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Transient increase in ligand binding to quisqualate and kainate sites in cerebral cortex of immature rats
Neuroscience Letters, 104 (1989) 161-166
161
Elsevier Scientific Publishers Ireland Ltd. NSL 06298
Transient increase in ligand binding to quisqualate and kainate sites in cerebral cortex of immature rats Sfindor L. Erd6 and Joachim R. Wolff Department of Anatomy, DevelopmentalNeurobiology Unit, Georg August University, G6ttingen ( F.R.G.) (Received 28 March 1989; Revised version received 9 May 1989; Accepted 15 May 1989)
Key words: Excitatory amino acid receptor; Quisqualate; Kainate; Cerebral cortex; Immature rat; Postnatal development The postnatal changes in the specific binding of [3H]kainate and [3H]AMPA (RS-~-amino-3-hydroxy-5methyl-4-isoxasolopropionic acid, an agonist of quisqualate receptors) were studied in cerebral cortex of rats, aged 2- 360 days. The binding of the two ligands was assayed in whole-tissue homogenates. Similar developmental time courses were found for kainate and AMPA binding, characterized by high perinatal levels, a further increase during the first few days after birth, an early maximum value around the age of one week, and a gradual decrease to adult values which were attained at an age of 3 4 weeks. As revealed by Scatchard analysis, the transient elevation of ligand binding was derived from an increased density of binding sites, which, in the case of AMPA, was accompanied also by an increase in binding affinity. The results indicate that, in the immature cerebral cortex, kainate and quisqualate receptors may play a role other than in synaptic transmission.
Glutamate and aspartate are the major excitatory amino acid (EAA) transmitters in the central nervous system (CNS) of vertebrates [14]. These amino acids act through at least three distinct receptor populations, named after the selective agonists N-methyl-D-aspartate (NMDA), quisqualate and kainate (e.g. ref. 10). Agonists of these receptor subclasses, applied in higher doses, exert characteristic neurotoxic effects (e.g. ref. 8). Recent studies have revealed that the neurotoxicity of NMDA transiently increases in the immature rat brain [14]. This increase shows a close correspondence with an early maximum in the density of NMDA-sensitive binding sites in the developing rat hippocampus [18]. In the present study, we attempted to investigate whether transient developmental changes may also occur in ligand binding to cerebral quisqualate and kainate receptor sites. Therefore the binding of selective agonists, i.e., [3H]AMPA (RS-~-amino-3hydroxy-5-methyl-4-isoxasolopropionic acid) and [3H]kainic acid was examined in Correspondence." S.L. Erd6, Department of Anatomy, Developmental Neurobiology Unit, Georg August University, Kreuzbergring 36, D-3400 Gfttingen, F.R.G. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
162 whole homogenates of the cerebral cortex, a region known to contain n o n - N M D A type EAA receptor subpopulations [13]. Sprague-Dawley rats (Charles River, Wiga GmbH) were used. Each age-group (2.-360 days) consisted of at least 5 rats. Animals were killed by cervical dislocation and the brains were quickly removed. The cortex was cleaned of white matter and placed into liquid nitrogen within 3 min after killing. Whole homogenates of the tissues were prepared as follows: cortices were homogenized in 20 vols. of ice-cold distilled water and the homogenates sedimented at 20,000 g and 4°C for 15 min. The resulting pellets were washed 3 times by repeated resuspension in distilled water and centrifugation. The final pellet was suspended in ice-cold assay buffers as indicated below. [3H]Kainate (spec. act. 3.6 Ci/mmol, New England Nuclear) binding was assayed in a 50 mM Tris-citrate buffer, pH 7.1, with a slightly modified version of the method of" London and Coyle [I 1]. After 10 min equilibration, incubations were performed at O C for 60 rain, in the presence of a high ligand concentration (50 nM), unless otherwise indicated. The reaction was terminated by vacuum-filtration through Whatman G F / C filters. Non-specific binding was defined as binding in the presence of I mM L-glutamate or I mM unlabeled kainate, and never exceeded 45% of the total binding. The binding of [3H]AMPA (spec. act. 27.6 Ci/mmol, New England Nuclear) was examined in a Tris-citrate buffer, pH 7.4, without added chaotropic ions. Incubations were carried out at 0 ' C for 30 min in the presence of 10 nM ligand (unless stated otherwise). Non-specific binding was determined in the presence of 1 mM L-glutamate or 0.5 mM unlabeled AMPA. The two compounds caused the same degree of displacement. Depending on age, non-specific binding varied between 25 and 55% of the total binding. Protein contents of the membrane homogenates were estimated according to the method of Lowry et al. [12], and were found to be in the range of 0.3-0.5 mg/ml. TABLE 1 DISPLACEMENT OF SPECIFIC [3H]KAINATE(20 nM) AND [3H]AMPA (10 nM) BINDING FROM CORTICALMEMBRANESOF ADULT (180-DAY-OLD)RATS Four Io 6 different concentrations of each compound were tested. Those producing 50% displacement of specific binding (ICs0values) were estimated by linear regression analysis. Values are mean + S.D., n = 3 N M DA, N-methyl-D-aspartate,AMPA RS-alpha-amino-3-hydroxy-5-methyl-4-isoxasolo-propionicacid. Compound
L-Glutamate NMDA AMPA Quisqualic acid Kainic acid
IC~ value (,uM) [3H]Kainate
pH]AMPA
0.47 4-0.08 > 100 85+ 14 0.38 +0.06 0.009 + 0.002
2.6 ± 0.4 > 100 0.71 _+0.14 0.081 +0.009 18+ 4
163 i
"0.3
1.5" .E
.E @
/~~,KAINATE
o
Q.
2n o~
Ca
0,2 E
E
< O
0 E Q. .s -o .=
E O.
0.~
~__,__~
.....
~ ..........
.. . . . . . ~ - : ; - . ~ . . . . ~ _ - ÷ - - _ ~ . _ .
AMPA
es
0: .E u
o
2b
4b
postnatal age;
do
"
,t 18o " S o
~o
days
Fig. 1. Postnatal changes of [3H]kainate (50 nM) and [3H]AMPA (10 nM) binding to membranes of the cerebral cortex. Points and vertical bars represent average values _ S.D. obtained in 3 duplicate experiments. AMPA, RS-c<-amino-3-hydroxy-5-methyl-4-isoxasolo-propionic acid.
In cortical homogenates of adult rats, the specific binding of [3H]kainate and [3H]AMPA was displaced by agonists of the different EAA receptor populations in a manner typical for kainate and quisqualate sites, respectively (Table I). The IC50 values estimated for the compounds are in a good agreement with earlier data [10]. The age-dependence of the binding was examined in a range of 2-360 days after birth (Fig. 1). The binding of the two ligands showed a similar pattern of postnatal changes. At day 2, both [3H]kainate and [3H]AMPA showed a high specific binding,
C
40
[3H]KAINATE
3
E
[3HIAMPA ,
0
/6
E 20 ~-
D
D
2
@
'0
18o
~\\ 500
1000
radioligand
bound;
150
360
fmol:mg protein
Fig. 2. Representative Scatchard plots showing saturable binding of [3H]kainate (1-400 nM) and [3H]AMPA (0.9-362 nM) to cortical membranes of 6- and 180-day-old rats. In these concentration ranges, single binding components were detected for both ligands and age-groups. For Kd and Bmaxvalues, see table 2. AMPA, RS-c<-amino-3-hydroxy-5-methyl-4-isoxasolo-propionic acid.
164
i.e., approximately the same as the respective levels in adults (day 180). The binding of both AMPA and kainate increased during the first few postnatal days to reach maximum values at an age of 6 days, and gradually declined to the adult levels (day 180), which were attained around the age of 4 weeks. To elucidate whether the early maximum values derived from changes in the affinity and/or density of binding sites, the binding parameters were determined for [3H]AMPA and [3H]kainate in adult (180-day-old) and 6-day-old rats. The binding of the two ligands proved to be saturable with increasing ligand concentrations, and the linear Scatchard plots obtained revealed the presence of single binding components for the two ligands in both age-groups in the examined ranges of ligand concentrations (Fig. 2). The binding parameters estimated by linear-regression analysis are summarized in Table II. The development of EAA receptor binding sites in the brain of rats have already been examined in some detail [3, 4, 6, 7, 16]. In these studies, the non-selective ligand, i.e., [3H]glutamate [3, 16], or [3H]kainate [6, 7] were used and synaptic membrane fractions were obtained from whole brains [16] or several cerebral regions [3, 6, 7, 16]. The developmental patterns were usually characterized by a low level of perinatal binding, a rapid increase from the second postnatal week, followed by a slower elevation to nearly adult levels having been attained around the age of 4-6 weeks after birth (e.g. refs. 4, 6, 16]. In our experiments, the developmental time courses of kainate and AMPA binding were found to be completely different from that described above. This discrepancy may result from the choice of our experimental conditions. We used whole homogenates of the cortex, as excitatory synapses are practically absent in the newborn cortex [I], and therefore synaptic membrane preparations would not have any real meaning in early postnatal ages. Thus, the use of whole homogenates, which contain both synaptic and non-synaptic receptor binding sites, may explain dissimilarities between our results and those reported for synaptic membrane preparations, or even P2 fractions (e.g. refs. 3, 16). Moreover, it has been shown that, depending on the conditions
TABLE II BINDING PARAMETERS FOR [3H]KAINATE AND [3H]AMPA IN VISUAL CORTICAL MEMBRANES OF 6- AND 180-DAY-OLD RATS Values are mean + S.D., n=3. Binding parameter
Kd (nM) Bmax (fmol/mg prot.) *P
Age (days)
6 180 6 180
Radioligand [3Hlkainate
[3H]AMPA
37+ 4 39+_ 3 1348+41 879+- 18"
148+14 67+ 9* 437+ 19 129+ 16"
165 of membrane preparation and binding assay, kainate sites may be present in two distinct forms. The medium-affinity kainate binding site (detected in our experiments) may dissociate to high- and low-affinity sites (see ref. 10) which may exhibit different developmental patterns (see ref. 4). The developmental time course of quisqualate type receptor sites in the brain has not been studied by others to our knowledge. Thus, the ontogenetic pattern of A M P A binding, demonstrated in our experiments has no direct correlates in the literature. It should be noted that, under the conditions of our experiments, namely in the absence of chaotropic ions, only one form of the quisqualate site is detected (see ref. 10). This may also explain the considerably lower density of A M P A than kainate binding sites found in our experiments. The transient increase in kainate and quisqualate binding does not seem to correlate with the development of excitatory synapses in the neocortex [1], or with the ontogeny of kainate neurotoxicity [4, 6]. However, the increased susceptibility of immature rat brain (day 7) to the neurotoxic effect of quisqualic acid [17], and the transient stimulation of inositol phospholipid metabolism by quisqualic acid in brains of 6- to 8-day-old rats [15], are in line with our findings for A M P A binding. This correspondence is consistent with an increase in the number and sensitivity (affinity) of quisqualate receptors in the immature cerebral cortex. The functional role (if any) of the high density of kainate and quisqualate receptor sites in the immature brain is not yet understood. Nevertheless, some role in the mediation of trophic actions [2], rather than in synaptic transmission, seems plausible, even though a transient perinatal glutamatergic innervation [9] has been suggested for subcortical structures. In summary, our results provide evidence that not only NMDA, but also quisqualate and kainate receptor sites (or, at least, certain forms of the latter two subtypes) show a transient increase in the immature cerebral cortex. As around the age of one week only few synapses are developed, a t r o p h i c role, rather than a synaptic transmitter role, is suggested for quisqualate and kainate sites. This work was supported by the Deutsche Forschungsgemeinschaft (Wo 279/6-3 and 6-5). 1 Aghajanian, G.K. and Bloom, F.E., The formation of synaptic junctions in developing rat brain: a quantitative electron microscopicstudy, Brain Res., 6 (1967) 716~727. 2 Aruffo, C., Freszt, R., Hildebrandt, A.G. and Cevros-Navarro, J., Low doses of L-monosodiumglutamate promote neuronal growth and differentiation in vitro, Dev. Neurosci., 9 (1987) 228-239. 3 Baudry, M., Arst, D., Oliver, M. and Lynch, G., Development of glutamate binding sites and their regulation by calcium in rat hippocampus, Dev. Brain Res., 1 (1981) 37-48. 4 Ben-Ari,¥., Tremblay, E., Berger, M. and Nitecka, L., Kainic acid seizure syndrome and binding sites in developingrats, Dev. Brain Res., 14 (1984) 284-288. 5 Ben-Ari,Y., Cherubini, E. and Krnjevic, K., Changes in voltage dependence of NMDA currents during development, Neurosci. Lett., 94 (1988) 88-92. 6 Berger, M.L., Tremblay, E., Nitecka, L. and Ben-Ari, Y., Maturation of kainic acid seizure-brain damage syndrome in the rat. III. Postnatal development of kainic acid binding sites in the limbic system, Neuroscience, 13 (1984) 1095 1104.
166 7 Campochiaro, P. and Coyle, J.T., Ontogenetic development of kainate neurotoxicity: correlates with glutamatergic innervation, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2025-2029. 8 Choi, D.W., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623-634. 9 Greenamyre, T., Penney, J.B., Young, A.B., Hudson, C., Silverstein, F.S. and Johnston, M.V., Evidence for transient perinatal glutamatergic innervation of globus pallidus, J. Neurosci., 7 (1987) 1022 1030. 10 Honore, T., Excitatory amino acid receptor subtypes and specific antagonists, Med. Res. Rev., 9 (1989) t 23. I 1 London, E.D. and Coyle, J.T., Cooperative interactions at [3H]kainic acid binding sites in rat and human cerebellum, Eur. J. Pharmacol., 56 (1979) 287-290. 12 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 13 Mayer, M.L. and Westbrook, G.L., The physiology of excitatory amino acids in the vertebrate central nervous system, Prog. Neurobiol., 28 (1987) 197 276. 14 McDonald, J.W., Silverstein, F.S. and Johnston, M.V., Neurotoxicity of N-methyl-D-aspartate is markedly enhanced in developing rat central nervous system, Brain Res., 459 (1988) 200 203. 15 Nicoletti, F., ladarola, M.J., Wroblewski, J.T. and Costa, E., Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: Developmental changes and interactions with alphat adrenoceptors, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 193l 1935. 16 Sanderson, C. and Murphy, S., Glutamate binding in the rat cerebral cortex during ontogeny, Dev. Brain Res., 2 (1982) 329 -339, 17 Silverstein, F.S., Chen, R. and Johnson, M.V., The glutamate analogue quisqualic acid is neurotoxic in striatum and hippocampus of immature rat brain, Neurosci. Lett., 71 (1986) 13 18. 18 Tremblay, E., Roisin, P.M., Represa, A., Charriaut-Martangue, C. and Ben-Ark Y.. Transient increased density of N MDA binding sites in the developing rat hippocampus, Brain Res., 461 (1988) 393 396.
161
Elsevier Scientific Publishers Ireland Ltd. NSL 06298
Transient increase in ligand binding to quisqualate and kainate sites in cerebral cortex of immature rats Sfindor L. Erd6 and Joachim R. Wolff Department of Anatomy, DevelopmentalNeurobiology Unit, Georg August University, G6ttingen ( F.R.G.) (Received 28 March 1989; Revised version received 9 May 1989; Accepted 15 May 1989)
Key words: Excitatory amino acid receptor; Quisqualate; Kainate; Cerebral cortex; Immature rat; Postnatal development The postnatal changes in the specific binding of [3H]kainate and [3H]AMPA (RS-~-amino-3-hydroxy-5methyl-4-isoxasolopropionic acid, an agonist of quisqualate receptors) were studied in cerebral cortex of rats, aged 2- 360 days. The binding of the two ligands was assayed in whole-tissue homogenates. Similar developmental time courses were found for kainate and AMPA binding, characterized by high perinatal levels, a further increase during the first few days after birth, an early maximum value around the age of one week, and a gradual decrease to adult values which were attained at an age of 3 4 weeks. As revealed by Scatchard analysis, the transient elevation of ligand binding was derived from an increased density of binding sites, which, in the case of AMPA, was accompanied also by an increase in binding affinity. The results indicate that, in the immature cerebral cortex, kainate and quisqualate receptors may play a role other than in synaptic transmission.
Glutamate and aspartate are the major excitatory amino acid (EAA) transmitters in the central nervous system (CNS) of vertebrates [14]. These amino acids act through at least three distinct receptor populations, named after the selective agonists N-methyl-D-aspartate (NMDA), quisqualate and kainate (e.g. ref. 10). Agonists of these receptor subclasses, applied in higher doses, exert characteristic neurotoxic effects (e.g. ref. 8). Recent studies have revealed that the neurotoxicity of NMDA transiently increases in the immature rat brain [14]. This increase shows a close correspondence with an early maximum in the density of NMDA-sensitive binding sites in the developing rat hippocampus [18]. In the present study, we attempted to investigate whether transient developmental changes may also occur in ligand binding to cerebral quisqualate and kainate receptor sites. Therefore the binding of selective agonists, i.e., [3H]AMPA (RS-~-amino-3hydroxy-5-methyl-4-isoxasolopropionic acid) and [3H]kainic acid was examined in Correspondence." S.L. Erd6, Department of Anatomy, Developmental Neurobiology Unit, Georg August University, Kreuzbergring 36, D-3400 Gfttingen, F.R.G. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
162 whole homogenates of the cerebral cortex, a region known to contain n o n - N M D A type EAA receptor subpopulations [13]. Sprague-Dawley rats (Charles River, Wiga GmbH) were used. Each age-group (2.-360 days) consisted of at least 5 rats. Animals were killed by cervical dislocation and the brains were quickly removed. The cortex was cleaned of white matter and placed into liquid nitrogen within 3 min after killing. Whole homogenates of the tissues were prepared as follows: cortices were homogenized in 20 vols. of ice-cold distilled water and the homogenates sedimented at 20,000 g and 4°C for 15 min. The resulting pellets were washed 3 times by repeated resuspension in distilled water and centrifugation. The final pellet was suspended in ice-cold assay buffers as indicated below. [3H]Kainate (spec. act. 3.6 Ci/mmol, New England Nuclear) binding was assayed in a 50 mM Tris-citrate buffer, pH 7.1, with a slightly modified version of the method of" London and Coyle [I 1]. After 10 min equilibration, incubations were performed at O C for 60 rain, in the presence of a high ligand concentration (50 nM), unless otherwise indicated. The reaction was terminated by vacuum-filtration through Whatman G F / C filters. Non-specific binding was defined as binding in the presence of I mM L-glutamate or I mM unlabeled kainate, and never exceeded 45% of the total binding. The binding of [3H]AMPA (spec. act. 27.6 Ci/mmol, New England Nuclear) was examined in a Tris-citrate buffer, pH 7.4, without added chaotropic ions. Incubations were carried out at 0 ' C for 30 min in the presence of 10 nM ligand (unless stated otherwise). Non-specific binding was determined in the presence of 1 mM L-glutamate or 0.5 mM unlabeled AMPA. The two compounds caused the same degree of displacement. Depending on age, non-specific binding varied between 25 and 55% of the total binding. Protein contents of the membrane homogenates were estimated according to the method of Lowry et al. [12], and were found to be in the range of 0.3-0.5 mg/ml. TABLE 1 DISPLACEMENT OF SPECIFIC [3H]KAINATE(20 nM) AND [3H]AMPA (10 nM) BINDING FROM CORTICALMEMBRANESOF ADULT (180-DAY-OLD)RATS Four Io 6 different concentrations of each compound were tested. Those producing 50% displacement of specific binding (ICs0values) were estimated by linear regression analysis. Values are mean + S.D., n = 3 N M DA, N-methyl-D-aspartate,AMPA RS-alpha-amino-3-hydroxy-5-methyl-4-isoxasolo-propionicacid. Compound
L-Glutamate NMDA AMPA Quisqualic acid Kainic acid
IC~ value (,uM) [3H]Kainate
pH]AMPA
0.47 4-0.08 > 100 85+ 14 0.38 +0.06 0.009 + 0.002
2.6 ± 0.4 > 100 0.71 _+0.14 0.081 +0.009 18+ 4
163 i
"0.3
1.5" .E
.E @
/~~,KAINATE
o
Q.
2n o~
Ca
0,2 E
E
< O
0 E Q. .s -o .=
E O.
0.~
~__,__~
.....
~ ..........
.. . . . . . ~ - : ; - . ~ . . . . ~ _ - ÷ - - _ ~ . _ .
AMPA
es
0: .E u
o
2b
4b
postnatal age;
do
"
,t 18o " S o
~o
days
Fig. 1. Postnatal changes of [3H]kainate (50 nM) and [3H]AMPA (10 nM) binding to membranes of the cerebral cortex. Points and vertical bars represent average values _ S.D. obtained in 3 duplicate experiments. AMPA, RS-c<-amino-3-hydroxy-5-methyl-4-isoxasolo-propionic acid.
In cortical homogenates of adult rats, the specific binding of [3H]kainate and [3H]AMPA was displaced by agonists of the different EAA receptor populations in a manner typical for kainate and quisqualate sites, respectively (Table I). The IC50 values estimated for the compounds are in a good agreement with earlier data [10]. The age-dependence of the binding was examined in a range of 2-360 days after birth (Fig. 1). The binding of the two ligands showed a similar pattern of postnatal changes. At day 2, both [3H]kainate and [3H]AMPA showed a high specific binding,
C
40
[3H]KAINATE
3
E
[3HIAMPA ,
0
/6
E 20 ~-
D
D
2
@
'0
18o
~\\ 500
1000
radioligand
bound;
150
360
fmol:mg protein
Fig. 2. Representative Scatchard plots showing saturable binding of [3H]kainate (1-400 nM) and [3H]AMPA (0.9-362 nM) to cortical membranes of 6- and 180-day-old rats. In these concentration ranges, single binding components were detected for both ligands and age-groups. For Kd and Bmaxvalues, see table 2. AMPA, RS-c<-amino-3-hydroxy-5-methyl-4-isoxasolo-propionic acid.
164
i.e., approximately the same as the respective levels in adults (day 180). The binding of both AMPA and kainate increased during the first few postnatal days to reach maximum values at an age of 6 days, and gradually declined to the adult levels (day 180), which were attained around the age of 4 weeks. To elucidate whether the early maximum values derived from changes in the affinity and/or density of binding sites, the binding parameters were determined for [3H]AMPA and [3H]kainate in adult (180-day-old) and 6-day-old rats. The binding of the two ligands proved to be saturable with increasing ligand concentrations, and the linear Scatchard plots obtained revealed the presence of single binding components for the two ligands in both age-groups in the examined ranges of ligand concentrations (Fig. 2). The binding parameters estimated by linear-regression analysis are summarized in Table II. The development of EAA receptor binding sites in the brain of rats have already been examined in some detail [3, 4, 6, 7, 16]. In these studies, the non-selective ligand, i.e., [3H]glutamate [3, 16], or [3H]kainate [6, 7] were used and synaptic membrane fractions were obtained from whole brains [16] or several cerebral regions [3, 6, 7, 16]. The developmental patterns were usually characterized by a low level of perinatal binding, a rapid increase from the second postnatal week, followed by a slower elevation to nearly adult levels having been attained around the age of 4-6 weeks after birth (e.g. refs. 4, 6, 16]. In our experiments, the developmental time courses of kainate and AMPA binding were found to be completely different from that described above. This discrepancy may result from the choice of our experimental conditions. We used whole homogenates of the cortex, as excitatory synapses are practically absent in the newborn cortex [I], and therefore synaptic membrane preparations would not have any real meaning in early postnatal ages. Thus, the use of whole homogenates, which contain both synaptic and non-synaptic receptor binding sites, may explain dissimilarities between our results and those reported for synaptic membrane preparations, or even P2 fractions (e.g. refs. 3, 16). Moreover, it has been shown that, depending on the conditions
TABLE II BINDING PARAMETERS FOR [3H]KAINATE AND [3H]AMPA IN VISUAL CORTICAL MEMBRANES OF 6- AND 180-DAY-OLD RATS Values are mean + S.D., n=3. Binding parameter
Kd (nM) Bmax (fmol/mg prot.) *P
Age (days)
6 180 6 180
Radioligand [3Hlkainate
[3H]AMPA
37+ 4 39+_ 3 1348+41 879+- 18"
148+14 67+ 9* 437+ 19 129+ 16"
165 of membrane preparation and binding assay, kainate sites may be present in two distinct forms. The medium-affinity kainate binding site (detected in our experiments) may dissociate to high- and low-affinity sites (see ref. 10) which may exhibit different developmental patterns (see ref. 4). The developmental time course of quisqualate type receptor sites in the brain has not been studied by others to our knowledge. Thus, the ontogenetic pattern of A M P A binding, demonstrated in our experiments has no direct correlates in the literature. It should be noted that, under the conditions of our experiments, namely in the absence of chaotropic ions, only one form of the quisqualate site is detected (see ref. 10). This may also explain the considerably lower density of A M P A than kainate binding sites found in our experiments. The transient increase in kainate and quisqualate binding does not seem to correlate with the development of excitatory synapses in the neocortex [1], or with the ontogeny of kainate neurotoxicity [4, 6]. However, the increased susceptibility of immature rat brain (day 7) to the neurotoxic effect of quisqualic acid [17], and the transient stimulation of inositol phospholipid metabolism by quisqualic acid in brains of 6- to 8-day-old rats [15], are in line with our findings for A M P A binding. This correspondence is consistent with an increase in the number and sensitivity (affinity) of quisqualate receptors in the immature cerebral cortex. The functional role (if any) of the high density of kainate and quisqualate receptor sites in the immature brain is not yet understood. Nevertheless, some role in the mediation of trophic actions [2], rather than in synaptic transmission, seems plausible, even though a transient perinatal glutamatergic innervation [9] has been suggested for subcortical structures. In summary, our results provide evidence that not only NMDA, but also quisqualate and kainate receptor sites (or, at least, certain forms of the latter two subtypes) show a transient increase in the immature cerebral cortex. As around the age of one week only few synapses are developed, a t r o p h i c role, rather than a synaptic transmitter role, is suggested for quisqualate and kainate sites. This work was supported by the Deutsche Forschungsgemeinschaft (Wo 279/6-3 and 6-5). 1 Aghajanian, G.K. and Bloom, F.E., The formation of synaptic junctions in developing rat brain: a quantitative electron microscopicstudy, Brain Res., 6 (1967) 716~727. 2 Aruffo, C., Freszt, R., Hildebrandt, A.G. and Cevros-Navarro, J., Low doses of L-monosodiumglutamate promote neuronal growth and differentiation in vitro, Dev. Neurosci., 9 (1987) 228-239. 3 Baudry, M., Arst, D., Oliver, M. and Lynch, G., Development of glutamate binding sites and their regulation by calcium in rat hippocampus, Dev. Brain Res., 1 (1981) 37-48. 4 Ben-Ari,¥., Tremblay, E., Berger, M. and Nitecka, L., Kainic acid seizure syndrome and binding sites in developingrats, Dev. Brain Res., 14 (1984) 284-288. 5 Ben-Ari,Y., Cherubini, E. and Krnjevic, K., Changes in voltage dependence of NMDA currents during development, Neurosci. Lett., 94 (1988) 88-92. 6 Berger, M.L., Tremblay, E., Nitecka, L. and Ben-Ari, Y., Maturation of kainic acid seizure-brain damage syndrome in the rat. III. Postnatal development of kainic acid binding sites in the limbic system, Neuroscience, 13 (1984) 1095 1104.
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