Modulatory role of dopamine on excitatory amino acid receptors

Pmg. NeumPsycho~-

&Bid.

Psych&t Cowaght

1996, Vol. 20. pp. 659-671 8 1996 E1sevte.r 6dence

PIintedIntheush

0276~5646/96

ELSEVIER

Inc.

Aurtghts$32.00

+ .OO

PII 90279-6s4s(99)ooo39-6

MODUTGRY

ROLE OF DOPAMINE ON EXCITATORY AMINO ACID RECEPTORS

OTTAVIO

GANDOLFI

and ROSSELLA

DALL’OLIO

Department of Pharmacology, University of Bologna, Italy

(Final form, February 1996)

Gandolfi Ottavio and Rossella Dall’Olio: Modulatory role of dopamine on excitatory amino acid receptors. F’rog.Neuro-Psychopharmacol. & Biol Psychiat. 1996 20, pp. 659-671 1. In extensively washed synaptic membrane preparations from rat prefrontal cortex, the “in vitro” addition of either the Dl (SKF 38393) or the D2 (LY 171555) specific agonists markedly decreased the apparent affinity of the NMDA receptor antagonist [3H]-MK801 specific binding. In the same membrane preparation, the concentration of L-glutamate required to produce half maximal enhancement of [3H]-MK801 binding was approximately the same both in the presence or in the absence of dopaminergic drugs. 2. I.c.v. administration of the neurotoxin 6-OHDA resulted in a dramatic reduction of dopamine (DA) prefrontal cortex levels, whilst repeated administrations (21 consecutive days) with either the Dl (SCH 23390) or the D2 (YM 09151-2) selective antagonist failed to change DA and DOPAC contents. 3. Repeated administrations with the Dl receptor blocker SCH 23390 selectively increased the Bmax values of [3H]-SCH 23390 binding while [3H]-spiroperidol binding was increased both by repeated administrations of YM 09151-2 and by i.c.v. injection of 6-OHDA. 4. Although both chronic D2 blockade and 6-OHDA lesions consistentely increased D2 receptor number, in extensively washed synaptic plasma membranes (SPM) of rats repeatedly administered with YM 09151-2 but not with 6-OHDA, the [3H]-MK801 binding was increased. 5. It is concluded that the effects of NMDA receptor activation could not be directely mediated by stimulation of DA release, but are highly dependent upon the presence of DA axon terminals. Kevwor&: catecholamines content, N-methyl-D-aspartate, administrations, specific dopamine receptors.

neurotoxic

lesion,

rats, repeated

. . Ddopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), excitatory amino acid (EAA), beta,gamma-imidoguanosine-5’-tryphosphate (Gpp(NH),), N-methyl-D-aspartate (NMDA), 6-hydroxydopamine (COHDA), synaphic plasma membrane (SPM).

Recently, the full comprehension of the molecular mechanisms underlying the interactions between dopamine (DA) and excitatory amino acidis (EAA) represents a scientific and clinical goal 659

660

0.

Gandolft and R Dall’Olio

since abnormalities in these neurotransmitter actions have been implicated in the clinical symptoms of Parkinson disease and schizophrenia (Carlsson and Carlsson, 1990, Deutch et al., 1989; Kim et al., 1980). Several biochemical studies focused on the modulatory effects of NMDA-sensitive glutamate receptors on DA release in tissue slices (Asencio et al., 1991; Bowyer et al., 1992; Dwoskin et al., 1992; Jhmandas and Marien, 1987; Marien et al., 1983; Ohmori et al., 1992; Roberts and Sharif, 1978; Roberts and Anderson, 1979; Snell and Johnson, 1986) and in dissociated cell cultures of rat mesencephalon (Mount et al., 1990). Extra-cellular levels of DA evaluated by microdyalisis studies did not generate univocal results: intrastriatal infusions with NMDA increased (Muck et al., 1992; Morari et al., 1993) or did not modify (Imperato et al., 1990) extra-cellular DA levels, and in our laboratory, D-cycloserine (3 mg/kg i.p.), a partial agonist of the strichnine-insensitive

glycine recognition site failed to change

either DA and DOPAC extra-cellular output twenty-four hours after the implantation

of the

transstriatal probe in freely moving rat striatal dialysates (Gandolfi et al., 1994). Electrophysiologic studies obtained with quantitative single-unit recording, show that in rat the NMDA uncompetitive receptor antagonist MK801 produces dose-dependent increases both in the burst firing of ventral tegmental Ato DA neurons and in dopaminergic tone within mesolimbicmesocortical regions (French et al., 1993). Behavioral studies in rodents show that MK801 antagonizes dopamine D2 receptor mediated catalepsy induced by different DA receptor antagonists (Papa et al., 1993; Schmidt et al., 1991) activates dopaminergic transmission (Hiramatsu et al., 1989; Liljequist et al., 1991; Rao et al., 1990) and induces hypermotility either in rats (Dall’Olio et al., 1995) or in monoamine-depleted mice (Carlsson and Carlsson, 1989). In this bulk of evidences, that not always generated consistent results, the authors planned biochemical experiments to evaluate the modulation of NMDA-sensitive glutamate receptors in two different experimental conditions: i) prolonged Dt or D2 receptor blockade-induced receptor super-sensitivity, and ii) DA receptor supersensitivity following specific neurotoxin (6-OHDA) lesions. The authors followed such dual approach in the light of strengthening

information

that

comparing the two experimental models, rats withdrawn from repeated treatments with DA blockers show the same degree of D2 receptor supersensitivity as rats lesioned with 6-OHDA which distroys most of DA axon terminals. The authors planned “ex vivo” [3H]- MK801 radiolingand binding studies, serving as an index of functional activation of NMDA receptors (Loo et al. 1986, Fagg 1987, Foster and Wong 1987), in well washed synaptic plasma membrane preparation (SPM) from the prefrontal cortex of tats either repeatedly treated with Dt/D2 blockers or in 6-OHDA lesioned animals. Other [3H]-MK801 radiolingad binding experiments after the “in vitro” addition of different DA agonists, or drugs acting at the G-protein level (Gpp(NH)p) were undertaken to evaluate whether DA-ergic drugs, at least in this simplified system could interfere by simply direct interaction with NMDA-sensitive glutamate receptor complex.

DA-NMDA

661

Interaction in CNS

Male Sprague Dawley rats (150-175 g, Charles River Italia SpA, Como, Italy) were housed in groups of four under controlled conditions of light (07:OOa.m. - 07:OO p.m.), temperature (22 rt 2’C) and humidity (65%) and were allowed free access to standard laboratory diet and tap water. Experimental protocols were approved by a local bioethical committee while the procedures and the animal confort were controlled by the University Veterinary Service.

[3H]-MK801 (15 Ci/mmol), [3H]-SCH 23390 (70 Ci/mmol) and [3H]-spiroperidol (15 Ci/mmol) were obtained from NEN, Boston, MA, USA. (+)-Butaclamol HCl, (+)-SCH 23390 ((R)-(+)-8chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-lH-3-benzazepine-7-maleate),

MK801 (dizocilpine)

and SKP 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-l-phenyl-lH-benzazepine)

were from RBI,

Natick, MA, USA. YM 09151-2 (Cis-N-(benzyl-2-metylpyrrolidin-3-yl)-5-chloro-2-methoxy-4methylamino-benzamide)

from Yamanouchi,

Tokyo, Japan. LY 171555 (Trans-(-)-4a-R-4-

,4a,5,6,7,8,8a,9-octahydro-5-propyl-2H-pyrazolo-(3,4)-quinoline Indianapolis,

IN, USA. G,(NH),

dihydroxy-phenyl-ethyl-amine),

HCl),

(beta, gamma-imidoguanosine-5, DOPAC

(3,4dihydroxyphenylacetic

from

Eli

Lilly

Co.,

triphosphate), DA (3,4acid),

6-OHDA

(6-

hydroxydopamine), (-)-sulpiride, L-glutamate and haloperidol, were from Sigma, St.Louis, MO, USA. ed

Drup Treatme-

Group of animals (n= I/group) received daily injections for 21 consecutive days of the following pharmacological treatments: the Dl dopamine receptor blocker SCH 23390 (0.05 mg/kg S.C.twice daily), the Dz specific antagonist YM 09151-2 (0.02 mg/kg i.p.), the “atypic neuroleptic” (-)sulpiride (20 mg/kg i.p.), haloperidol (0.125 mg/kg i.p.), or saline. Three days after the withdrawal of 21-day repeated administrations when behavioral supersensitivity to a subthreshold dose of apomorphine was observed (Dall’Olio et al., 1990) rats were killed by decapitation, the brains removed, rinsed in ice-cold isotonic saline, and frontal cortices dissected over ice were stored at -80°C until assays. $-HvdrQ.g&pamme (6-OHDA) - Lesiom

Groups of rats (n= lo/group) were anesthetized with pentobarbital. Lesions were produced by injecting 6-OHDA (200 pg) stereotaxically in the lateral ventricle. Sham-lesioned animals were given i.c.v. injections of the vehicle solution, 0.01% (wt/vol) ascorbic acid in saline. Animals of both groups were injected intraperitoneally with desmethylimipramine

(25 mg/kg) 40 minutes

before the injection of neurotoxin (or vehicle) to prevent the uptake of the neurotoxin into noradrenergic axons and thus minimize the degeneration of these terminals. Lesioned and shamlesioned rats were killed by decapitation 24 days after surgery, the brains were removed and prefrontal cortices dissected over ice were stored at -80°C until assays.

662

0. GandoIfi and R. Dall’Olio

.

.

Dopamine (DA) and Dihydroxvahenvlacehc acrd (DOPAC) Determinatiotg The concentrations and Salama

of DA and DOPAC in rat frontal cortices were measured according

(1984) using high-performance-liquid

detector (HPLC-ED). homogenates

centrifuged

the HPLC-ED

system. The mobile-phase

(pH 4.0-4.3) consisted

0.1 mM ethylendiamine

were carried out with minor modifications

buffer pH 7.4, the homogenates

resuspended,

and the homogenates

pellets were resuspended

together with different spiroperidol

according

were centrifuged

receptor

to Billard et al. (1984) and

with Polytron in ice-cold 50 mM

(30,000 x g, 10 min), pellets were

in 50 mM Tris-HCl buffer containing of membrane

concentrations

120 mM NaCl, 5 mM KCl, 2 mM

suspensions

were incubated

(30 min, 37°C)

of [3H]-SCH 23390 (ranging from 0.25 to 6 nM) or [3H]-

through Watman GF/C filters. The specific

between

the total binding

and the binding

were determined

in the presence

respectively

under by the

of a specific

for [3H]-SCH 23390 and

binding.

The [3H]-MK801

binding assays were carried out according to Stirling et al. (1989). Prefrontal

were homogenized

homogenizer.

bindings

remaining

1 pM cold SCH 23390 or 1 pM (+)-butaclamol

[3H]-spiroperidol

cortices

to Dl and D2 dopamine

(ranging from 0.02 to 0.5 nM). The reaction was stopped by rapid filtration

pressure

displacer,

column (C-

were pelleted twice in the same buffer. The final membrane

CaC12 and 1 mM MgC12. Aliquots

difference

on a reverse-phase

detector (Waters M-460) was operated at +0.65 mV.

and Langer (1978). Briefly, cortices were homogenized

negative

to

Bindin-

respectively,

Tris-HCl

acid, the

were applied diictely

of 70 mM NaI-IzPOd, 0.65 mM

The binding assays for [3H]-SCH 23390 and [3H]-spiroperidol

Briley

to electrochemical

tetracetic acid (EDTA), 0.035 M citric acid and

Separation of DA and DOPAC was accomplished

18; 25 cm) and the electrochemical &g&&and

coupled

in 0.1 M perchloric

and samples (20 pl) of the resulting supematant

sodium octyl sulphate, 35% acetonitrile.

chromatography

Briefly, prefrontal cortices were homogenized

to Saller

The

in 10 vol ice-cold 0.32 M sucrose using a motor-driven

homogenate

sucrose, and centrifuged

again. The supematants

pellets. These were resuspended pellets were resuspended

was centrifuged

glass-teflon

at 1,000 x g, the pellet resuspended were pooled, centrifuged

in 0.32 M

at 45,000 x g to yield P2

in 20 vol 5 mM Tris-HCl pH 7.7 at 4°C and centrifuged.

in ice-cold distilled water and centrifuged.

This procedure

The

was repeated

three times and the pellets were frozen at -80°C for at least 18 hours. On the day of the assay pellets were thawed, centrifuged Tris-HCl

suspended

in 20 volumes

of 5 mM Tris-HCl

pH 7.7 at 4”C, homogenized

at 30,000 x g. This wash step was repeated four more times in 100 volumes buffer and the final suspension

was incubated at room temperature

centrifugation

at 30,000 x g. The final pellets were resuspended

concentration

of 0.8-l mg/ml. Aliquots of synaptic plasma membranes

prefrontal

cornices

were incubated with different concentrations

to 100 nM). In some experiments,

membranes

concentrations

5 mM

for 20 min prior to

in the same buffer to give a protein @PM) prepared

from the

of [3H]-MK801

(ranging from 2

were incubated with [3H]-MK801

(25 nM) and Dl

agonist SKF 38393, the D2 agonist LY 171555 (both 100 pM) either in the presence absence of different

and

of L-glutamate

or in the

(ranging from lo-lo to 10” M). In different

DA-NMDA Interaction in CNS

663

series of experiments, [3H]-MK801 (25 nM) specific binding was run in the presence of the non hydrolizable GTP analogue G&NH&

(100 pM) to activate the G-protein coupler of adenylate

cyclase system. The specific binding was determined between the total binding and the binding left in the presence of 1 uM cold MK801. After incubation for 2 hr at 25°C (equilibrium), the mixture was rapidly filtered through Watman GF/C filters presoaked in 0.05% polyethyleneimine radioactivity remaining on the filters was counted by liquid scintillation

and the

spectrometry using

Atomlight (NEN, Boston, MA, USA). The kinetic characteristics of the specific binding were analyzed according to the method of Scatchard (1949) using the program of Munson and Rodbard (1980). Proteins were measured using BSA as standard according to Lowry et al. (1951). Data a42.lM

Radioligand binding data were analysed by Students t test, and the concentrations of DA and DOPAC

were analysed by means of ANOVA followed by single comparisons of the means.

In extensively washed SPM from rat frontal cortex, [3H]-MK801 labeled a single population of sites (Bmax = 6.92 f 0.67 pmol/mg prot) with good affinity (Kd = 19.5 f 2.1 nM). The “in vitro” addition of either the Dl receptor agonist SKF 38393 or the D2 agent LY 171555 (both 100 JIM) markedly decreased the affinity of [3H]-MK801 binding (Table 1) while failed to change the maximum number of binding sites. Lower DI or D2 agonists concentrations did not show any statistically different changes in the kinetic characteristics of [3H]-MK801 specific binding (data not shown). Table 1

Influence of DI and D2 DA Agonists “in vitro” Addition, on the Kinetic Characteristics of [3H]MK801 Specific Binding to Extensively Washed Rat Prefrontal Cortex Membranes. Bmax (pmol/mg prot) 6.9240.67

Control SKF 38393

100 FM

5.86M.51

( LY 171555

100 PM

6.15k0.49

Kd (nM) 19.5dz2.1 47.6&3.6*

38.4+2.7* [3H]-MK801 binding was performed either in the absence of any drug (control) and in the presence of 100 pM SKF 38393 or 100 pM LY 171555. Mean values f SEM are reported. The apparent Kd values following the “in vitro” addition of either agonists were significantly different (*p c.05 Student r-lest) from those obtained in control.

In

the same preparation, [3H]-MK801 (25 nM) specific binding has been enhanced by the

addition of L-glutamate (10-a - 1O-4M). Figure 1 shows that in the presence of maximally active concentration of L-glutamate (10m4M) the [3HJ-MK801 specific binding was increased by about

664

0. Gandolfi

and R Dall’Olio

300%. The interactions of the D2 agent LY 171555 with [3H]-MK801 binding sites were of uncompetitive nature; increasing concentrations of L-glutamate did not alter the inhibitory effect of the DA-ergic agent on [3H]-MK801 binding in extensively washed SPM prepared from rat prefrontal cortex. The concentration of L-glutamate required to produce half maximal enhancement of [3H]-MK801 binding was approximately the same both in the presence (EC50 = 0.9 pM) or in the absence (EC50 = 0.7 pM) of LY 171555.

. :: 0-1 0

/

-a

I

-7

-6

L-Glutamate

-5

-4

-3

[M]

Fig 1. Basal [3H]-MK801 specific bindings (25 nM) in the absence (*) and in the presence (0) of LY 171555 (100 @I) were 2.4 f 0.18 (SEM) and 1.2 f 0.08 @EM) pmol/mg prot respectively. Specific binding was over 70% of total binding in basal conditions and was greater than 80% of total binding under conditions of maximal stimulation. Comparable results have been obtained in at least three separate experiments.

The ability of the G-protein non-hydrolysable activator G,(NH),

(10e4 M) to modulate the [3H]-

MK801 (25 nM) specific binding to SPM preparations is shown in Table 2. The “in vitro” addition of the stimulating G protein nucleotide inhibited by more than 50% [3H]-MK801 specific binding. Maximally active concentrations of L-glutamate, although dramatically increasing [3H]-MK801 binding were unable to reverse guanine nucleotide-induced inhibition. Table 2. Inhibition of ]3H]-MK801 (25 nM) Specific Binding by the Non Hydrolizable GTP Analogue to Extensively Washed SPM prepared from Rat Prefrontal Cortex

Basal G,(NH), (to(J@0 Resultsare expressedas mean f SEM

[3H]-MK 801 specific binding (25 nM) (pmol/mg pro0 -L-Glut. + 100 pM L-Glut. 8.=.6* 2.5M. 18 3.4k0.2* l.kbO.07

values from at least three experiments each performed in triplicate. *p 1.05 (Student r-test) when compared to the respective basal value.

DA-NMDA

.

Interaction in CNS

665

.

Neurotomc Lesron and Repeated Drue Administrations

Table 3 shows that the i.c.v. injection of neurotoxin 6-OHDA resulted in a dramatic reduction of DA and DOPAC levels in rat prefrontal cortex. Consistent decreases (70-80%) of [3HJ-DA uptake by cortical synaptosomes (not shown) used as a further index of the degree of neurotoxic lesion were found. In contrast, consistently with Finlay et al. (1987) and Essig and Kilpatrick (1991) DA and DOPAC cortical levels were not modified by repeated administrations of Dl or D2 blockers. Consistenly with previous results (Cmese and Chen 1985, Gandolfi et al. 1988, Duncan et al. 1993, Giorgi et al. 1993), Scatchard analysis of the saturation data of [3H]-SCH 23390 and [3H]spiroperidol bindings to SPM prepared from repeatedly treated rats showed that the D, blocker increased (about 30%) the number but not the apparent affinity values of Dl binding sites labelled by [3H]-SCH 23390. The same repeated treatment with the Dt receptor blocker failed to modify the kinetic characteristics of [3H]spiroperidol specific binding to SPM. The kinetic characteristics of [3H]-Spiroperidol binding to SPM of rats repeatedly administered with YM 09151-2 revealed large increases in Bmax values (about 48%) while Dl receptors were unaffected. Table 3 shows also that in SPM prepared from the 6-OHDA lesioned rats there is a statistically significant increase (about 40%) in the maximum number (Bmax) of [3H]-spiroperidol specific binding, while the kinetic characteristics of [3H]-SCH 23390 binding to Dl receptors did not change. Table 3 Effects of 6-OHDA or Repeated Administrations with Either Dl and D2 Dopamine Receptor Blockers on DA and DOPAC Cortical Levels and on the Kinetic Characteristics of DI and D2 Receptors in SPM. DA (pmol/w) Non-lesioned 6-OHDA

62W2 4w*

DGPAC (pmdmg) 72k3 g&0.8*

[3H]-SCH 23390

[ 3HI-spiroperidol Bmax(fmol/mg) Kd(nM)

Kd(nM)

Billax (fmol/mg)

0.9fo.03

396+17

0.16M.01

1lm.6

1.2M.05

42W3 1

0.15~.01

16W.l*

SCH 23390

596&68

68f5

l.lXl.04

53X27**

0.17+0 - .01

103*4.1

YM 09151-2

65 lzh66

83s

0.8M.04

352&20

0.21fl.03

177&9**

DA and DOPAC levels, like c3H]-SCH 23390and [3Hj-spiroperidol specific bindings were determined following a 72 hr washout period from rats repeatedly treated (21 days) with SCH23390 (0.05 mg/kg i.p. twice daily) or with YM 09151-2 (0.02 mg/kg i.p.) or 24 days following 6-OHDA (200 pg i.c.v.) administration. *~.05 when compared to sham-lesioned rats **~.05 significantly different from repeatedly saline-treated rats

Figure 2 shows the normalized data transformed to % control of [3H]-MK801 specific binding to extensively washed SPM. Statistically significant increase of [3H]-MK801 binding was found only following repeated administrations of either YM 09151-2 (3389 f 174 fmol/mg Vs 2394f 102 fmol/mg pc.05 Student’s t test) or haloperidol (3009 f 178 fmol/mg p<.O2 Student’s t test). Repeated administrations either of SCH 23390 or the atypic neuroleptic (-) sulpiride failed to change [3H]-MK801 specific binding (pannel B).

666

0. GandolB

Although

in SPM of either repeatedly

there are consistent prepared

increases

from DA depleted

and R. DaU’Olfo

YM 09151-2 treated rats or 60HDA-lesioned

of D2receptor

binding (Table 3), in prefrontal

rats no changes in [3H]-MK801

Moreover in the same membrane preparation

binding were observed

from DA-depleted

animals

cortex membranes (Pannel A).

animals the “in vitro”, addition of

high doses of DA (10 PM) failed to modify [3H]-MK801 specific binding.

A

l3

0

control

@j

6-OHDA

c]

control

•j

SCH

23390

YM

09151-2

i.c.v.

q 6-DHDA

+

DA

=I-sulp

@halo

10“H 100

/

-

0

-_ i ** *

Fig 2. [3Hj-MK801

specific binding (25 nM) to extensively-washed SPM prepared from rat prefrontal cortex was performed as described in “Radiolingand Binding Studies”. Animals were killed either 72 hr following repeated (21 days) administration of SCH 23390 (0.05 mg/kg i.p. twice daily), (-)-sulpiride (2Omg/kg i.p.), YM 09151-2 (0.02 mg/kg i.p.), haloperidol (0.125 mg/kg i.p.) or saline (Part B), or 24 days following 6-GHDA (200 pg i.c.v.) (Part A). The results are expressed as % change of control [3H]-MK801 specific binding. Non transformed data were: control=2394 f102 fmol/mg; YMO9151-2=3389+174 fmol/mg; haloperidol =3OfI9*178 fmobmg. Bach value represents the means of at least three experiments done in triplicate. SEM were less than 10%. *p<.O5 (Student’s ttest) when compared to control. **p<.O2 (Student’s t-test) when compared to control.

In this study, the L-glutamate the general hypothesis

enhancement

that the non-competitive

activated state of the NMDA receptor-cation serving as an index of functional Loo et al. (McDonald

1986).

induced

enhancements different

This

et al. 1987)

linked ion channel binding

of [3H]-MK801 binding gives supportive evidence to

receptor,

by repeated

antagonist

MK801 binds to an

(Wong et al., 1986) and therefore

activation of the receptor (Fagg, 1987; Foster and Wong, 1987;

interpretation, that MK

NMDA receptor

channel complex

taken

together

with

electro-physiological

evidencies

801 is an open channel blocker that binds within the NMDA-

allows us to assume that at equilibrium administrations

increases

with YM 09151-2 or haloperidol

of [3H]-MK801 are indicative

of

in NMDA receptor function. These results agree with other data obtained both with

ligands ([3H]-TCP)

or with quantitative

autoradiography

techniques

NMDA receptor function following repeated haloperidol or eticlopride

showing

administrations

increased (Byrd et al.

1987; Ulas et al., 1994) but are at variance with others (Creese et al. 1994; Lang et al. 1992).

DA-NMDA Interaction in CNS

Different

techniques

(autoradiography

vs radioreceptor binding

667

studies), labels, membrane

preparation procedures, doses, times and washout periods could be responsible for this apparent discrepancy. Interuretation of “ex vivo” Radioliand

Bindi@&&

The present data show that in rat prefrontal cortex, a cerebral area having one of the highest concentrations of DA-ergic nerve fibers, there is an enhancement of [3H]-MK801 binding only in SPM prepared from D2 supersensitive rats following D2 receptor blockade. In 6-OHDA-lesioned animals where DA axon terminals are almost completely absent, the increase in D2 receptor number did not parallel the increase [3H]-MK801 specific binding. It is unlike that the DA paucity following 6-OHDA lesion (about 10%) is insufficient to activate D2 receptors and to increase [3H]MK801 specific binding, since the “in vitro” addition of DA-ergic drugs (10-S M) in SPM of shamlesioned rats decreased [3H]-MK801 binding, while in synaptic membranes of rats lesioned with 6OHDA DA failed to affect [3H]-MK801 binding. Intremetation qf “in vitro” Radio&gmd Bind& &&Q

The D2 agent LY 171555 produced similar inhibitions of [3H]-MK801 specific binding either in the absence or in the presence of different concentrations of L-glutamate, theoretically suggesting that the interactions between NMDA and DA receptors, non mediated via the NMDA recognition site, could be of uncompetitive nature. In contrast to our “ex vivo” results showing [3H]-MK801 binding increases in rats repeatedly administered with D2 blockers, the “in vitro” addition of high (but not low) doses of DA-ergic agents decreases affinity (Kd values increases) of [3H]-MK801 specific binding in well washed SPM of untreated animals suggesting that, at least in this system, DA-ergic drugs could modify glutamatergic function. This effect however was not abserved when DA was added in SPM prepared from 6-OHDA lesioned rats. To verify whether the failure of DAergic drugs to modify [3H]-MK801 binding in 6-OHDA lesioned rats could be due to the uncoupling of DA receptors with the G protein following the lesion, the authors performed “in vitro” [3H]-MK801 binding studies after activation of G protein by the non-hydrolizable analogue G,,(NH),.

GTP

Our results indicated that the inhibiting effet of this drug on [3H]-MK801

binding is unrelated to a direct effect of guanine nucleotides on the NMDA-glutamate recognition site since the inhibition of [3H]-MK801 binding failed to change after the addition of 10-d M Lglutamate. Other studies (Baron et al. 1989) that showed that guanine nucleotides reduced both antagonists ([3H]-CPP) and agonist ([3H]-glutamate) binding sites are inconsistent with all we know about GTP-binding protein-linked receptors where guanine nucleotides selectively decrease agonist but not antagonist

binding sites by converting high affinity to low affinity sites

(Sokolovsky et al. 1980) and suggest that the inhibition of [3H]-MK801 binding could not be related to the activation of GTP binding proteins. All these data taken together suggest that the DA-elicited modulation of NMDA receptors could not be simply a function of DA release from nerve terminals at this site. Endogenous DA, via presynaptic action on supersensitive receptors, through asso-assonic interaction could modulate glutamate or another substance (peptide) release from a DA-controlled neuron that appears to play

668

0.

Gandolfi and R Dall’Olio

an important role in modulating the NMDA receptor function. However, when DA neurons are absent, the lack of DA-ergic terminals could not induce the DA-controlled (glutamate ?) neuron to activate and therefore to increase [3H]-MK801 specific binding.

All these results taken together with other data showing the incapacity of MK801 and other glutamatergic drugs (cycloserine) to modulate extracellular levels of DA (Gandolfi et al. 1992, 1994) indicate that the effects of NMDA receptor activation could not be directely mediated by stimulation of DA release, however this response is highly dependent upon the presence of DA axon terminals, subserving therefore the complex modulatory role of dopamine in EAA receptor activation. The present results rather suggest trans-synaptic mechanisms, and do not allow to assume a direct or a G-protein mediated control of DA-ergic agonists on NMDA receptor function. The exact mechanisms of this interaction is still obscure; electrophysiological

studies utilizing

membrane patch techniques could represent the most suitable approach to examine the regulation of NMDA responses in both experimental conditions of Dz-receptor supersensitivity.

The present work was supported by Grants of Italian Ministry for University. The authors thank Mr. R. Laghetti and Mrs. C. Cappelletti for typing the manuscript.

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