Distribution of AMPA selective glutamate receptors in the thalamus of adult rats and during postnatal development. A light and ultrastructural immunocytochemical study

DEVELOPMENTAL BRAIN RESEARCH ELSEVIER

Developmental Brain Research 82 (1994) 231-244

Research

report

Distribution of AMPA selective glutamate receptors in the thalamus of adult rats and during postnatal development. A light and ultrastructural immunocytochemical study R. Spreafico

a.,, C . F r a s s o n i

a, p . A r c e l l i b, G . B a t t a g l i a a, R . J . W e n t h o l d

c, S. D e B i a s i u

a Dipartimento di Neurofisiologia, Istituto Nazionale Neurologico 'C. Besta; via Celoria, 11, 20133 Milano, Italy, b Dipartimento di Fisiologia e Biochimica Generali, Sezione di lstologia e Anatomia Umana, Universit?t di Milano, Italy, c Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892, USA

Accepted 21 June 1994

Abstract

The regional, cellular and subcellular distribution of AMPA receptors was demonstrated immunocytochemically within the thalamus of adult and young (from 1 to 20 days postnatal, P1-P20) rats. The antipeptide antibodies used 37 recognize individual subunit proteins of the AMPA-preferring glutamate receptor, i.e., GluR1, GluR2-3 and GIuR4. Our results demonstrate that these AMPA receptor subunits are generally not highly expressed in the thalamus, as compared to other brain areas and that they are enriched differentially within different thalamic nuclei. GIuR1 is mostly found in intralaminar and midline nuclei throughout life, whereas GluR2-3 is moderately expressed in the thalamus, with no major developmental changes. GIuR4 is the predominant subunit expressed in the reticular nucleus in adult rats, but not in young animals, where until P9 it is instead present in the ventrobasal complex. Samples of paraventricular and lateral geniculate nuclei stained with GIuR1 and of reticular nucleus as well as ventrobasal complex stained with GIuR4 were used for the ultrastructural study. In all the samples, labelling was in the somatic and dendritic cytoplasm, with dense patches of reaction product apposing post-synaptic densities of terminals with round clear vesicles and asymmetric specializations. Glial staining was observed only with the GluR1 antiserum and there was no evidence of labelled synaptic terminals. The differential distribution of GIuR subunits in the thalamus suggests that certain subunits may participate more than others in mediating post-synaptic responses in distinct neuronal populations and also that other GIuR types may be involved in the thalamic networks. Keywords: Receptor; Glutamate; Non-NMDA; Ontogenesis; Thalamus; Electron microscopy

1. Introduction

Electrophysiological and neurochemical data show that glutamate and aspartate are the most important excitatory neurotransmitters in the brain, playing also an important role in developmental synaptic plasticity and neurogenesis [8,9,30]. The immunocytochemical visualization of these excitatory neurotransmitters has been performed in different regions of the CNS [10,18,35] and several lines of

* Corresponding author. Fax: (39) (2) 70600775. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-3806(94)00116-H

evidence suggest that neurons within the thalamus receive excitatory glutamergic or aspartergic innervation from a variety of regions [11,41,51]. The identification of neuronal circuits by means of specific antibodies against amino acids has been often challenged by the widespread involvement of these molecules in intermediary metabolism. For this reason the identification and localization of receptors mediating excitatory transmission is considered a more refined approach for studying the organization of excitatory systems. The excitatory amino acid receptors can be divided into metabotropic G-protein coupled receptors [4,27, 29,46] and into two distinct subtypes of ionotropic receptors as identified on the basis of pharmacological and physiological responses to selective antagonists:

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the N-methyl-D-aspartate (NMDA) and the nonNMDA [17,46,58]. The non-NMDA group includes kainate (KA) and a-amino-3-hydroxy-5-methyl-4-isoxazolone propionic acid (AMPA) receptors. Although several subunits of non-NMDA receptors have been identified, only four, designed as GluRI-4, are known to bind 3H-AMPA with high affinity and this binding is inhibited by CNQX. These four subunits of AMPA receptors are thought to be integrated into heteromeric receptor complexes of variable subunit composition and are found in many excitatory synapses mediating the majority of the fast excitatory transmission with low Ca 2+ permeability [25,46,58]. In addition, sequence variants of these subunits exist that may impart different channel properties. For instance, adjacent exons encode two alternative versions (flip-flop) of the GIuR1-4 amino acidic sequences [48]. Distribution of AMPA receptors in the mature and developing CNS has been investigated by radio-ligand binding studies and by in situ hybridization histochemistry [22,34,36]. The former studies, however, indicate only the presence of a ligand binding entity, whereas the latters indicate sites or cells expressing receptor subtypes and both methods have an intrinsical low resolution. The regional distribution of AMPA receptor subtypes within the cells expressing them has recently been investigated at light and ultrastructural level by means of immunocytochemical methods, using anti-peptide antibodies that recognize individual subunit proteins of the AMPA-preferring glutamate receptor, i.e., GluR1, GluR2-3 and GluR4 [28,32,37]. Although this approach is generally considered to give a much better resolution, as compared to both autoradiographic binding and in situ hybridization methods, the immunocytochemical studies so far performed were mainly focused on selected brain regions with a very high level of labelling, such as neocortex, hippocampus and cerebellum, while little attention was paid to the thalamus. Moreover, although several lines of evidence suggest that receptors for excitatory amino acids are involved in a variety of physiological processes during development [30], both binding and in situ hybridization studies have only marginally considered this diencephalic center during the ontogenesis [22,31,33,36] and no immunocytochemical investigations have been performed. The aims of this study are therefore to evaluate (a) the expression of AMPA receptor subunits in different thalamic nuclei of adult rats, and (b) whether differences exist in the regional and cellular localization of AMPA receptors during postnatal development of the thalamus, using immunocytochemical methods at light and electron microscopy. Preliminary results have been presented in abstract form [16].

2. Materials and methods

Animals used were Wistar albino rats at postnatal (P) days 1, 2, 5, 7, 9, 10, 15 and 20 (11 animals) and adults (200-250 gr., six animals). Pups from P~ to P~ were cryoanesthetized with ice; older pups and adults were anesthetized with chloral hydrate (4%; 1 ml/100 g b/w). All the animals were than perfused intra-aortically with 1% paraformaldehyde in 0:tM phosphate buffer (PB) pH 7.4 followed by 4% paraformaldehyde/0.05% glutaraldehyde in PB. The brains were removed from the skull and post-fixed overnight in 4% paraformaldehyde in PB at 4°C. Serial, 50 /xm-thick, vibratome sections were collected in phosphate buffered saline solution (PBS). Representative sections throughout the rostrocaudal extent of the dorsal thalamus were processed for immunocytochemistry, while the adjacent ones were mounted on coated slides and counterstained with thionine. After quenching of free aldehyde groups with 0.05M NH4C1 in PB (30'), sections intended solely for light microscopy were permeabilized (30 min) in 0:4% Triton X-100 in PBS, whereas sections for electron microscopy were frozen in liquid nitrogen and thawed in PB-sucrose 20%, after appropriate cryoprotection in 10% and 20% sucrose (1 h each). Sections were incubated for t h in 10% normal goat serum (NGS) in PBS and then overnight at 4°C in the primary antisera solutions containing rabbit anti GIuR1 (1:2,000), anti GluR2-3 (1:750) and anti GIuR4 (1:500) antisera [37] diluted in PBS containing 1% NGS. The antibodies were made in rabbits against synthetic peptides corresponding to the C-terminal portions of AMPA-receptor subunits, conjugated to BSA with glutaraldehyde [56,57]. All antibodies were shown to be specific for their receptor subunits by Western blot analysis of membranes of cultured cells transfected with GIuR1,2 and 3 or 4 cDNAs [56,57]. After washing in PBS, sections were incubated in biotinylated goat anti-rabbit IgG (Vector) followed by avidin-biotin peroxidase complex (ABC; Vector) for 75' each. A solution of 0.05% diaminobenzidine tetrahydrochloride (DAB) and 0.015% H20 2 in PB was used to reveal the immunocytochemical reaction. For ultrastructural analysis some sections were treated also with cobalt chloride and nickel ammonium sulfate to intensify the DAB reaction product [1]. In control experiments, performed by omitting the primary antiserum in the incubation cycle, no detectable labelling was observed. Sections for light microscopy were mounted onto gelatine-coated slides and coverslipped with DPX. Sections selected for electron microscopy were post-fixed with 2.5% glutaraldehyde (15') and with 1% osmium tetroxide in PB (1 h), dehydrated in ethanol and flat embedded in Epon-Spurr. Sample areas from different

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J

Fig. 1. Low magnification survey of coronal sections trom adult brain immunolabelled with antisera to GIuR1 (A,B), G l u R 2 - 3 (C) and GIuR4 (D,E). For abbreviations see attached list. Scale bars: A - D = 10 p.m; E = 100/xm.

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thalamic nuclei were trimmed out under a dissection microscope, mounted on blank cured blocks of resin with cyanoaerylate cement and then sectioned with a Reichert ultramicrotome. Ultrastructural analysis was performed on reticular nucleus, ventrobasal complex, ventrolateral geniculate and paraventricular thalamic nuclei. Semithin (1 tzm-thick) sections were collected on glass slides and observed unstained with a Zeiss III photomicroscope. Thin sections were collected on copper grids, counterstained with uranyl acetate and lead citrate, with lead citrate only or left unstained, and examined with a Jeol T8 electron microscope. Although different concentrations of primary antisera were tested in different animals, the results will be described with reference to the experiments with the lowest concentration, in which no labelling (background) was observed in the major fiber bundles. Therefore the staining in the internal capsule and in the external medullary lamina were considered as a reference to which compare the neuropil staining in the different thalamic nuclei. The intensity of staining is reported according to a relative scale ranging from (0) for the nuclei in which the labelling is similar to the background observed in the above-mentioned fiber bundles, to (5) for the most intensely stained thalamic nuclei. The relative scale [37] reported in Table 1 has

been obtained by matching the evaluation of direct microscopic observation performed by two different observers.

3. Results

The distribution of GluR1, GluR2-3 and GIuR4 immunoreactivity (ir) will be first described in the adult thalamus and then in representative developmental stages of postnatal rats (P2, P7 and P15)- These ages were chosen because in the other stages of postnatal development no significative variations of the ir were observed with respect to these representative animals. In the description of the pattern of thalamic labelling the habenular complex (Hb), that belongs to the epithalamus, will be mentioned as it is present in the figures and it is intensely immunoreactive particularly during development. 3.1. Adult animals

Incubation of tissue sections with anti GtuR antisera generated reproducible patterns of staining within discrete thalamic nuclei. Individual GIuR subunits showed specific and unique patterns of ir within different regions of rat thalamus. The distribution of GIuR ir in adjacent non-thalamic areas (hippocampus, neocortex,

Table 1 Relative scale of the intensity of immunostaining for the three A M P A subunits within different nuclei in adult thalamus and during representative postnatal (P) stages Thalamic Nuclei

GIuR1

GluR2-3

GIuR4

P2

P7

P15

Adult

P2

P7

P15

Adult

P2

P~

Pt5

Adult

Hbm Hbl PV CM Rh Re MD CL Pt Pc Pf AV AM AD VL VPM VPL VM dLG vLG MG PO LP LD Rt

5 3 3 3 4 2 2 3 2.5 3 1 0.5 1 2.5 0.5 0.5 1.5 3 0.5 3 0 1 2.5 3.5 0.5

5 4 3 3 4 2 1.5 3 3 3 2.5 0 1 2.5 0 0 0 3.5 0.5 2.5 0 0.5 2 3 0

5 4.5 3.5 2.5 3.5 2 2 3 3 3 1.5 0 1.5 2.5 0 0 0 3 1.5 3 0 0.5 1 3 0

4 3 3 2.5 3.5 1.5 1.5 2.5 2 2.5 3 0 1 2.5 0 0 0 2.5 1.5 2.5 0 0 0 2 1

2.5 3 2.5 2.5 1.5 2 1.5 2.5 2.5 2.5 1.5 1 1 1 2 2.5 3 2.5 2 2.5 1.5 1.5 2.5 2.5 3

2 2 2 1 2 1.5 1.5 2 1.5 1.5 1 1 1 1 1.5 2.5 2.5 2.5 1.5 2 1.5 1 2.5 2 3

2 2 2.5 1.5 1.5 1.5 1.5 2 2 2 1,5 1,5 2 1 1 0.5 0.5 1.5 1 2.5 1 0.5 1.5 2.5 2

1.5 1.5 2 1.5 1 1 1.5 1,5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1 1 1.5 1.5 2 1 1.5 1.5 1.5 1.5

3 2 1 I 1 1 1 1 1 1 1 1 1 1,5 I 4,5 5 1 2.5 2 1 1 1.5 1 1,5

2.5 2 1 1 l 1 1 1 1 l 1 1 1 1 1 4 4 1 2 1.5 1 1 1 1 1.5

3 2.5 1 1.5 1 2 l 1 1 1 1 1 1 1.5 1 2 2 1 1.5 1.5 1 1 l 1 3

3 2.5 1 t.5 1 1 1,5 1.5 1,5 I I 1 t 2 J I 1 t 1.5 1.5 1 [ t.5 0.5 4

The epithalamic habenular nuclei (Hbl and H b m ) are included as they are p r e s e n t in the figures and are intensely immunoreactive.

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hypothalamus) was comparable to that reported in previous studies using similar antisera [28,37]. The immunolabelling for all antisera was mainly

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observed in the thalamic neuropil, where it was clearly confined within the cytoarchitectonic boundaries of selected thalamic nuclei.

Fig. 2. Coronal sections at the level of midline thalamus of rats at P2 (A) and Pl5 ( B - E ) immunolabelled with the antiserum to GIuR1. A,B: intense labelling is present in the same nuclei at both ages. For abbreviations see attached list. Scale bars = 0.5 mm. C: detail of PV nucleus showing labelling in the neuropil and in several neurons (arrows), one of which (arrowhead) is shown at higher magnification in D. Scale bar = 100 tzm. D: in neurons the immunoreaction product is in the cytoplasm of perikaryon and dendrites, but not in the nucleus. Scale bar = 25 /xm. E: a labelled astrocyte in PV nucleus. Scale bar = 25/xm.

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In sections processed with the GIuR1 antiserum intense ir was observed t h r o u g h o u t all the anteroposterior extent of most of the intralaminar and midline nuclei (Fig. 1A, Table 1). Particularly evident was the labelling also in ventromedial (VM) and anterodorsal ( A D ) nuclei (Table 1). A less intense labelling was present in mediodorsal (MD), laterodorsal (LD) and reuniens nuclei (Re) (Fig. 1A, Table 1). All the sensory-motor relay nuclei and the reticular thalamic nucleus (Rt) were virtually untabelled, with the exception of the ventrolateral geniculate nucleus (vLG) (Fig. 1B, Table 1). In the Hb the staining was particularly intense and present also in several neurons. I m m u n o s tained n e u r o n s were also f o u n d in vLG, r h o m b o i d (Rh), paraventricular (PV) and parafascicular (PD nuclei. In the lateroposterior (LP), P V and dorsolateral geniculate ( d L G ) nuclei a few small stained cells were found with a m o r p h o l o g y similar to that of glial elements.

In sections processed with the G I u R 2 - 3 antiserum ~ more diffuse and far less intense pattern ,ff labelling through the thalamus was observed with respecl to GluR1 (Fig. 1C, Table 1). Using the antibody against the G l u R 4 subunit, Rt was the most intensely stained nucleus in the thalamus (Fig. 1D). Both its neuropil and neurons were labelled (Fig. I E). In all the other nuclei the staining was just above the background with the exception of the H b where several neurons were labelled, particularly in the medial component: (Fig. 1D, Table 1). 3.2. N e o n a t a l a n d y o u n g a n i m a l s

In sections of neonatal and young rats incubated with GIuR1 and G l u R 2 - 3 antisera the distribution pattern of ir in the thalamus was generally similar to that observed in adults (Fig. 2C,D, Table 1). L a b e l l e d neurons were present in the same nuclei as in adult

Fig. 3. Coronal sections at the level of Rt and VB nuclei of rats at P2 (A), P7 (B,C) and P15 (D,E) immunolabelled with the antiserum to GIuR4. A-C: in young animals very intense GIuR4 ir is present in VB and absent in Rt. C is a higher magnification of VB showing that both VPL and VPM, but not Rt, are labelled. D: at P15 only the Rt nucleus is labelled. E: higher magnification of D, showing intensely labelled neurons and neuropil in Rt nucleus. Scale bars: A,B,D = 1 mm; C = 250 tzm: E = 100/xm.

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(Fig. 2C,D) but in addition several labelled glial ceils were observed in LP and dLG at P~0 and Pt5 (Fig. 2E). The intensity of labelling was higher in animals at P2

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and P7 for the GIuR1 subunit and it progressively decreased with age until P15-Pz0 when the adult configuration was achieved. A particularly intense ir for

Fig. 4. Electron micrographs of thalamic nuclei of adult rats immunolabelled with antisera to GIuR1 (A-C) and to GIuR4 (D-G). A,B" PV nucleus; C: Rh nucleus; D - G : Rt nucleus. A,D: with both antisera clumps of immunoreaction product (arrows) are present in the neuronal cytoplasm, but not in the nucleus (N). Lead citrate staining. B,E-G: labelling is also found in dendrites (D) of all sizes, sometimes in areas postsynaptic (arrowheads) to a terminal (T) with round clear vesicles and asymmetric synaptic specialization. Uranyl acetate and lead citrate staining. C: with GIuR1 antiserum, immunoreactivity (arrow) is also present in astrocytic profiles. Uncounterstained section. Scale bars = 0.5 ~m.

238

~il i¸¸¸ ~

R. Spreafico et al. / Det,elopmental Brain Research 82 (1994) 23I 244

j~! !! i~ !

. . . . . .

Fig. 5. Electron micrographs of VB nucleus of rats at P2 ( A - C ) and P7 ( D - F ) immunolabetted with t h e antiserum to GluR4. All the pictures are from uncounterstained sections. A,D: at both ages, clumps of immunoreaction product (arrows) are present in the neuronal cytoplasm, but not in the nucleus (N). B,C,E: immunoreactivity (arrows) is also present in dendritic profiles postsynaptic to immature terminals (T) with round clear vesicles. D: at P7 immunoreactivity (arrow) is found postsynaptic to a more mature terminal (T). A similar adjacent terminal (t) with round clear vesicles and asymmetric specialization (arrowhead) contacts an unlabelled dendrite (d). Scale bars = 0.5 /xm.

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GIuR1 was present at P2 in most of the intralaminar and midline nuclei. Their intensity of labelling was similar to that of the Hb which remained the most intensely stained area throughout all the postnatal period (Fig. 2A,B, Table 1). Only in Pf an increase of GIuR1 ir was observed during the postnatal life. The sensory-motor nuclei were, as in the adult, the less intensely immunoreactive areas within the dorsal thalamus, with the exception of vLG. GluR2-3 ir was more homogeneously distributed in the different thalamic nuclei with respect to GIuR1, as in the adult (Table 1) and its intensity showed a general decrease during postnatal development. The decrease was particularly evident in VB and Rt that at P2 were intensely labelled. With the GluR4 antiserum no significative developmental variations in the pattern of labelling were detected in the dorsal thalamus, with the exception of VB and Rt. A very dense staining was in fact present in both ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei of VB complex from P2 (Fig. 3A) to P7 (Fig. 3B,C), while in Rt the ir was virtually absent during the first postnatal week. After this age, a progressive decrease of labelling in VB was paralleled by an increasing ir in Rt, until P~5 when adult configuration was reached with intense labelling in both neurons and neuropil (Fig. 3D, see also Table 1). 3.3. Electron microscopy

Cellular and subcellular localizations of labellings were visualized by electron microscopic analysis of vibratome sections processed using preembedding immunocytochemistry. At the ultrastructural level GIuR ir was identified by the presence of the electron-dense reaction product of DAB. The subcellular distribution of GIuR1 ir was investigated in PV and vLG nuclei of adult animals. In all the samples, dense patches of immunoreaction product were observed in the cytoplasm and along the cytoplasmic side of the plasma membrane of both neuronal cell bodies and dendrites (Fig. 4A,B). Nuclei were always devoid of reaction product (Fig. 4A) and no labelling was visualized within presynaptic axon terminals. Dense aggregates or clusters of reaction product were also found on or near the plasma membrane, in areas post-synaptic to unlabelled terminals with asymmetric synaptic specialization and round clear vesicles, but also in non-synaptic areas (Fig. 4C). GluR1 ir was also found in astrocytic processes (Fig. 4D). The subcellular distribution of GIuR4 ir was investigated in the Rt nucleus of adult animals and in the VB complex of young (P2 and P7) rats. In Rt of adults, immunolabelling was found in neuronal cell bodies (Fig. 4E) and in dendrites of all sizes (Fig. 4E-G), with a general pattern of distribution similar to that de-

239

scribed for GIuR1, but there was no evidence for GIuR4 ir within glial cells. In VB of young animals, clumps of GIuR4 ir were present in neuronal cell bodies (Fig. 5A, D) and in immature dendrites (Fig. 5B,C,E,F) lacking a well differentiated cytoskeleton. Dense patches of reaction product were often found in areas postsynaptic to immature terminals of small size, containing few round, clear vesicles and no mitochondria (Fig. 5B,C,E). The morphological immaturity of the terminals and the density of reaction product generally prevented unambiguous identification of synaptic specializations. In P7 animals, however, some of the terminals apposing patches of reaction product had more mature features, such as higher number of synaptic vesicles and presence of mitochondria and their postsynaptic specialization more closely resembled that of asymmetric synapses in adults (Fig. 5F).

4. Discussion

The antisera used in the present study have been extensively characterized in adult brain tissues [37]. Bands of GluR1, GluR2-3 and GIuR4 obtained in Western blot analysis preformed on P5 rat forebrain were similar to those found in adults (Wenthold, unpublished observations). Our immunocytochemical results demonstrate that AMPA receptors ir is present in the rat thalamus from P1 to adulthood, in line with several data showing that glutamate is a major excitatory neurotransmitter in this region [41,51] and that AMPA receptors subunits are enriched differentially within different thalamic nuclei and also during postnatal development. However, compared to other brain areas of the rat, such as neocortex and hippocampus [28,32,37], AMPA receptors ir is, on the whole, not very highly expressed in the thalamus, thus suggesting that thalamic neurons may use other types of excitatory amino acids receptors (see below). 4.1. Distribution o f labelling

In the dorsal thalamus of adult rats, intralaminar and midline nuclei were intensely labelled with GluR1 antiserum, but only moderately or lightly with the other two antisera. The Rt nucleus was intensely labelled with the GIuR4 antiserum and faintly with GluR1 and GluR2-3 antisera, whereas most of the sensory relay nuclei and particularly the VB complex (VPL and VPM) showed low immunostaining. Overall, the distribution of AMPA receptors immunolabelling during the postnatal development largely matched that described in adults, with only a few differences. In many thalamic nuclei, the intensity of labelling for both the GluR1 and GluR2-3 subunits

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was transiently higher during the first postnatal week, relative to adult. Moreover, a major difference was found in the distribution of labelling for the GIuR4 subunit. During the first postnatal week, GIuR4 ir was very intense in VPL and VPM nuclei, that in adults are virtually unlabelled and weak in the Rt nucleus, that in adults is instead intensely labelled. This modification of GluR4 labelling pattern between P2 and adults suggests that a rearrangement of the different subunits of AMPA-selective glutamate receptors takes place during postnatal development. Both functional properties and ontogenetic development of VB and Rt are, however, too different to suggest that a temporal coincidence in the different expression of the GluR4 links the two thalamic nuclei. While VB receives inputs from spinal cord and brainstem and from primary somato-sensory cortex, the Rt is primarily recipient of afferents from widespread cortical areas and from all the dorsal thalamic nuclei. In addition, the ir was present throughout all the Rt extension and not restricted to selected sensory recipient areas of the nucleus. In VB the temporal modification of ir pattern for GluR4 is coincident with the ephemeral cellular segmentation and related to high synaptic rearrangement observed in the sensory thalamus during the first postnatal week [23,40]. It is also interesting to note that the pattern of GluR4 labelling resembles the transient expression of AChE in VB, that is very intense in the first postnatal week and then declines [26]. 4.2. Correlation with other immunocytochemical studies Our results in adult animals are in general agreement with those previously reported using the same [37] or similar [28] antisera against AMPA receptors subunits. The lower labelling intensity detected in our samples with respect to that reported by Petralia and Wenthold [37] is likely due to the lower concentration of the antisera used in our study. Test sections incubated with higher concentrations of primary antisera (unpublished observations) gave results comparable to those obtained by Petralia and Wenthold [37] but with a general increase in background staining. The cellular distribution of labelling in the thalamus differs, however, from that observed in other brain regions, such as neocortex and hippocampus [28,37]. In most of the thalamic nuclei in fact the immunostaining is mainly found in the neuropil and only few scattered neurons are stained. An exception is represented by PV and Rt nuclei, where several perikarya are intensely labelled. Astrocytic staining such as that observed in some thalamic nuclei with the GIuR1 antiserum has been reported with other GIuR subunits in different brain areas [28,37] and is in line with the demonstration of

Glu receptors on glial cells obtained with difterent approaches [6]. The subcellular localization of ir is als~ in agreement with previous ultrastructural studies in other brain areas [14,28,37]. At the electron microscopic level the immunoprecipitate was restricted to somatic and dendritic neuronal profiles, with no evidence of labelled synaptic terminals. The intracellular accumulation of reaction product may represent epitope sites associated with various stages of synthesis, transport, assembly and degradation of receptor subunits. A similar possibility has been previously proposed for the immunocytochemical localization of A M P A receptors in other brain areas [37] and also for other types of receptors [21,39,49,52]. The localization of GluR1 and GIuR4 ir to post-synaptic densities of terminals with round clear vesicles and asymmetric specializations is in line with the proposed excitatory nature of these terminals. Moreover, morphologically similar terminals in PV and Rt nuclei were shown to be enriched in glutamate ir [3,51]. The fact that in every microscopical field only a fraction of the asymmetrical synapses contacted labelled post-synaptic profiles may be due to technical problems, such as low penetration of the antibodies in the tissue. In addition, it may indicate that A M P A receptors subunits are selectively distributed at the synaptic level and that other excitatory amino acid receptors play a role in the thalamus. To our knowledge, the distribution of AMPA receptors has not been so far investigated with immunocytochemical methods during development. Immunocytochemistry has, however, demonstrated that also the GABA A receptor is expressed early in the thalamus and that it undergoes a considerable rearrangement during the first postnatal weeks [5]. 4.3. Correlation with binding and in situ hybridization studies In general, our immunocytochemical results are consistent for the most part with previous studies showing that 3H-AMPA binding and mRNA levels for all the subunits are relatively low in the thalamus of adult rats, with respect to other brain regions [22,25,33,36]. Moreover, particularly high levels of GIuR4 gene are found in Rt. In neonatal and young animals, the correlation of our results with radioligand binding and in situ hybridization studies is more difficult, mostly because of the inherent lower sensitivity of the non i m m u n ~ t o chemical methods that hampers the identification of individual thalamic nuclei. On the whole, however, our results are compatible with the low 3H-AMPA binding and the low expression of AMPA receptors mRNAs described across development [22,33,36].

R. Spreafico et al. / Deuelopmental Brain Research 82 (1994) 231-244

Kein~inen et al. [25] reported that the GluR4 gene is the most ubiquitously expressed in the adult thalamus, with particularly high levels in Rt. PellegriniGiampietro et al. [36] did not find detectable signal through the thalamus at any age for GluR1. Conversely GluR2 expression was abundant at P4 and P7 but undetectable at Pt4 and P21 and in adult animals, while a faint appearance of GluR3 mRNA occurred at P14. No mention of GIuR4 expression in the thalamus at any age is present in their paper. It is, however, possible that some thalamic nuclei could not be identified because of the use of sagittal sections. Sommer et al. [48] found that GluR1,2 and 3 mRNAs are prominently expressed and widely distributed in the CNS, but did not mention the thalamus. Also Monyer et al. [33] focused their attention to neocortical, hippocampal and cerebellar areas, showing flip and flop variants in the adult and developing brain. At P1 low levels of both GIuR1 and GIuR2 mRNAs are found, whereas GluR3 flip and GluR4 flop mRNAs are expressed at pronounced levels in some thalamic nuclei. However, in the absence of counterstained Nissl sections it is difficult to identify the thalamic nuclei from their figures. Nevertheless the thalamic areas with highest signals at P~ for GIuR3 flip and GluR4 flop mRNAs could be recognized as Rt nucleus. The fact that either flip or flop variants are not expressed in VB during the first postnatal week and by contrast that at P15 no signal is present in Rt are conflicting with our data and with those reported by Kein~inen et al. [25]. We have no explanation for these discrepances and more detailed data, using high resolution analysis of the thalamus, are needed to elucidate this problem. 4.4. Functional considerations

The differential distribution of GluR subunits in different thalamic nuclei suggests that certain subunits may contribute more than others in mediating postsynaptic responses in distinct neuronal populations. As different combinations of subunits show differing channel kinetics, dose-response characteristics or ion selectivities, the functional differences between these subtypes may be taylored to fit the requirements of the particular neuronal circuit in which they are activated. The GluR2 subunit, for instance, dominates the properties of ion flow and in particular the Ca 2+ permeability of the channel [7,19,20,54]. AMPA receptor heteromeric configurations containing the GIuR2 subunit have low divalent ion permeabilities, whereas those lacking the GIuR2 subunit are Ca 2+ permeable [7,58]. In the thalamus of the adult rat, most of the nuclei show only a moderate or low GIuR2-3 ir. It may be suggested that these thalamic nuclei operate through AMPA receptors with higher Ca 2+ permeability with

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respect to other brain areas where the GluR2 subunit is more highly expressed. Thalamic regions that are particularly enriched in GIuR1 and are also moderately labelled with GluR2-3, include the intralaminar and midline nuclei. These areas are interconnected with non primary cortices and particularly with the limbic system, that show high concentrations of all AMPA receptors. Since these (GluR1-4) receptors mediate fast excitatory postsynaptic potentials, it seems reasonable to assume that their presence and their operating mechanisms are important in the elaboration of signals concerning autonomic, learning and memory functions. These properties, however, do not seem to fulfill the requirements of somatosensory relay nuclei, such as VPL and VPM, where no GluR1 and only sparse GIuR2-3 and GluR4 ir were detected. The Rt nucleus deserves special mention, as it has ontogenetic, hodological as well as physiological properties different from all the other thalamic nuclei and it plays a pivotal role in the reciprocal thalamo-cortical connections [2,12,50]. Interestingly, this nucleus has a moderate GluR2-3 ir and it is the thalamic nucleus with the highest GluR4 ir. Although no conclusive data are available on the functional significance of GluR4 subunit, one can speculate that the peculiar properties of Rt could be related to the presence of this molecule. It is also worth mentioning that two Ca 2+ binding proteins (Calbindin and Parvalbumin) have a topographic distribution in the thalamus that closely matches that of GluR1 and GIuR4 subunits respectively [15]. The generally higher expression of AMPA receptors observed in the first postnatal days seems to be a general phenomenon, as an overshoot of glutamate receptors has been described with other methods in several brain areas [30,31]. It may indicate that glutamate receptors are somehow involved in synapse formation and maturation, as in the case of nicotinic ACh receptor, where the embryonic-type receptor contributes the synapse formation of the neuromuscular junction [24]. Preliminary electron microscopic data on VB synaptogenesis indicate that although the major afferents to this area are already present at birth, the morphological differentiation of their terminals is achieved about two weeks after birth and myelinated fibers are present only from P8 [53]. A further complication in the functional interpretation of the immunocytochemical data comes from the demonstration that AMPA receptors containing either the flip or flop forms or a combination of both subunit types, exhibit different functional properties, although the physiological significance of this remains unknown [48]. It is also interesting to mention that in neonatal rats a differential expression of the flip and flop version of GluR1-4 was found in various thalamic nuclei

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during development [33]. As flip versions of GluRs give greater current responses when expressed in mammalian cells [48], the predominant expression of one of the two versions may have important functional consequences.

4.5. Other types of excitatory amino acids receptors As previously mentioned, the generally low expression of AMPA receptors in the thalamus contrasts with the demonstration of a major role of excitatory amino acids in this region [11,41,51] and suggests that other types of excitatory amino acids receptors may be used by thalamic neurons. This possibility is supported by several lines of evidence. In situ hybridization studies show that Rt contains high levels of mRNA encoding only one of the five high affinity subunits of KA receptors, whereas no signal was detected in VB for KA receptors [59]. High levels of metrabotropic G l u R l a protein and of its mRNA transcripts are expressed in the thalamus, but not in Rt [4,27,47], in line with pharmacological and electrophysiological results [45]. Physiological studies have shown that both NMDA and non-NMDA receptors are involved in the synaptic response of VB neurons to sensory stimulation, depending on the mode of stimulation of the afferent pathway [13,42,43,44]. A distinct distribution of five NMDA receptor subunits in the thalamus has been recently shown by Watanabe et al. [55]. It is interesting to note that, with the exception of one subunits that is highly expressed in all thalamic nuclei, the distribution of the 61_4 subunits is specular to the AMPA subunits in most thalamic nuclei and particularly in VB and Rt. Moreover intense immunostaining for the NMDA receptor subunit NMDA R1 is present in the ventral thalamic nuclear group and in Rt [38]. NMDA and non-NMDA receptors operate with different mechanisms related to ions flux and gating low or fast excitatory responses and the physiological responses are related to the different subunit composition. Thus the data obtained by both in situ and immunocytochemical studies indicate that signal transmission and modulation, specific for each thalamic nucleus, depend on the type of receptors present in this area, further refined by the different subunit composition typical for each receptor in that specific area.

Acknowledgements This work has been supported by 'P. Zorzi Association for Neuroscience' and MURST 40%. Thanks are due to Mrs. M. Denegri for typing the manuscript and to Mrs. M.C. Regondi for the skilful technical assistance.

Abbreviations AD AM AV CL CM dLG Hb Hbl Hbm ir LD LP MD MG Pc Pf PO Pt PV Re Rh Rt VB VL vLG VM VPL VPM

anterodorsal nucleus anteromedial nucleus anteroventral nucleus centrolateral nucleus centromedian nucleus dorsolateral geniculate habenular complex lateral habenular nucleus medial habenular nucleus immunoreactivity laterodorsal nucleus lateroposterior nucleus mediodorsal nucleus medial geniculate nucleus paracentral nucleus parafascicular nucleus posterior thalamic nucleus paratenial nucleus paraventricular nucleus nucleus reuniens rhomboid nucleus reticular nucleus ventrobasal complex ventrolateral nucleus ventrolateral geniculate ventromedial nucleus ventroposterolateral nucleus ventroposteromedial nucleus

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