefore, it is pos-sible that a GABAA - D2 DA receptor-receptor inter-action may take place on the somatodendritic surface ofthe striatopallidal neurons. The possibility that an inter-action of GABAA receptors takes place presynapticallywith D2 DA autoreceptors seems unlikely in view of the

1054 Perez dela Mora, Ferre, and Fuxe

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