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Postnatal development of NMDA, AMPA and kainate receptors in individual layers of rat visual cortex and the effect of monocular deprivation
@ Pergamon
hr. J Drvi Nelfri~.\~f~~~rcl~. Vol. 12.No.]. pp. 3111.
IYW Elsevier Science Ltd. Copyright 0 lYY4ISDN Printed in Great Britain. All rights reserved 0736-5748194 $h.oo+o.m
POSTNATAL DEVELOPMENT OF NMDA, AMPA AND KAINATE RECEPTORS IN INDIVIDUAL LAYERS OF RAT VISUAL CORTEX AND THE EFFECT OF MONOCULAR DEPRIVATION ASHOK I&MAR, REI~HARD SCHLIEBS* and VOLKER BIGL University of Leipzig, Paul Flechsig Institute for Brain Research. Department of Neurochemistry, Leipzig, Germany (Received 5 April 1993; revised 16 September 1993; accepted 22 September 1993)
Abstract-The postnatal development of the ligand binding to N-metbyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-S-methylisoxazole-4-propionic acid (AMPA), and kainate receptor sites was studied in individual layers of rat visual cortex with [3H]MK-801, 13H]CNQX and [3H]kainate, respectively. The studies were performed by quantitative receptor autoradiography in the different visual cortical layers from normally raised rats and from monocularly deprived animals. In normally raised rats, in all visual cortical layers, [3H]MK-S01 and [“HICNQX binding increased significantly from birth to around postnatal day 20, at which age already the adult values are reached. In contrast, the increase in [3H]kainate binding from day 1 to day 15 is followed by a significant decrease in binding sites up to day 25; at this age the adult binding level is nearly attained. Monocular deprivation, by unilateral eyelid suture at the age of 11 days onwards, leads to a transient decrease in [“HJCNQX binding in all visual cortical layers, whereas [ Hlkamate binding results to a permanent decrease in AMPA receptor sites in cortical layers II to Vl in both sides of the cortex. In contrast, only long-term monocular deprivation until the age of 90 days resulted in decreased [1H]MK-801 binding levels as compared to age-matched controls. The data suggest that the laminar development of glutamate receptor subtypes is differentially affected by visual experience during early brain maturation. Key words: ontogeny, [3H]MK-801 binding, [3H]CNQX binding, receptor autoradiography, binding, monocular deprivation, image analysis, mammalian brain.
radioligand
The visual system in the rat undergoes postnatal functional and structural changes which are expected to be correlated with the development of the functionai properties of pattern re~gnition. Visual stimuli have been proposed to participate in this development and to influence the final tuning of the functional and/or structural neuronal connections within the visual system.31 It is becoming now increasingly evident that excitatory amino acid receptors play an important The effects of role in plasticity mechanisms in the developing and mature brain. 2,3,6&11,12,2uZ3 excitatory amino acids are mediated by a variety of different receptor types: N-methyl-D-aspartate (IDA) receptor, alpha-amino-3-hydroxy-5-methyl-isoxazole acid (A~PA) receptor, kainate receptor and the metabotropic receptor (for review, see e.g. Refs 18,20). Neurotransmitter receptors are one of the decisive links in the chain of synaptic information processing. Due to their adaptive properties in response to altered levels of neurotransmitter as a consequence of changes in neuronal activity,30 they are of p articular importance for functional-adaptive processes occurring during the maturation of the visual system with respect to pattern recognition. This is emphasized by studies demonstrating the permissive role of NMDA receptors2+11$22and nonNMDA receptorsr* in modification of synaptic transmission in young animals. The postnatal development of excitatory amino acid receptors in membrane fractions of the rat visual cortex has recently been examined4 suggesting that in the immature visual cortex NMDA but not quisqualate and kainate receptors may be primarily involved in synaptic transmission. However, there is still a lack of more quantitative data concerning the postnatal maturation of the laminar pattern of distinct glutamate receptor subtypes in rat visual cortex. Due to the essential role of excitatory ammo acid receptors in developmental plasticity, it should be important to determine *Author to whom correspondence should be addressed: Dr Reinhard Schliebs, Paul Flechsig Institute for Brain Research, Department of Neurochemistry, Faculty of Medicine, University of Leipzig, Jahnallee 59, D-04109 Leipzig, Germany. Tel: (+49341) 7974601; Fax: (+49341) 2114492.
whether the establishment of a laminar pattern of distinct glutamate receptor classes during the postnatal development is driven by a requirement for normal physiological light stimulation. The present paper describes the effect of different periods of monocular visual deprivation (unilateral eyelid suture from postnatal day 11 onwards) on the development of NMDA. AMPA, and kainate receptors in distinct layers of rat visual cortex using quantitative receptor autoradiography. Here we report ontogenetic differences in the developmental pattern of different glutamate receptor subtypes as well as differential effects of visual deprivation upon the laminar devel~)pmcnt of the receptors examined.
EXPERIMENTAL
PROCEDURES
Materials
[3-3H]( +)MK-801 (specific activity 951 GBq/mmol), [5-3H]CNQX (577 GBq/mmol), and [vinylidene3H]kainicacid were purchased from NEN Research products, Germany. MK-801 (RBI). L-glutamate, and kainic acid (SIGMA) were used for nonspecific binding; all other chemicals used were commercial products of highest purity available.
For deveIopmentalstudies, Wistar rats at agesof 1,7,10,15,18,21,25,35,and90dayspostnata11y were used. For visual deprivation studies, Wistar rats were subjected to unilateral eyelid closure as previously described29 on postnatal day 11 (just some days before the natural eyelid-opening usually on postnatal day 14), and were further raised with their untreated littermates until ages of 18,21. 25,35 and 90 days. Some control rats received the anaesthetic (Nembutal sodium solution, Abbott Lab., Chicago, 40 mg/kg body weight) only; no effects compared to untreated control rats could be observed. Rats were killed by decapitation at the ages indicated and the brains rapidly removed. Brains were frozen within 2 min in isopentane at -70°C. Ten micrometre thick coronal sections were cut between bregma -4.3 and -6.3 mm (according to the atlas by Zilleg4) at -16 to -20°C with a cryostat microtome (Miles, USA) and thaw-mounted on gelatin coated slides.
E3H]MK-801 binding to NMDA receptors was carried out as follows (using a slightly modified procedure according to Sakurai et al.26): sections were prewashed for 45 min in 50 mM Tris-acetate buffer (pH 7.4) at 4°C and blown dry under a stream of air at room temperature before performing the binding assay. After preincubation, the dried slides were covered with 50 mM Tris-acetate (pH 7.4) containing 50 nM [3H]MK-801 and incubated in humid chambers for two hrs at room temperature. Following incubation, slides were dipped quickly into ice-cold buffer, then rinsed for 80 min in ice-cold buffer and blown dry under warm air. Nonspecific binding was estimated in adjacent sections by adding 100 PM unlabeled MK-801 to the incubation buffer, and represented 6% of total binding. For 13H]CNQX binding to AMPA receptors2r sections were prewashed for 45 min in 50 mM Tris-acetate buffer (pH 7.2) at 4°C and blown dry under a stream of warm air prior to the binding assay. After preincubation, the dried slides were flooded with 50 mM Tris-HCt (pH 7.2) containing 48 nM [3H]CNQX and incubated in humid chambers for 45 min at 2°C. Following incubation, sections were rinsed quickly three times of 3 set each with ice-cold buffer, and blown dry under warm air. Nonspecific binding was estimated in adjacent sections in the presence of 1 mM L-glutamate in the incubation buffer, and represented <5% of total binding. For [3H]kainate binding (slightly modified after Nielson et d21), sections were prewashed for 45 min in 50 mM Tris-citrate buffer (pH 7.2) at 2°C and blown dry under a stream of room temperature air prior to the binding assay. After preincubation, the dried slides were flooded with 50 mM Tris-&rate (pH 7.2) containing 25 nM f3H]kainate and incubated in humid chambers for 45 min at 2°C. Following incubation, sections were rinsed three times of 20 set each with ice-cold buffer, and blown dry under warm air. Nonspecific binding was estimated in adjacent sections, co-incubated with 100 FM unlabeled kainic acid and did normally not exceed 15% of total binding.
Laminar ontogeny of glutamate receptors in visual cortex
33
~~l~~ary experiments were performed with each radioligand to characterize binding sites and to determine optimal binding and washing #nditions (reaching equ~ibrium at the time indicated; binding was displaceable by unlabeled drugs used; checking for optimal buffer systems and rinsing times). Receptor autoradiography
The labelled and dried tissue sections were apposed to tritium-sensitive film (3H-Hyperfihn, Amersham) at 4°C together with slides containing standards of known radioactivity level (“H-microscale, Amersham). After exposure for three to five weeks, the films were developed with a Kodak D19 developer for 5 min at 20”, fixed, rinsed and dried. Exaltation of autoradi~gra~
Quantitative analysis of the autoradio~ams was done on video camera-based, computer assisted imaging device (SIGNUM) using the autoradiographic software package Image Pro Plus, version For calibration of grey values (optical density) 3H-microscale standards (Amersham) co-exposed with labelled sections were used. The density of binding sites was calculated from the mean grey level determined in the corresponding tissue region using a calibration curve plotted from the radioactivity of the tissue standards (kBq/mg tissue equivalent) and the densitometrically determined optical density values of the respective autoradiograms. Receptor densities were expressed in femtomoles of specifically bound ligand per milligram of tissue weight.32 Optical density readings were performed in the visual cortex between bregma -4.3 and -6.3 (according to the atlas of Zilles”4) including occipital cortex, area Vl and VZ. For measuring the laminar distribution of receptor binding sites, the variation of grey levels along a 1 mm thick band over the entire cortical depth (from the pial surface to the cortex-white matter boldly) was determined (binding density thick profile) and corrected for nonspecific binding (for details, see also Kumar and Schliebsi4). Measurements were made on 3-5 sections from each animal (including two optical density readings of each section) and the data averaged. The mean binding profiles through the visual cortex from four different animals belonging to each experimental group, were estimated and expressed graphically. The mean variance of the averaged binding profiles in both control and monocularly deprived rats did not normally exceed 15%. To compare binding profiles through the visual cortex between animals of different ages, the cortical thickness was standardized to 100% for each age. In order to estimate binding levels for individual cortical layers, the data points of each binding profile were grouped into five subdivisions (bins), and the data in each bin were averaged. The size of these bins was so selected to be directly related to the laminar a~angement of the visual cortex (layers I, II/III, IV, V, and VI, see also Fig. 1B). The thickness of each cortical layer was estimated at every age from cresyl violet-stained coronal sections of the visual cortex. Note that some imprecisions may arise due to individual differences in laminar thickness. The average was calculated for each of these bins (=cortical layer) from scanning data of 3-5 sections from four different animals. Statistical analysis
A one-way analysis of variance (ANOVA) was used to examine differences in the neurochemical parameters measured between all layers at each age and also within each layer as a function of developmental age. To test for statistically significant differences between cortical layers or binding levels from control and experimental animals, the two-tailed Student’s t-test was applied. RESULTS In Fig. 1 representative examples of binding profiles through the visual cortex of adult rats obtained by quantitative image analysis are displayed demonstrating the laminar distribution of NMDA, AMPA, and kainate receptors. The density profile through the depth of the visual cortex displays a rather homogeneous distribution for [3H]MK-801 binding. In contrast, [3H]CNQX binding is more labeled in the upper visual cortical layers, whereas [3H]kainate binding sites are more concentrated in the deeper ones.
34 Visulll corlex Thick profile
Cortical
layer
Fig. I. (A) Representative autoradiograms of [“HIMK-801 (top), [%]CNQX. and [“Hlkainate binding (bottom) showing the size of the cortical region used to measure the variations in density levels throughout the entire cortical depth {thick profile). (B) Representative examples of binding profiles of f3H]MK-Xt?i. [“HfCNQX, and FHjkainate throughout ~hevisualcort~ca1 depth as indicated in (a&The thickness ofvisual cortical tayers i through VI is inserted in each figure. The values are the meansL-tS.D. from four ‘IO-day-old control rats.
~~~~~~~~~~~~
of&e
~~~~a~ p~~ern ofg~~~a~ate receptor subtypes in rut mkdaf cortex
The development of glutamate receptor subtype in individual layers of rat visua’t cortex ot normally raised rats is summarized in Figs 211. In all visual cortical layers of normal rats there was a sharp increase in [‘H]MK-801 binding (about 3-fold) from day 1 up to day 20; at this age the adult values were practically attained (Fig. 2). Similar to NMDA receptor ontogeny, [3H]CNQX binding to AMPA receptors increased from day 1 up to day 20. but at a more moderate rate (about 2-fold), in all visual cortical layers examined; at which age it already equals the level found in the adult (Fig. 3). f%fkainate bind’mg increased moderately (about 1.5-fold) in all visual cortical layers, also from day 1 to day 15. However, between postnatal days 15 and 20, there was a considerable decrease in kainate receptor binding: thereafter this decline continued until adulthood but at a more moderate rate (Fig. 4). Effect of visual deprivation on laminur development of glutamate receptor subtypes
Monocular deprivation from postnatal day 11 up to day 35 did not alter [3H]MK-801 binding in any of the visual cortical layers examined (Figs 2 and 5). However, when monocular deprivation persisted until postnatal day 90. significantly decreased binding levels by about tS-20% wcrc
Laminar
ontogeny
of glutamate
Postnatal -
CONTROL
--e-s
receptors
age
in visual cortex
35
(days)
m
**,*@**t
p&J
1
contrafat.
ipr;ilat.
Fig. 2. Postnatal development of [3H]MK-801 to NMDA receptors in individual Layers of rat visual cortex and the effect of monocular deprivation from postnatal day 11 onwards. Data points are the means5CE.M.. obtained by evaluation of3-5 sections from four different animals at each developmenral age. For relative changes (inclusive statistical significance) in binding sites in each visual cortical layer following monocular deprivation until the ages of 18 and 90 days, see Fig. 5.
detected in all cortical layers both ipsilateral and contralateral to the sutured eye as compared to age-matched controls, ~ouocuiar deprivation up to day 18 resulted in decreased [3H]CNQX boding level by about 40% in ali visual cortical layers both contra- and ipsilateral to the sutured eye as compared to that
36
Postnatal age kiays) ---c
CONTROL
-..t)...” h”rD contralat.
-**““‘, MD, ipsilat.
Fig. 3. Fostnataf deveiopm~nt of [3H]CNQX to AMPA receptors in i~di~~du~~ iayers of rat visuaf cortex and the effect of monocular deprivation (MD) from postnatal day onwards. Data points are the meansltS.E.M., obtained by evaluation of 3-5 sections from four different animals at each developmental age. FQT relative changes (inclusive statistieaf significance) in binding sites in each visual cortical layer follo~ng monocular deprivation until the ages of 18 and 90 days, see Fig. 5.
taminar
ontogeny
of glutamate
receptors
in visual cortex
3
--a---
CONTROL_
-+-
MD.
contralat.
.,,s@*..
No. ipsilat.
Fig. 4. Postnatal development of f3H]kainate to k&rate receptors in individual layers of rat visual cortex and the effect of monocular deprivation from postnatal day 11 onwards. Data points are the means?S.E.M., obtained by evaluation of 3-5 sections from four different animals at each developmental age. For relative changes (inclusive statistical significance) in binding sites in each visual cortical layer following monocular deprivation until the ages of 18 and W days, see Fig. 5.
37
l
90 DAYS
J. Cortical
U
MD, contralateral
layer -
MD, ipsilateral
Fig. 5. Relative changes (expressed in percentage over control) in binding sites in each visual cortical layu following monocular deprivation (MD) until the ages of IX and 90 days. Data represent the means+ S.E.M.. obtained by evaluation of 3-5 sections from four different animals at each experimental group. *P
detectable in normally raised rats (Fig. 5). Visual deprivation for a longer period of time leads to an approach of binding levels to control values in all visual cortical layers (Fig. 3). Monocular deprivation from day 11 up to day 18 leads to significantly decreased [“Hlkainate binding (by about 20%) in cortical layers II to VI both contra- and ipsilaternl to the closed eye in comparison to age-matched control animals. Following visual deprivation for longer periods of time ~~H~kainate binding remained slightly decreased, an effect which was more ~ronoun~d in layers IV and V (Figs 4 and 5). DISCUSSION The present study was undertaken to investigate the postnatal development of glutamate receptor subtypes in individual visual cortical layers, and to reveal whether the establishment of laminar pattern of these binding sites is driven by a requirement for normal physiological light stimulation. The major findings reported here are that (i) NMDA and AMPA receptors have similar postnatal laminar developmental time-courses which sharply contrast to those of kainate receptors, and (ii) the development of glutamate receptor subtypes is seemingly regulated by visual experience at an early period of brain maturation, but in a differential manner, There are several studies in various species demonstrating that the postnatal ontogeny of glutamate receptors in the visual cortex is developmentally regulated 3&53XL27(for review, see Ref. 17).
In the present study the postnatal ontogeny of NMDA receptors was found to be similar in all visual cortical layers reaching adult values already three to four weeks after birth. This compares
Laminar ontogeny of glutamate receptors in visual cortex
39
rather well to similar studies performed in cat visual cortex with respect to laminar ontogeny and developmental patterns. 3,7.23[3H]MK-801 is assumed to bind to a site within the NMDA receptor ion channel and thus can be considered as a marker of the agonist-bound, open state of the channel (for review, see Ref. 20). In contrast to the ontogenetic course of NMDA receptors, the developmental patterns of [3H]kainate binding displayed a transient increase in binding sites up to postnatal day 15 followed by a significant decrease until day 25. Similar developmental patterns were observed in homogenate suspensions of rat visual cortex using [“H]CPP and 13H]kainate to label NMDA and kainate receptors, respectively.4 [“H]CNQX bind’mg to AMPA receptors was found to moderately increase from birth up to day 20 in all visual cortical layers at which age the adult level was practically attained. This developmental time-course does not compare to that observed in homogenates of rat visual cortex using [3H]AMPA to label AMPA receptors.4 The reason for this discrepancy is yet unclear but might be partly due to the different methodological approaches applied. However, it should be noted that the developmental time-courses of AMPA and kainate receptors in rat visual cortical layers reported here are similar to those found in rat frontal cortex using also quantitative receptor autoradiography.‘Tr9 In contrast to the ontogenetic pattern of kainate binding sites, the developmental time-courses of AMPA and NMDA receptors in the visual cortical layers correlate well with the temporal maturation of the excitatory innervation in the rat visual cortex which seems to be complete around the postnatal age of 20 days.5 The early postnatal maximum in ligand binding to kainate receptors around day 15 coincides with the time of natural eye-opening and is reached during a period of high cortical plasticity. The sharp decrease of [3H]kainate binding between days 15 and 20 might then be considered as a degradation of those binding sites not effectively involved in the ongoing formation of excitatory amino acid synapses in individual visual cortical layers. It is interesting to note that the developmental time-course of kainate receptors in visual cortical layers of normally raised rats is similar to that obtained in a previous study assaying [3H]glutamate binding in membrane fractions of rat visual cortex during postnatal ontogenesis. The different developmental time-courses of NMDA, AMPA and kainate receptors in rat visual cortical layers suggest specific roles during a critical period of the functional maturation of the visual cortex. This is emphasized by our findings that monocular deprivation influences differentially and selectively the development of NMDA, AMPA and kainate receptors in individual visual cortical layers during the postnatal period which is associated with synaptogenesis and the final tuning of connectional patterns in the visual cortex. There are several reports emphasizing the important role of glutamate in experience-dependent plasticity of the visual cortex (see e.g. Ref. 13). In particular, it was suggested that the strengthening of coactive synapses is mainly mediated by activation of NMDA-receptor-mediated Ca2+ flux over a critical level.‘~2.8~11,2”However, recently also the involvement of non-NMDA receptors in modification of synaptic transmission was described in kitten visual cortex.12 Neurotransmitter receptors may be regulated in response to their level of activation.‘O Thus changes in glutamate receptor subtypes might reflect alterations in receptor sensitivity as a result of altered glutamatergic activity following visual deprivation. The transient changes in f3H]CNQX binding might suggest that AMPA receptors have a modulatory function only during a certain period of early postnatal development which is highly susceptible to visual experience. In contrast, long-term monocular deprivation until adulthood results in decreased binding levels of NMDA receptors in all visual cortical layers of both hemispheres, whereas the binding sites of kainate receptors seem to be persistently decreased restricted to the deeper layers. The changes in glutamate receptor subtypes following monocular deprivation are detectable on both sides of the visual cortex, a phenomenon which was also observed in similar studies on other transmitter receptors14~15 and corresponding receptor mRNA transcripts. 24,25Whether the altered binding in the contralateral cortex represents a non-specific influence of eye-closure or whether an interhemispheric cortico-cortical information transfer plays an important role (as concluded from a different approach”) cannot be derived from this investigation and needs further clarification. In contrast to the metabotropic glutamate receptor, NMDA, kainate and AMPA receptors appear to be ligand-gated ion channels and seem to mediate different cellular events. When
A. Kurnar et al.
40
stimulated, kainate and AMPA receptors activate an ion channel that is permeable to Na ’ and K ’ ions but not for Ca*+, thus mediating fast excitatory transmission at glutamatergic synapses (for review, see e.g. Ref. 33). Activation of NMDA receptors leads directly to a large Ca2+ influx into neurons. l6 Therefore, the differential effects of visual deprivation on glutamate receptor subtypes suggest that distinct cellular events are initiated to control experience-dependent visual cortical maturation. At this stage of investigation. however. it is difficult to say whether the alterations in receptor binding are the result of receptor desensitization or whether they can be considered as changes in the homeostatic equilibrium of glutamatergic transmission at the synaptic level to strengthen certain synaptic connections. Acknowledgemenrs-The Deutscher Akademischer
authors acknowledge the expert technical Austauschdienst for providing a two-year
assistance of Mrs Renata IBRO-scholarship.
Jendrek.
A. K. 1s pratetul
to
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hr. J Drvi Nelfri~.\~f~~~rcl~. Vol. 12.No.]. pp. 3111.
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POSTNATAL DEVELOPMENT OF NMDA, AMPA AND KAINATE RECEPTORS IN INDIVIDUAL LAYERS OF RAT VISUAL CORTEX AND THE EFFECT OF MONOCULAR DEPRIVATION ASHOK I&MAR, REI~HARD SCHLIEBS* and VOLKER BIGL University of Leipzig, Paul Flechsig Institute for Brain Research. Department of Neurochemistry, Leipzig, Germany (Received 5 April 1993; revised 16 September 1993; accepted 22 September 1993)
Abstract-The postnatal development of the ligand binding to N-metbyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-S-methylisoxazole-4-propionic acid (AMPA), and kainate receptor sites was studied in individual layers of rat visual cortex with [3H]MK-801, 13H]CNQX and [3H]kainate, respectively. The studies were performed by quantitative receptor autoradiography in the different visual cortical layers from normally raised rats and from monocularly deprived animals. In normally raised rats, in all visual cortical layers, [3H]MK-S01 and [“HICNQX binding increased significantly from birth to around postnatal day 20, at which age already the adult values are reached. In contrast, the increase in [3H]kainate binding from day 1 to day 15 is followed by a significant decrease in binding sites up to day 25; at this age the adult binding level is nearly attained. Monocular deprivation, by unilateral eyelid suture at the age of 11 days onwards, leads to a transient decrease in [“HJCNQX binding in all visual cortical layers, whereas [ Hlkamate binding results to a permanent decrease in AMPA receptor sites in cortical layers II to Vl in both sides of the cortex. In contrast, only long-term monocular deprivation until the age of 90 days resulted in decreased [1H]MK-801 binding levels as compared to age-matched controls. The data suggest that the laminar development of glutamate receptor subtypes is differentially affected by visual experience during early brain maturation. Key words: ontogeny, [3H]MK-801 binding, [3H]CNQX binding, receptor autoradiography, binding, monocular deprivation, image analysis, mammalian brain.
radioligand
The visual system in the rat undergoes postnatal functional and structural changes which are expected to be correlated with the development of the functionai properties of pattern re~gnition. Visual stimuli have been proposed to participate in this development and to influence the final tuning of the functional and/or structural neuronal connections within the visual system.31 It is becoming now increasingly evident that excitatory amino acid receptors play an important The effects of role in plasticity mechanisms in the developing and mature brain. 2,3,6&11,12,2uZ3 excitatory amino acids are mediated by a variety of different receptor types: N-methyl-D-aspartate (IDA) receptor, alpha-amino-3-hydroxy-5-methyl-isoxazole acid (A~PA) receptor, kainate receptor and the metabotropic receptor (for review, see e.g. Refs 18,20). Neurotransmitter receptors are one of the decisive links in the chain of synaptic information processing. Due to their adaptive properties in response to altered levels of neurotransmitter as a consequence of changes in neuronal activity,30 they are of p articular importance for functional-adaptive processes occurring during the maturation of the visual system with respect to pattern recognition. This is emphasized by studies demonstrating the permissive role of NMDA receptors2+11$22and nonNMDA receptorsr* in modification of synaptic transmission in young animals. The postnatal development of excitatory amino acid receptors in membrane fractions of the rat visual cortex has recently been examined4 suggesting that in the immature visual cortex NMDA but not quisqualate and kainate receptors may be primarily involved in synaptic transmission. However, there is still a lack of more quantitative data concerning the postnatal maturation of the laminar pattern of distinct glutamate receptor subtypes in rat visual cortex. Due to the essential role of excitatory ammo acid receptors in developmental plasticity, it should be important to determine *Author to whom correspondence should be addressed: Dr Reinhard Schliebs, Paul Flechsig Institute for Brain Research, Department of Neurochemistry, Faculty of Medicine, University of Leipzig, Jahnallee 59, D-04109 Leipzig, Germany. Tel: (+49341) 7974601; Fax: (+49341) 2114492.
whether the establishment of a laminar pattern of distinct glutamate receptor classes during the postnatal development is driven by a requirement for normal physiological light stimulation. The present paper describes the effect of different periods of monocular visual deprivation (unilateral eyelid suture from postnatal day 11 onwards) on the development of NMDA. AMPA, and kainate receptors in distinct layers of rat visual cortex using quantitative receptor autoradiography. Here we report ontogenetic differences in the developmental pattern of different glutamate receptor subtypes as well as differential effects of visual deprivation upon the laminar devel~)pmcnt of the receptors examined.
EXPERIMENTAL
PROCEDURES
Materials
[3-3H]( +)MK-801 (specific activity 951 GBq/mmol), [5-3H]CNQX (577 GBq/mmol), and [vinylidene3H]kainicacid were purchased from NEN Research products, Germany. MK-801 (RBI). L-glutamate, and kainic acid (SIGMA) were used for nonspecific binding; all other chemicals used were commercial products of highest purity available.
For deveIopmentalstudies, Wistar rats at agesof 1,7,10,15,18,21,25,35,and90dayspostnata11y were used. For visual deprivation studies, Wistar rats were subjected to unilateral eyelid closure as previously described29 on postnatal day 11 (just some days before the natural eyelid-opening usually on postnatal day 14), and were further raised with their untreated littermates until ages of 18,21. 25,35 and 90 days. Some control rats received the anaesthetic (Nembutal sodium solution, Abbott Lab., Chicago, 40 mg/kg body weight) only; no effects compared to untreated control rats could be observed. Rats were killed by decapitation at the ages indicated and the brains rapidly removed. Brains were frozen within 2 min in isopentane at -70°C. Ten micrometre thick coronal sections were cut between bregma -4.3 and -6.3 mm (according to the atlas by Zilleg4) at -16 to -20°C with a cryostat microtome (Miles, USA) and thaw-mounted on gelatin coated slides.
E3H]MK-801 binding to NMDA receptors was carried out as follows (using a slightly modified procedure according to Sakurai et al.26): sections were prewashed for 45 min in 50 mM Tris-acetate buffer (pH 7.4) at 4°C and blown dry under a stream of air at room temperature before performing the binding assay. After preincubation, the dried slides were covered with 50 mM Tris-acetate (pH 7.4) containing 50 nM [3H]MK-801 and incubated in humid chambers for two hrs at room temperature. Following incubation, slides were dipped quickly into ice-cold buffer, then rinsed for 80 min in ice-cold buffer and blown dry under warm air. Nonspecific binding was estimated in adjacent sections by adding 100 PM unlabeled MK-801 to the incubation buffer, and represented 6% of total binding. For 13H]CNQX binding to AMPA receptors2r sections were prewashed for 45 min in 50 mM Tris-acetate buffer (pH 7.2) at 4°C and blown dry under a stream of warm air prior to the binding assay. After preincubation, the dried slides were flooded with 50 mM Tris-HCt (pH 7.2) containing 48 nM [3H]CNQX and incubated in humid chambers for 45 min at 2°C. Following incubation, sections were rinsed quickly three times of 3 set each with ice-cold buffer, and blown dry under warm air. Nonspecific binding was estimated in adjacent sections in the presence of 1 mM L-glutamate in the incubation buffer, and represented <5% of total binding. For [3H]kainate binding (slightly modified after Nielson et d21), sections were prewashed for 45 min in 50 mM Tris-citrate buffer (pH 7.2) at 2°C and blown dry under a stream of room temperature air prior to the binding assay. After preincubation, the dried slides were flooded with 50 mM Tris-&rate (pH 7.2) containing 25 nM f3H]kainate and incubated in humid chambers for 45 min at 2°C. Following incubation, sections were rinsed three times of 20 set each with ice-cold buffer, and blown dry under warm air. Nonspecific binding was estimated in adjacent sections, co-incubated with 100 FM unlabeled kainic acid and did normally not exceed 15% of total binding.
Laminar ontogeny of glutamate receptors in visual cortex
33
~~l~~ary experiments were performed with each radioligand to characterize binding sites and to determine optimal binding and washing #nditions (reaching equ~ibrium at the time indicated; binding was displaceable by unlabeled drugs used; checking for optimal buffer systems and rinsing times). Receptor autoradiography
The labelled and dried tissue sections were apposed to tritium-sensitive film (3H-Hyperfihn, Amersham) at 4°C together with slides containing standards of known radioactivity level (“H-microscale, Amersham). After exposure for three to five weeks, the films were developed with a Kodak D19 developer for 5 min at 20”, fixed, rinsed and dried. Exaltation of autoradi~gra~
Quantitative analysis of the autoradio~ams was done on video camera-based, computer assisted imaging device (SIGNUM) using the autoradiographic software package Image Pro Plus, version For calibration of grey values (optical density) 3H-microscale standards (Amersham) co-exposed with labelled sections were used. The density of binding sites was calculated from the mean grey level determined in the corresponding tissue region using a calibration curve plotted from the radioactivity of the tissue standards (kBq/mg tissue equivalent) and the densitometrically determined optical density values of the respective autoradiograms. Receptor densities were expressed in femtomoles of specifically bound ligand per milligram of tissue weight.32 Optical density readings were performed in the visual cortex between bregma -4.3 and -6.3 (according to the atlas of Zilles”4) including occipital cortex, area Vl and VZ. For measuring the laminar distribution of receptor binding sites, the variation of grey levels along a 1 mm thick band over the entire cortical depth (from the pial surface to the cortex-white matter boldly) was determined (binding density thick profile) and corrected for nonspecific binding (for details, see also Kumar and Schliebsi4). Measurements were made on 3-5 sections from each animal (including two optical density readings of each section) and the data averaged. The mean binding profiles through the visual cortex from four different animals belonging to each experimental group, were estimated and expressed graphically. The mean variance of the averaged binding profiles in both control and monocularly deprived rats did not normally exceed 15%. To compare binding profiles through the visual cortex between animals of different ages, the cortical thickness was standardized to 100% for each age. In order to estimate binding levels for individual cortical layers, the data points of each binding profile were grouped into five subdivisions (bins), and the data in each bin were averaged. The size of these bins was so selected to be directly related to the laminar a~angement of the visual cortex (layers I, II/III, IV, V, and VI, see also Fig. 1B). The thickness of each cortical layer was estimated at every age from cresyl violet-stained coronal sections of the visual cortex. Note that some imprecisions may arise due to individual differences in laminar thickness. The average was calculated for each of these bins (=cortical layer) from scanning data of 3-5 sections from four different animals. Statistical analysis
A one-way analysis of variance (ANOVA) was used to examine differences in the neurochemical parameters measured between all layers at each age and also within each layer as a function of developmental age. To test for statistically significant differences between cortical layers or binding levels from control and experimental animals, the two-tailed Student’s t-test was applied. RESULTS In Fig. 1 representative examples of binding profiles through the visual cortex of adult rats obtained by quantitative image analysis are displayed demonstrating the laminar distribution of NMDA, AMPA, and kainate receptors. The density profile through the depth of the visual cortex displays a rather homogeneous distribution for [3H]MK-801 binding. In contrast, [3H]CNQX binding is more labeled in the upper visual cortical layers, whereas [3H]kainate binding sites are more concentrated in the deeper ones.
34 Visulll corlex Thick profile
Cortical
layer
Fig. I. (A) Representative autoradiograms of [“HIMK-801 (top), [%]CNQX. and [“Hlkainate binding (bottom) showing the size of the cortical region used to measure the variations in density levels throughout the entire cortical depth {thick profile). (B) Representative examples of binding profiles of f3H]MK-Xt?i. [“HfCNQX, and FHjkainate throughout ~hevisualcort~ca1 depth as indicated in (a&The thickness ofvisual cortical tayers i through VI is inserted in each figure. The values are the meansL-tS.D. from four ‘IO-day-old control rats.
~~~~~~~~~~~~
of&e
~~~~a~ p~~ern ofg~~~a~ate receptor subtypes in rut mkdaf cortex
The development of glutamate receptor subtype in individual layers of rat visua’t cortex ot normally raised rats is summarized in Figs 211. In all visual cortical layers of normal rats there was a sharp increase in [‘H]MK-801 binding (about 3-fold) from day 1 up to day 20; at this age the adult values were practically attained (Fig. 2). Similar to NMDA receptor ontogeny, [3H]CNQX binding to AMPA receptors increased from day 1 up to day 20. but at a more moderate rate (about 2-fold), in all visual cortical layers examined; at which age it already equals the level found in the adult (Fig. 3). f%fkainate bind’mg increased moderately (about 1.5-fold) in all visual cortical layers, also from day 1 to day 15. However, between postnatal days 15 and 20, there was a considerable decrease in kainate receptor binding: thereafter this decline continued until adulthood but at a more moderate rate (Fig. 4). Effect of visual deprivation on laminur development of glutamate receptor subtypes
Monocular deprivation from postnatal day 11 up to day 35 did not alter [3H]MK-801 binding in any of the visual cortical layers examined (Figs 2 and 5). However, when monocular deprivation persisted until postnatal day 90. significantly decreased binding levels by about tS-20% wcrc
Laminar
ontogeny
of glutamate
Postnatal -
CONTROL
--e-s
receptors
age
in visual cortex
35
(days)
m
**,*@**t
p&J
1
contrafat.
ipr;ilat.
Fig. 2. Postnatal development of [3H]MK-801 to NMDA receptors in individual Layers of rat visual cortex and the effect of monocular deprivation from postnatal day 11 onwards. Data points are the means5CE.M.. obtained by evaluation of3-5 sections from four different animals at each developmenral age. For relative changes (inclusive statistical significance) in binding sites in each visual cortical layer following monocular deprivation until the ages of 18 and 90 days, see Fig. 5.
detected in all cortical layers both ipsilateral and contralateral to the sutured eye as compared to age-matched controls, ~ouocuiar deprivation up to day 18 resulted in decreased [3H]CNQX boding level by about 40% in ali visual cortical layers both contra- and ipsilateral to the sutured eye as compared to that
36
Postnatal age kiays) ---c
CONTROL
-..t)...” h”rD contralat.
-**““‘, MD, ipsilat.
Fig. 3. Fostnataf deveiopm~nt of [3H]CNQX to AMPA receptors in i~di~~du~~ iayers of rat visuaf cortex and the effect of monocular deprivation (MD) from postnatal day onwards. Data points are the meansltS.E.M., obtained by evaluation of 3-5 sections from four different animals at each developmental age. FQT relative changes (inclusive statistieaf significance) in binding sites in each visual cortical layer follo~ng monocular deprivation until the ages of 18 and 90 days, see Fig. 5.
taminar
ontogeny
of glutamate
receptors
in visual cortex
3
--a---
CONTROL_
-+-
MD.
contralat.
.,,s@*..
No. ipsilat.
Fig. 4. Postnatal development of f3H]kainate to k&rate receptors in individual layers of rat visual cortex and the effect of monocular deprivation from postnatal day 11 onwards. Data points are the means?S.E.M., obtained by evaluation of 3-5 sections from four different animals at each developmental age. For relative changes (inclusive statistical significance) in binding sites in each visual cortical layer following monocular deprivation until the ages of 18 and W days, see Fig. 5.
37
l
90 DAYS
J. Cortical
U
MD, contralateral
layer -
MD, ipsilateral
Fig. 5. Relative changes (expressed in percentage over control) in binding sites in each visual cortical layu following monocular deprivation (MD) until the ages of IX and 90 days. Data represent the means+ S.E.M.. obtained by evaluation of 3-5 sections from four different animals at each experimental group. *P
detectable in normally raised rats (Fig. 5). Visual deprivation for a longer period of time leads to an approach of binding levels to control values in all visual cortical layers (Fig. 3). Monocular deprivation from day 11 up to day 18 leads to significantly decreased [“Hlkainate binding (by about 20%) in cortical layers II to VI both contra- and ipsilaternl to the closed eye in comparison to age-matched control animals. Following visual deprivation for longer periods of time ~~H~kainate binding remained slightly decreased, an effect which was more ~ronoun~d in layers IV and V (Figs 4 and 5). DISCUSSION The present study was undertaken to investigate the postnatal development of glutamate receptor subtypes in individual visual cortical layers, and to reveal whether the establishment of laminar pattern of these binding sites is driven by a requirement for normal physiological light stimulation. The major findings reported here are that (i) NMDA and AMPA receptors have similar postnatal laminar developmental time-courses which sharply contrast to those of kainate receptors, and (ii) the development of glutamate receptor subtypes is seemingly regulated by visual experience at an early period of brain maturation, but in a differential manner, There are several studies in various species demonstrating that the postnatal ontogeny of glutamate receptors in the visual cortex is developmentally regulated 3&53XL27(for review, see Ref. 17).
In the present study the postnatal ontogeny of NMDA receptors was found to be similar in all visual cortical layers reaching adult values already three to four weeks after birth. This compares
Laminar ontogeny of glutamate receptors in visual cortex
39
rather well to similar studies performed in cat visual cortex with respect to laminar ontogeny and developmental patterns. 3,7.23[3H]MK-801 is assumed to bind to a site within the NMDA receptor ion channel and thus can be considered as a marker of the agonist-bound, open state of the channel (for review, see Ref. 20). In contrast to the ontogenetic course of NMDA receptors, the developmental patterns of [3H]kainate binding displayed a transient increase in binding sites up to postnatal day 15 followed by a significant decrease until day 25. Similar developmental patterns were observed in homogenate suspensions of rat visual cortex using [“H]CPP and 13H]kainate to label NMDA and kainate receptors, respectively.4 [“H]CNQX bind’mg to AMPA receptors was found to moderately increase from birth up to day 20 in all visual cortical layers at which age the adult level was practically attained. This developmental time-course does not compare to that observed in homogenates of rat visual cortex using [3H]AMPA to label AMPA receptors.4 The reason for this discrepancy is yet unclear but might be partly due to the different methodological approaches applied. However, it should be noted that the developmental time-courses of AMPA and kainate receptors in rat visual cortical layers reported here are similar to those found in rat frontal cortex using also quantitative receptor autoradiography.‘Tr9 In contrast to the ontogenetic pattern of kainate binding sites, the developmental time-courses of AMPA and NMDA receptors in the visual cortical layers correlate well with the temporal maturation of the excitatory innervation in the rat visual cortex which seems to be complete around the postnatal age of 20 days.5 The early postnatal maximum in ligand binding to kainate receptors around day 15 coincides with the time of natural eye-opening and is reached during a period of high cortical plasticity. The sharp decrease of [3H]kainate binding between days 15 and 20 might then be considered as a degradation of those binding sites not effectively involved in the ongoing formation of excitatory amino acid synapses in individual visual cortical layers. It is interesting to note that the developmental time-course of kainate receptors in visual cortical layers of normally raised rats is similar to that obtained in a previous study assaying [3H]glutamate binding in membrane fractions of rat visual cortex during postnatal ontogenesis. The different developmental time-courses of NMDA, AMPA and kainate receptors in rat visual cortical layers suggest specific roles during a critical period of the functional maturation of the visual cortex. This is emphasized by our findings that monocular deprivation influences differentially and selectively the development of NMDA, AMPA and kainate receptors in individual visual cortical layers during the postnatal period which is associated with synaptogenesis and the final tuning of connectional patterns in the visual cortex. There are several reports emphasizing the important role of glutamate in experience-dependent plasticity of the visual cortex (see e.g. Ref. 13). In particular, it was suggested that the strengthening of coactive synapses is mainly mediated by activation of NMDA-receptor-mediated Ca2+ flux over a critical level.‘~2.8~11,2”However, recently also the involvement of non-NMDA receptors in modification of synaptic transmission was described in kitten visual cortex.12 Neurotransmitter receptors may be regulated in response to their level of activation.‘O Thus changes in glutamate receptor subtypes might reflect alterations in receptor sensitivity as a result of altered glutamatergic activity following visual deprivation. The transient changes in f3H]CNQX binding might suggest that AMPA receptors have a modulatory function only during a certain period of early postnatal development which is highly susceptible to visual experience. In contrast, long-term monocular deprivation until adulthood results in decreased binding levels of NMDA receptors in all visual cortical layers of both hemispheres, whereas the binding sites of kainate receptors seem to be persistently decreased restricted to the deeper layers. The changes in glutamate receptor subtypes following monocular deprivation are detectable on both sides of the visual cortex, a phenomenon which was also observed in similar studies on other transmitter receptors14~15 and corresponding receptor mRNA transcripts. 24,25Whether the altered binding in the contralateral cortex represents a non-specific influence of eye-closure or whether an interhemispheric cortico-cortical information transfer plays an important role (as concluded from a different approach”) cannot be derived from this investigation and needs further clarification. In contrast to the metabotropic glutamate receptor, NMDA, kainate and AMPA receptors appear to be ligand-gated ion channels and seem to mediate different cellular events. When
A. Kurnar et al.
40
stimulated, kainate and AMPA receptors activate an ion channel that is permeable to Na ’ and K ’ ions but not for Ca*+, thus mediating fast excitatory transmission at glutamatergic synapses (for review, see e.g. Ref. 33). Activation of NMDA receptors leads directly to a large Ca2+ influx into neurons. l6 Therefore, the differential effects of visual deprivation on glutamate receptor subtypes suggest that distinct cellular events are initiated to control experience-dependent visual cortical maturation. At this stage of investigation. however. it is difficult to say whether the alterations in receptor binding are the result of receptor desensitization or whether they can be considered as changes in the homeostatic equilibrium of glutamatergic transmission at the synaptic level to strengthen certain synaptic connections. Acknowledgemenrs-The Deutscher Akademischer
authors acknowledge the expert technical Austauschdienst for providing a two-year
assistance of Mrs Renata IBRO-scholarship.
Jendrek.
A. K. 1s pratetul
to
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