Treatment with NMDA receptor antagonists does not affect developmental changes in NMDA receptor properties in vivo

Neurochem. Int. Vol. 27, No. 4/5, pp. 355 366, 1995

~ ) Pergamon

0197-0186(95)00017-8

Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0197~0186/95 $9.50+0.00

TREATMENT WITH N M D A RECEPTOR ANTAGONISTS DOES NOT AFFECT DEVELOPMENTAL CHANGES IN N M D A RECEPTOR PROPERTIES IN VIVO M E N N O V A N L O O K E R E N C A M P A G N E * , J O L A N D A P. V E R M E U L E N , G E R A R D J. B O E R t and R O B E R T BAL./~ZS~ Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam ZO, The Netherlands (Received 15 October 1994 ; accepted 26 January 1995)

Abstract--Effects of acute and long-term treatment of neonatal rats with N-methyl-o-aspartate (NMDA) receptor antagonists on changes in NMDA receptor properties were examined. Animals received either on postnatal day 6 a single dose of the antagonists MK-801 (1 mg/kg), or D-CPPene (2 mg/kg) or during the period from postnatal day 5 to 14, two daily injections of MK-801 (0.25 mg/kg) or D-CPPene (0.75 mg/kg). Control littermates received saline injections. In both cases animals were sacrificed one day after the last injection. NMDA receptor properties were examined in membrane preparations derived from the cerebral cortex by studying the modulation of [3H]MK-801 binding by glutamate, Mg 2+ and D-CPPene. The density of agonist- and antagonist-binding sites in the CA1 region of the hippocampus were determined by autoradiography, using [3H]CGP39653 or [~H]glutamate as ligands. A significant developmental increase in NMDA receptor binding sites was detected both in preparations of cerebral cortical membranes and in the CA1 area of the hippocampus, In addition, the Mg 2+ sensitivity of [3H]MK-801 binding was significantly higher in membrane preparations from the cerebral cortex of postnatal day 15 compared to postnatal day 7 animals. Neither the single nor the subchronic treatment with NMDA receptor antagonists exerted a significant influence on the density of antagonist binding sites or on the modulation of [3H]MK-801 binding by glutamate, Mg 2+ or D-CPPene. We conclude therefore that neonatal treatment with NMDA receptor antagonists in vivo does not involve significant alterations in the properties and the densities of NMDA receptors in the brain regions studied, i.e., during the period when expression of these receptors is subject to pronounced developmental regulation.

Glutamate is the most abundant excitatory neurotransmitter in the mammalian brain. Responses to glutamate are mediated by two major classes of receptors, namely ionotropic and metabotropic receptors. The ion channel-forming receptors can be subdivided based on their pharmacological and structural proper-

ties into N-methyl-o-aspartate(NMDA)-, amino-3hydroxy-5-methylisoxazole-4-proprionic acid(AMPA)and kainate-preferring glutamate receptors. N M D A receptors constitute a channel permeable to both monovalent cations and Ca 2+, and they can be allosterically modulated by discrete receptor-ligand interactions involving glutamate, glycine and polyamines. Dizolcipine (MK-801) is a non-competitive N M D A receptor * Present address : F. Hoffmann-La Roche Ltd, PRPN, Bau 69/402, Grenzacherstrasse 124, CH-4002 Basel, Swit- antagonist which binds to a site in the open receptor zerland. channel (Wong et al., 1986). It seems therefore that ~-Author to whom all correspondence should be addressed. binding of labelled MK-801 is a useful means to study :~Present address : University of California, Irvine, IRU in Brain Aging, Biosciences II, 2205 Irvine, CA 92717, modulation of the N M D A receptor channel by compounds acting at various sites on the N M D A receptor U.S.A. Abbreviations: D-CPPene. D-(E)-4-(3-phosphono-2-pro(Reynolds and Miller, 1988 ; Williams et al., 1991 ; Wong penyl)-2-piperazine-carboxylic acid; EC50. concen- et al., 1986). Indeed, it has been observed that binding tration of drug which increases binding of the ligand of labelled MK-801 is enhanced by glutamate, glycine by 50% ; MK-801. (+)-5-methyl-10,11-dihydro-5Hdibenzo (a,d)cyclohepten-5,10 imine; IC50. concentration and certain polyamines and inhibited by M g 2÷, Zn 2+ and open channel blockers (Foster and Wong, 1987). of drug which inhibits binding of the ligand by 50%. 355

356

Menno Van Lookeren Campagne et al.

N M D A receptors are thought to play a crucial role in the development of the central nervous system (CNS). There is evidence that N M D A can promote the survival and maturation ofcerebellar granule cells depending on their maturational state (Balfizs et al., 1988 : Cambray-Deakin and Burgoyne, 1992 : Resink et al., 1992). It has also been shown recently that N M D A receptor activation exerts an influence on the migration of immature cerebellar granule cells (Komuro and Rakic, 1993). Similar findings were obtained concerning immature dentate granule neurons, whose generation and death are also affected by N M D A (Gould et al., 1994). Furthermore, it seems that N M D A receptors are involved in the shaping of neuronal cytoarchitecture and in the development of neuronal connections (Kleinschmidt et al., 1987). Perinatal asphyxia is still an important health problem that affects in various degrees of severity a relatively great number of babies. It seems that the neonatal/infant brain is especially vulnerable to elevated levels of glutamate, and there is evidence that N M D A receptor activation may play a critical role in neuronal death induced by neonatal ischaemia (Olney, 1993). Treatment with N M D A receptor antagonists can attenuate damage resulting from neonatal cerebral ischemia in experimental animals (McDonald et al., 1987: Olney et al., 1989) and thus might provide an approach to counteract injury in human perinatal asphyxia. However, in view of the importance of N M D A receptor activity in nerve cell survival, maturation and synaptic plasticity (see above), it is necessary to examine whether or not treatment with N M D A receptor antagonists interferes with the normal development of the CNS. Alterations in the properties and/or the density of N M D A receptors may be among the direct consequences of N M D A receptor blockade during ontogeny. It is well known that long-term treatment with transmitter receptor antagonists often leads to the development of receptor supersensitivity, involving receptor upregulation. Such alterations have indeed been described to occur in developing neurons after N M D A receptor blockage (McDonald et al., 1990b: Williams et al., 1993). Furthermore, it has been reported that treatment of rat pups with MK-801 from postnatal day 9 (P9) to P21, elicits certain changes in the properties of N M D A receptors in hippocampal nerve cells, assessed by iontophoretic techniques (Gorter et al., 1992b). Finally, lasting behavioural and biochemical alterations are detectable in adult rats after subchronic, neonatal treatment with N M D A receptor antagonists (Facchinetti et al., 1993; Gorter et al., 1992a). Therefore, although N M D A receptor antagonists are promising can-

didates for the treatment of perinatal asphyxia/ ischemia, it is essential to gather more information on the possible long-term effects of neonatal treatment with N M D A receptor antagonist. In the present study we examined whether acute or subchronic treatment with N M D A receptor antagonists can induce specific alterations in the characteristics of the N M D A receptors. Since previous investigations have shown that the behavioural and biochemical consequences of subchronic treatment with competitive and non-competitive antagonists are different (Facchinetti et al., 1993, 1994), we included both a non-competitive (MK-801) and a competitive blocker (D-CPPene) in our study. Effects of N M D A receptor blockade were examined during the second postnatal week, since treatment during this period could give relevant information on the possible consequences of antagonist treatment of newborn children. Furthermore, N M D A receptors and developmental processes which may be influenced by the receptor activity show characteristic changes in the rat brain (McDonald and Johnston, 1990), and interference in these processes might perturb the development of normal brain function.

EXPERIMENTAL PROCEDURES

Experimental protocols were approved by the appropriate institutional review committee and met the guidelines of the responsible governmental agency. Pregnant Wistar rats were kept in separate cages until partum. Litter size was brought to 8 pups after birth. The day of birth was called postnatal day zero (P0). The pups of each litter were randomly divided over different experimental treatment groups and kept with their mother. Pups received daily injections of saline or NMDA receptor antagonists according to the following schedules: (a) acute treatment on P6 with a single subcutaneous injection of 1 mg/kg MK-801 (a gift from Merck, Sharp and Dohme, U.K. ; n = 8), 2 mg/kg D-CPPene (a gift from Sandoz Pharma Ltd, Switzerland ~n = 8) or 0.1 ml of the saline vehicle (n = 8) followed by sacrifice 24 h later ; (b) subchronic treatment from P5 to P14 with two subcutaneous injections per day of either 0.25 mg/kg MK-801 (n = 8), 0.75 mg/kg D-CPPene (n = 8) or saline (n = 8) followed by sacrifice on P15, 24 h after the last injection. The treatment schedules were empirically determined after preliminary trials with different drug doses. The treatment routinely adopted resulted in <5% mortality or <40% weight loss. Body weights were recorded every day. After sacrifice by decapitation, forebrains were removed, weighed and either immediately frozen on crushed dry ice for later processing for receptor autoradiography, or a large cortical slab was first dissected, approximately corresponding to the fronto-parietal region, which was also weighed and frozen in liquid nitrogen for later membrane binding studies. Tissues from each animal were separately processed.

Treatment with NMDA receptor antagonists

[ 3H ] M K-801 membrane binding assay [3H]MK-801 binding to triplicate membrane preparations was performed with slight modifications as described by Ransom and Stec (1988). Except for saturation experiments, all binding experiments were performed using 5 nM of [3H]MK801 (DuPont-NEN, U.S.A., specific activity 30.0 Ci/mmol). Saturation experiments and the influence of [Mg2+] on membrane binding was carried out in the presence of 50 #M glutamate and 50 #M glycine. The effect of D-CPPene on [3H]MK-801 binding was tested in the presence of 50 #M glutamate only, and the effect of glutamate on [3H]MK801 binding was performed without additions. Nonspecific binding was determined using 200 #M of the non-competitive NMDA receptor antagonist ketamine. Following a 1 h incubation, binding reaction was terminated by filtration through Whatman GF/B filters soaked in 0.1% polyethyleneimine using a Skatron Cell Harvester. Radioactivity was measured using PCS scintillation cocktail and a Packard Series 4000 instrument. Data from saturation binding studies (1.540 nM [3H]MK-801) were analyzed using the LIGAND program of Munson and Rodbard (1980) as modified for PC by McPherson (1985). Concentration response curves were analyzed using nonlinear regression with GRAPHPAD InPlot (v4.0, H. Motulsky).

Autoradiography with [3H]CGP39653 and [3H]glutamate Coronal 15 #m thick cryostat sections of the forebrains at the stereotactic plane A 3.2 (Sherwood and Timiras, 1970) were thaw-mounted on amino-alkylsylane-coated slides. Receptor autoradiography with [3H]CGP39653 (New England Nuclear, U.S.A., specific activity 42.0 Ci/mmol) was performed as described by Jaarsma et al. (1993). Non-specific binding was determined in the presence of 100 #M O,LAP5. [3H]glutamate autoradiography was executed using the procedure of McDonald et al. (1990b) with some modifications. Sections were washed in 50 mM Tris-HCl buffer, pH 7.2 at 0°C for 30 min, at 30°C for 10 rain and at 0°C for 5 rain. Then the tissue was exposed to [3H]glutamate (New England Nuclear, U.S.A., specific activity 48 Ci/mmol) in the presence of 5/aM quisqualate and 2 pM kainate to block binding to non-NMDA receptors. Non-specific binding was determined in the presence of 200 #M NMDA. The final step in both protocols was the rapid drying of sections under a stream of cold air. For each concentration of ligand used to construct the saturation curves, measurements were performed by integrating the optical density obtained from 2 sections per animal (n = 3) in a frame projected on the stratum radiatum of both the left and the right hippocampus (Fig. 1). Since there were no left-right differences, all four measurements for each concentration were averaged. Tissue standards were used as a reference for quantification and were prepared as 16 #m thick cryostat sections from frozen pieces of cerebral cortex homogenates mixed with known concentrations of [3H]D,L-aspartate (New England Nuclear, U.S.A., specific activity 50 Ci/mmol). Quantification of the films was performed on an IBAS KAT image processing system connected to a Zeiss microscope.

Statistical analysis Analysis of data on body- and brain weights and on binding parameters was performed by one-way ANOVA with a post-hoc Bonferroni's t-test. Developmental changes in NMDA receptor properties were tested using ANOVA and covariance with repeated measures.

357 RESULTS

Brain and body weights Chronic treatment with N M D A receptor antagonists resulted in a marked retardation in growth (Table 1; see also Facchinetti et al., 1993). Body weight was more severely affected (about 35% reduction compared to controls) than the weight of the total forebrain and the cerebral cortex weight (12 and 8% decrease respectively).

Developmental changes Receptor density and ligand affinity. Saturable specific [3H]MK-801 binding was detectable in membrane preparations prepared from the cerebral cortex of both P7 and P15 rats (Fig. 2 A ; Table 2). Analysis o f the data indicated the involvement of a single population of [3H]MK-801 binding sites. A more than twofold increase was observed in the density of [3H]MK801 binding sites in cerebral cortical membrane preparations from saline-treated rats from P7 to PI 5. The affinity constant for the ligand MK-801 (Kd) decreased with almost 50% over this time period. The CA1 striatum radiatum of the hippocampus is known to contain the highest density of N M D A receptors in the C N S (McDonald et al., 1990a ; Monaghan et al., 1983). This area was, therefore, selected as a representative area for evaluating the autoradiograms (Fig. 1). In addition, this area shows an homogenous distribution of C G P and glutamate binding sites, and therefore allows for reliable optic density measurements in an outlined area (see Fig. 1). Saturable specific binding of both [3H]CGP39653 and [3H]glutamate in the stratum radiatum of the C A I subfield was observed (Fig. 2 B, C). Scatchard analysis and the estimation of Hill coefficients of the data indicated the presence of a single population of [3H]CGP39653 and [3H]glutamate binding sites (results not shown). The number of binding sites for [3H]CGP39653 in the stratum radiatum of the hippocampus increased more than 4-fold during the period P7 to P15 (Fig, 1, 2; Bmax in Table 2), whereas significant changes with age were not detectable in the affinity of the receptor for the competitive receptor antagonist (Kd in Table 2). Glutamate-induced increase in [3H]MK-801 binding. As previously reported by Foster and Wong (1987), we also observed that L-glutamate exerted a profound effect on [3H]MK-801 binding (Fig. 3A). Characterization of the effect of glutamate on [3H]MK-801 binding in P7 and P15 cerebral cortical membranes revealed only small differences in absolute potency (EC50) (Table 2), but similar effects when

358

Menno Van Lookeren Campagne et al.

Fig. 1. Autoradiograms of [3H]CGP39554 binding (150 nM) in P7 and Pl5 saline-treated rats. The box indicates the area in which the number of binding sites were quantitatively measured. Scale bar = 500/~m.

359

Treatment with NMDA receptor antagonists Table 1. Effect of subchronicneonataltreatmentwith NMDA receptor antagonistsfrom postnatalday 5 to 14on bodyweightand the weightof certainbrainareas of the 15-days-old rat Treatments

Control

MK-801

D-CPPene

Bodyweight(g) Forebrain(rag) Cerebral cortex (mg) Hippocampus(rag)

40.2-t-0.5 1275_+8 543+ 17 95.7+ 3.0

25.2_+1.4" 27.5+0.4* 1085+27" 1148+8" 501+45* 496+ 10" 78.3+ 8.0 84.7+ 4.9

ANOVA P < 0.0001 P < 0.0001 P < 0.0001 N.S.

Valuesrepresentmean-+S.E.M. of 3 animals. *Significantlydifferentfromcontrols,one-wayANOVAand Bonferronit-test. expressed as percentages. One should note, however, that the levels of [3H]MK-801 binding in the absence of glutamate and glycine is more than twice as high in PI 5 compared to P7 cortical membrane preparations (0.56+_0.05 vs 0.22+_0.05 pmol/mg protein respectively). As a result, levels of [3H]MK-801 binding under saturating glutamate concentrations are also more than twice as high in P15 compared with P7 animals. M y 2+- and D-CPPene-induced inhibition o f MK-801 bindiny. Mg 2+ interfered, in a concentration dependent manner, with the specific binding of [3H]MK-801 to membrane preparations from the cerebral cortex of both P7 and P15 rats. However, there was a pronounced developmental change in the affinity for Mg 2+, the potency of Mg 2+ in inhibiting [3H]MK-801 binding being over 8-fold higher in the preparations from animals at P15 than at P7 (Figs 3B, 4, Table 2). Also here one should note that the level of [3H]MK801 binding without the addition of Mg 2+ is more than twice as high in P15 compared to P7 animals (see legends Fig. 2). D-CPPene also inhibited in a concentration dependent fashion [3H]MK-801 binding in these preparations, but the potency of this drug was not significantly affected by age (Fig. 3C, Table 2).

Effect o f sinyle and prolonyed treatment with N M D A receptor antayonists A single injection of MK-801 (1 mg/kg) or DCPPene (2 mg/kg) given at P6 had no significant effect on N M D A receptor binding examined 24 h later (Table 2). Kinetic parameters of [3H]MK-801 binding in cerebral cortical membrane preparations and the densities of N M D A receptors and their affinities to an agonist (glutamate) and a competitive antagonist (CGP39653) in the stratum radiatum of the CA1 subfield of the hippocampus were similar to control values. We also tested the effect of a relatively long, neonatal treatment with N M D A receptor antagonists, by giving two daily injections of MK-801 or D-CPPene

(2 × 0.25 mg/kg or 2 x 0.75 mg/kg) during the period P5-P14. However, even this treatment failed to exert a significant influence on the densities and properties of N M D A receptors, assessed 1 day after termination of the treatment, either in membrane preparations from the cerebral cortex or with autoradiography in the stratum radiatum (Table 2, Figs 2, 3). DISCUSSION

Developmental alterations in N M D A receptor properties Present kinetic estimates of the density of N M D A receptor channels and dissociation content were within the range obtained in some earlier published studies (Javitt and Zukin, 1989 ; Reynolds and Miller, 1988), but differed from others (Morin et al., 1989; Facchinetti, 1993). Variation in experimental conditions (ionic strength, concentrations of L-glutamate and glycine, presence of detergent, subcellular fraction used) as well as the age of the animals (neonatal vs adult) might account for these discrepancies. The density of N M D A receptor channels, estimated by [3H]MK-801 binding using thoroughly washed cerebral cortex membrane preparations, was found to increase markedly with age during the period from P7 to PI5. Autoradiographic studies in the CA1 area of the hippocampus also showed a massive increase with age in the number of [3H]CGP39653 binding sites. The developmental increase in the density of the antagonist recognition sites and of ion channels of N M D A receptors is consistent with previous findings in different brain regions of both rats (McDonald et al., 1990a; Morin et al., 1989; Insel et al., 1990; Tremblay et al., 1988) and cats (Gordon et al., 1991). Estimated Kd values appeared to be lower in P 15 compared to P7 animals. Since glutamate and glycine were added in the underlying saturation studies, the difference in Kd values might be explained by a different potency of glutamate at both ages, the EC50 values for glutamate in P7 animals being higher compared to those in P15 animals.

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Treatment with NMDA receptor antagonists

361

Table 2. Effectof acute or chronic treatment with NMDA receptor antagonists in neonatal rat on the density and certain properties of the NMDA receptor in the brain Postnatal day 15 (chronictreatment)

Postnatal day 7 (acute treatment) Treatments

Control

[~H]MK-801 bindin9 in cortical membranes Bm,x(pmol/mgprotein) 1.00_+0,05 Kd (nM) 2.71 _+0,78 EC50 glutamate (#M) 137_+64 IC50 D-CPPene(#M) 131_+31 1C50 Mg2+ (mM) 1.45_+0.01

MK-801

D-CPPene

1.19_+0.10 2.92_+0.61 117_+44 108_+15 1.16_+0.25

1.32_+0.21 3.4_+0.48 105_+50 136_+42 1.16_+0.47

[3H]CGP39653 binding on sections of the CA1 stratum radiatum Bm~x(fmol/mgwet weight) 77.0 _+4.9 75.4 _+4.3 72.0 _+5.0 Ka (nM) 34.6_+5.9 32.8_+2.5 27.4_+6.3 [3H]ylutamate binding on sections of the CA 1 stratum radiatum Bmax(fmol/mgwet weight) 129_+48 144_+3 Kd (nM) 63_+40 60_+ 10

Control

MK-801

ANOVA age

D-CPPene

2.32+0.10" 2.28+0.19 2.48+0.11 1.44_+0.06 1.25_+0.11 1.16_+0.03 51.3_+0.1 104_+29 77_+3.2 149_+24 188_+1 161_+25 0.18_+0.03" 0.16_+0.02 0~20_+0.03 302_+32* 33+ 16

306_+73 45+ 14

294_+ 17 29+6

< P value 0.0001 0.0040 0.21 0.16 0.0012 0.0002 0.64

127_+5 56_+25

Rats receivedeither a singleinjectionof MK-801 (1 mg/kg), D-CPPene(2.0 mg/kg) or the salinevehicle(0.1 ml, control) on P6 or subchronic treatment with MK-801 (0.25 mg/kg, twice daily) or D-CPPene (0.75 mg/kg, twice daily) and saline (0.1 ml) from P5 to PI4. Animals were killed 24 h after the last treatment at P7 and P15 respectively.Results are mean + S.E.M, of 3 animals. Data were subjected to two-way ANOVA statistics.No statisticallysignificantchangeswere found for the drug treatments, the P-values for age effectare indicated in the last column. Statistical significanteffectsbetweenthe P7 and PI 5 control values are indicated by asterisks (post-hoc testing).

M a r k e d age-dependent differences were found in the potency o f M g 2+ to block [3H]MK-801 binding in the cortex. The lower M g 2÷ potency at P7 as compared to P15 is consistent with some electrophysiological findings (Ben-Ari et al., 1988 ; Bowe and Nadler, 1990 ; Burgard and Hablitz, 1994 ; K a t o and Yoshimura, 1993; Kleckner and Dingledine, 1991; Morrisett et al., 1990). O t h e r studies have failed to detect such a change in the potency o f M g 2+ (LoTurco et al., 1991 ; Hestrin, 1992). However, in these investigations the effect o f M g 2+ on synaptic N M D A receptors in immature tissue has not been examined in the presence o f sufficiently low external M g 2+ concentration. F u r t h e r m o r e , care should be taken when comparing data on the M g 2÷ inhibition o f [3H]MK-801 binding to m e m b r a n e preparations with M g 2÷ responses measured electrophysiologically in live cells. The m e m b r a n e potential o f the vesicles in the membrane preparations is continuously depolarized, and

it is k n o w n from studies on intact cells that the strength o f the M g 2+ blockade is directly related to the m e m b r a n e potential. The developmental changes in the properties o f the M g 2÷ blockade observed in electrophysiological and m e m b r a n e binding studies may be accountable by alterations in the subunit c o m p o s i t i o n o f N M D A receptors ( M o n y e r et al., 1992; Pollard et al., 1993). A relatively low M g 2÷ potency for blocking N M D A receptor channels during the early postnatal period may also contribute to the greater vulnerability to N M D A o f the brain o f neonatal c o m p a r e d with the adult rats ( M c D o n a l d and J o h n s t o n , 1990).

E f f e c t o f neonatal t r e a t m e n t with N M D A antagonists

receptor

It has been previously reported that subchronic N M D A receptor blockade in the neonatal period has

Fig. 2. Receptor binding studies on rat cerebral cortex and hippocampus at P7 and P15 after acute and chronic treatment with MK-801 and D-CPPene. (A) Saturation binding curves of [3H]MK-801 to cerebral cortical membranes of P7 and P I 5 rats. Binding of [3H]MK-801 was performed in the presence of 50 #M glutamate and 50 #M glycine. Animals sacrificed at P7 received a single injection of MK-801 (1 mg/kg), D-CPPene (2 mg/kg) or saline (controls) on P6; animals sacrificed at P15 were treated twice daily with MK-801 (0.25 mg/kg) or D-CPPene (0.75 mg/kg) from P5 to P14. Values are mean+S.E.M. (n = 3). (B) [3H]CGP39954 saturation curves measured in autoradiograms of the CA1 area of P7 and PI5 rats treated as described for Fig. 2A (n = 3). (C) Saturation curves of [3H]glutamate binding in the CA1 area of the hippocampus assessed using autoradiography after single treatment with MK-801 (1 mg/kg), D-CPPene (2.0 mg/kg) or saline (n = 3).

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Treatment with NMDA receptor antagonists significant long-lasting behavioural consequences. Systemic administration of N M D A receptor antagonists to rats during the first three postnatal weeks produced long lasting locomotor hyperactivity (Facchinetti et al., 1993). In addition, it has been observed that neonatal blockade of N M D A receptors by MK801 results in a persistent impairment of spatial learning in rats (Gorter and de Bruin, 1992). It is conceivable that the perturbation of N M D A receptor function is involved in the behavioural changes induced by treatment with receptor antagonists through the modulation of N M D A receptor density. Previous studies have indicated that antagonist treatment can lead to the up-regulation of certain transmitter receptors and that this effect is often related to the blockade of the tonic influence of endogenous agonists. To test the effect of long-term exposure to antagonists, NMDA receptor studies have been conducted on nerve cells in culture, these permitting the maintenance of a constant concentration of antagonists and therefore a tonic blockade of the receptors. For example, Williams et al. (1993) reported that treatment of cultured neurons for 4 days with the N M D A receptor antagonist DAP5 (2-amino-5-phosphonopentanoicacid) results in a 65% increase in the density of NMDA receptors assessed by [~25I]MK-801 binding, without any change in the affinity of the receptors to [J25I]MK-801. Another study has shown that prolonged (2-20 weeks) exposure of cultured hippocampal neurons to the nonselective glutamate receptor antagonist kynurenic acid or to elevated concentrations of Mg 2÷ increased the excitability of neurons and their susceptibility to excitotoxicity after the removal of the antagonists. These observations also imply an increase in N M D A receptor density (Furshpan and Potter, 1989). In vivo observations on the effect of N M D A receptor antagonists on receptor density in the developing CNS are, however, ambiguous. It has been reported that treatment of neonatal rats with a single dose of MK-801 results in an increase in glutamate receptor binding in the CAI and CA3 subfields of the hippocampus, the striatum and the cingulate cortex. This effect is

363

accompanied by an increase in the extent of the excitotoxic damage induced by intrastriatally injected N M D A (McDonald et al., 1990b). However, the same study has claimed a 60-80% reduction in the density of binding sites for [3H]TCP, which is a ligand of the N M D A receptor channel. The authors have attributed this effect to differential modulation by antagonist treatment of the number of N M D A receptor channel sites and N M D A antagonist binding sites. In our studies we did not observe any change in N M D A receptor characteristics in the cerebral cortex and hippocampus 24 h after treatment of neonatal rats with MK-801. Differences in the experimental protocol of McDonald et al. (1990b) and ours, including the rat strain used (Sprague-Dawley vs Wistar) and the route of antagonist administration (i.p. vs s.c.), might partly explain the different results. So far, we have no additional explanation why our results differ from those of McDonald et al. (1990b). Also prolonged treatment with MK-801 and acute or prolonged treatment with the competitive N M D A receptor antagonist D-CCPene did not induce any alterations in N M D A receptor properties. In a recent study, Facchinetti et al. (1993) similarly showed no alterations in [3H]MK-801 binding in the hippocampus and the cerebral cortex 2 or 48 days after treating rats with either CGP39551 (a competitive N M D A receptor blocker) or MK-801 from Pi to P22. Whereas in most studies the characteristics of NMDA receptors were tested shortly after the period of antagonist treatment, in one study N M D A receptors were measured in the brain five months after ending a daily treatment with MK-801 from P8 to P19. Also in this case, no changes were found in the properties for [~25I]MK-801 and [3H]CGP39653 binding to hippocampal membranes (Gorter, 1992; Gorter J. A., Vermeulen J. P. and Boer G. J., unpublished observations). The difference between the effect of N M D A receptor blockade under in vitro and in vivo conditions may relate to the difference in maintaining a prolonged, effective antagonist exposure. In the in vivo studies N M D A receptor blockade may not have been

Fig. 3. Effect of age and singleor subchronic treatment with NMDA receptor antagonists on the modulation of [3H]MK-801 binding by glutamate (A), Mg2+ (B) and D-CPPene (C), using cerebral cortical membrane preparations. Treatment schedules were as described for Fig. 2A. Influence of Mg2+ (B) or D-CPPene (C) on [3H]MK-801 binding was determined in the presence of 50 #M L-glutamate plus 50 #M glycine or 50 #M glutamate. Levels of [3H]MK-801 binding (pmol/mg protein, mean_S.E.M.) in the absence of glutamate (A) were 0.22+0.05 for P7 and 0.56+0.05 for P15 animals respectively,in the absence of Mg2÷ (B) 0.83+ 0.05 and 2.23 + 0.13, and in the absence of D-CPPene (C) 0.62 ___0.06 and 2.28 + 0.07. There was no significant difference in basal values of [3H]MK-801 binding between the treatments within each age group. Data are means of triplicate determinations which varied by less than 10%.

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sufficient to induce alterations in the expression and properties of the receptor. The half life of [~25I]MK801 in adult rats after intravenous injection is about 60 min in all the brain regions studied (Gibson et al., 1992). If clearance of the drug in the adult rat brain was similar to that in the neonatal rat brain, it is conceivable that our treatment schedule failed to effect a sufficient tonic suppression of N M D A receptor activity in vivo comparable to that in vitro. There are no pharmacokinetic data available on the clearance of D-CPPene in the neonatal rat brain, but the halflife of the drug in blood plasma is relatively short, around 60 rain, after a subcutaneous injection in P7 rats (Guerret M. and Herrling P., personal communication). The efficacy of D-CPPene may have been also affected by relatively slow penetration into the brain. Because of its hydrophilic nature, it will take longer for this drug than for MK-801 to pass the blood brain barrier and to block N M D A receptors (van Lookeren Campagne et al., 1994). Treatment of neonatal rats with N M D A receptor antagonists applying a protocol, which differs in the dose and the age range of the treatment, induced electrophysiological (Gorter et al., 1992b), behavioural (Gorter and de Bruin, 1992) and biochemical alterations (Gorter et al., 1992a ; Contestabile et al., 1994 : Facchinetti et al., 1993, 1994). The absence of changes in N M D A receptor density in these studies (Facchinetti et al., 1993 ; G o r t e r , 1992 ; Gorter J. A., Vermeulen J. P. and Boer G. J., unpublished observations), suggest, together with the results from the present study, that the observed alterations do not result primarily from changes in the density or the properties of the N M D A receptors. Rather, changes in other neurotransmitter systems may be involved in the behavioural alterations resulting from neonatal N M D A receptor blockade. Indeed, it has been shown recently that chronic MK-801 blockade of N M D A receptors in adult rats (0.1-0.4 mg/kg p.o. for 50 days) can induce D2 receptor synthesis in the striatum (Micheletti et al., 1992). It has also been reported that chronic, neonatal N M D A receptor blockade with a competitive antagonist (CGP39551) can elicit a persistent increase in D2 receptor binding sites in the striatum of adult rats (Dall'Olio et al., 1994). In addition, Gorter et al. (1992b) have found that neonatal treatment with MK-801 evokes in striatum and frontal cortex longterm effects on monoamine metabolism, indicating elevated activity. It seems, therefore, that interactive effects involving the perturbed glutamatergic system and the dopaminergic and presumably other neurotransmitter systems may contribute to the behavioural alterations induced by

N M D A receptor blockade during the period when neuronal connections are developing.

Acknowledyements--The authors gratefully acknowledge Dr

P. L. Herrling, Dr D. A. Lowe and Dr M. Guerret from Sandoz Pharma Ltd for helpful discussions, for providing D-CPPene and for plasma estimations; Drs J. J. Van Heerikhuize and Dr C. W, Pool for their valuable advice with the quantification of the autoradiograms and Mr G. van der Meulen for photographical assistance. MK-801 was a generous gift of Merck, Sharp and Dohme, West Point, Pa, U.S.A. This research was supported by the Praeventiefonds (M.v.L.C. and J.P.V.) and carried out at the Netherlands Institute for Brain Research.

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