Colocalization of Glutamate Ionotropic Receptor Subunits in the Human Temporal Neocortex

María C. González-Albo and Javier DeFelipe

Instituto Cajal (CSIC), Madrid, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
{alpha}-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors represent major classes of glutamate receptors (GluR) which play fundamental roles in normal excitatory synaptic activity and, probably, in the etiology of several brain diseases. These receptors are composed of multiple receptor subunit proteins, and the differential expression of these subunits in cortical neurons is considered to be one of the substrates for the functional diversity of cortical excitatory circuitry. In the monkey neocortex, different subpopulations of neurons have been identified on the basis of immunocytochemical colocalization studies using subunit-specific antibodies, but no comparable investigations have been made in the human neocortex. The aim of the present study was to determine quantitatively GluR subunit combinations in the human temporal neocortex by double-labeling immunocyto- chemical experiments. We quantified the neuronal populations expressing different receptor subtypes with fluorescent tags visualizing them with confocal laser microscopy. We studied AMPA, kainate- and NMDA-receptor subunits, using antibodies against GluR1, GluR2, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1 subunits. A high degree of colocalization (93–100%) using combinations of antibodies against GluR2 with GluR2/3, GluR2/3 with GluR2/4, and GluR2 or GluR2/4 with NMDAR1 was found, whereas for other combinations the degree of colocalization varied between 38% and 88%. Some of the percentages reported here are similar to those found in the monkey cortex, whereas others differ considerably. These results emphasize the diversity of excitatory circuits in the human neocortex, and suggest species differences with regard to some of these GluR-mediated circuits.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Ionotropic glutamate receptors (GluR) represent a major class of receptors which play a fundamental role in normal excita- tory synaptic activity and, probably, in the etiology of several brain diseases (Choi, 1988Go; Hicks and Conti, 1996Go; Michaelis, 1998Go; Feldmeyer et al., 1999Go; Conti and Weinberg, 1999Go). They have been subdivided pharmacologically in three main groups: the {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), kainate, and N-methyl-D-aspartate (NMDA) subtypes. These receptors are composed of multimeric assemblies of functionally distinct receptor subunits. For example, AMPA receptors include combinations of four subunits (GluR1–4), while the kainate receptors include subunits GluR5–7 and two kainate-binding proteins (KA1 and KA2). The NMDA receptors are composed of NMDAR1 and NMDAR2A–D subunits. Further- more, the different combinations of these receptor subunits confer distinct functional properties (Monyer et al., 1992Go; Nakanishi, 1992Go; Sommer and Seeburg, 1992Go; Seeburg, 1993Go; Hollmann and Heinemann, 1994Go; Bettler and Mulle, 1995Go). Thus, the postsynaptic effects of glutamate depends on the subtype, and composition, of receptor subunits expressed in the post- synaptic neurons.

Glutamate receptor subunit distribution in the neocortex of a variety of species has been studied with immunocytochemical techniques, at both light and electron microscope levels (Petralia and Wenthold, 1992Go; Huntley et al., 1993Go, 1994aGo, bGo 1997Go; Vickers et al., 1993Go; Conti et al., 1994Go, 1997Go, 1999Go; Hof et al., 1996Go; Muñoz et al., 1999Go). These studies have shown that different subpopulations of neurons, glial cells and synaptic circuits can be identified by the expression of particular subunit com- binations. Moreover, differences may also be found according to cortical layers in a variety of species, including human neocortex.

On the basis of immunocytochemical colocalization studies, at least two distinct populations of NMDAR1-immunoreactive neurons have been identified in the monkey neocortex: one inmunostained for GluR2/3 and GluR5/6/7, and the other for GluR2/3 but not GluR5/6/7 (Vickers et al., 1993Go; Huntley et al., 1994aGo, 1997Go). Four types of neurons have been identified in the rat cortex, on the basis of the expression of differ- ent combinations of AMPA receptor subunits: GluR1/GluR2- expressing neurons, GluR1-negative/GluR2-expressing neurons, GluR1-expressing/GluR2-negative neurons and GluR1-negative/ GluR2-negative neurons (Kondo et al., 1997Go). The differential expression of GluR subunits in cortical neurons is considered to be one of the substrates for the functional diversity of cortical excitatory circuitry (Tsumoto, 1990Go; Huntley et al., 1994bGo; Thomson and Deuchars, 1994Go; Hicks and Conti, 1996Go; Thomson et al., 1997). Presently available data suggest the possibility of species differences in the colocalization, or in the composition, of certain receptor subunits (Meoni et al., 1998Go). However, no data are available on the colocalization of GluR subunits in the human neocortex.

The aim of the present study was to determine quantitatively the neuronal populations expressing different GluR subunit combinations in the human temporal neocortex by double- labeling immunocytochemical experiments. We quantified the differences of receptor subtypes with fluorescent tags and visualized them with confocal laser microscopy. We studied AMPA-, kainate- and NMDA-receptor subunits, including antibodies for GluR1, GluR2, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1 subunits. Preliminary results of this investigation have been published previously (González-Albo and DeFelipe, 1999Go).


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The human tissue was obtained post-operatively (Department of Neuro- surgery, Hospital de la Princesa, Madrid) from three male patients (H39, H57 and H59) suffering pharmaco-resistant right temporal lobe epilepsy (partial complex with secondary generalized tonic–clonic seizures). Tissue specimens were taken from biopsies collected for neuropatho- logical assessment. Informed consent was obtained from all patients prior to surgery. All portions of neocortex used in the present study were from the right anterolateral middle temporal gyri (Brodmann's area 21). As indicated below, and in the results section, this neocortical tissue was considered to be normal on the basis of electrophysiological and histopathological examination. Neurological and neuropsychological examinations revealed that the patients were normal.

Clinical History

The symptoms differed between patients, and are summarized briefly. The patients ranged in age from 25 to 30 years (H39, 25; H57, 27; H59, 30). The frequency of seizures differed between patients: H39 suffered two seizures per month (age of onset: 17 years); H57, daily seizures (age of onset: 13 years); and H59, 6–10 seizures per month (age of onset: 7 years). The neurological and neuropsychological examinations indicated that all patients were normal. All patients were right-handed, had normal IQs (H39, 103; H57, 110; H59, 101) and were left-hemisphere dominant. Magnetic resonance imaging of the brains showed no gross abnormalities in any of the patients. In all cases the epileptogenic region (most interictal epileptiform activity and all the ictal activity) was localized in mesial temporal structures (amygdalo-hippocampal region). Simultaneous intraoperative electrocorticographic recordings from mesial temporal structures and the lateral cortex were performed using grid electrodes (4 and 20 disc electrodes respectively). The cortical samples used in the present study were only from non-spiking (normal activity) regions that were removed to allow access to the amygdalo-hippocampal region. Surgical procedures consisted of the removal of the anterolateral temporal cortex, the amygdala, the anterior portion of the hippocampus (1–3 cm) and adjacent cortex (Olivier, 1992Go). After surgery, all patients were seizure-free except patient H59.

Tissue Preparation

Biopsies were immediately immersed in cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB) for 2–3 h, and then cut into small blocks and postfixed in the same fixative for 24 h at 4°C. Thereafter, the blocks were cryoprotected in 25% sucrose in PB and stored at –20°C in a solution of glycerol, ethylene glycol and PB. The blocks were cut at 100 µm on a Vibratome and the sections pretreated with a solution of ethanol and hydrogen peroxide in PB to remove endogenous peroxidase activity. Some of these sections were processed by immunoperoxidase procedures, whereas other sections were processed for double- immunocytochemical staining using immunofluorescence procedures and confocal laser microscopy. Adjacent sections were stained with thionin.

Single Immunostaining (Immunoperoxidase Procedure)

Sections were preincubated in 3% normal serum (horse or goat) in PB with Triton X-100 (0.25%) for 2 h at room temperature. Sections were then incubated for 24 h at 4°C in the above solution containing one of the following primary polyclonal (rabbit) or monoclonal (mouse) antibodies (Chemicon, Temecula, CA, USA): rabbit anti-GluR1 (1:50) (Petralia and Wenthold, 1992Go); mouse anti-GluR2 (1:200) (Vissavajjhala et al., 1996Go); rabbit anti-GluR2/3 (1:100) (Wenthold et al., 1992Go); mouse anti-GluR2/4 (1:200) (Siegel et al., 1995Go); mouse anti-GluR5/6/7 (1:100) (Huntley et al., 1993Go); and rabbit anti-NMDAR1 (1:200) (Petralia et al., 1994Go). The sections were subsequently washed in PB, incubated in biotinylated goat anti-rabbit IgG or horse anti-mouse (Vector, Burlingame, CA, USA) (diluted 1:200 in PB) for 1 h at room temperature. The sections were then processed by the avidin–biotin–peroxidase method, using the Vectastain ABC immunoperoxidase kit (Vector) and reacted histochemically with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 0.01% hydrogen peroxide. In some cases, DAB–nickel ammonium sulfate (2.5%) was used as the chromogen. Finally, the sections were mounted onto glass slides, dehydrated, cleared with xylene and coverslipped.

In addition, some sections were processed immunocytochemically for parvalbumin and the GABA transporter GAT-1 (see Discussion), using a mouse-anti-parvalbumin antibody (Swant, Bellinzona, Switzerland) and a rabbit anti-GAT-1 antibody (Chemicon), diluted 1:4000 in PB containing 3% normal serum and 0.25% Triton X-100 (24 h of incubation at 4°C). Thereafter, the sections were processed with the ABC kit, and DAB was used as the chromogen as above.

Double Immunocytochemical Staining (Confocal Laser Microscopy)

Sections were double-stained for GluR subunits, using the same rabbit polyclonal and mouse monoclonal antibodies and dilutions as indicated above. The following combinations were studied: GluR1 + GluR2; GluR1 + GluR2/4; GluR1 + GluR5/6/7; GluR2 + GluR2/3; GluR2 + NMDAR1; GluR2/3 + GluR2/4; GluR2/3 + GluR5/6/7; GluR2/4 + NMDAR1; GluR5/6/7 + NMDAR1. Then the sections were incubated for 1 h at room temperature in a solution containing biotinylated goat anti-rabbit IgG (Vector Laboratories), diluted 1:200. The sections were then incubated for 2 h at room temperature in a mixture of Cy5-conjugated goat anti-mouse IgG (diluted 1:200) and Cy2-conjugated streptavidin (diluted 1:1000) (Amersham Life Science, Arlington Heights, IL, USA). For each double-labeled combination, the sections from the three different cortical samples were processed simultaneously.

Afterwards, the sections were mounted and examined in a Leica TCS 4D confocal laser scanning microscope equipped with an argon/krypton mixed-gas laser and a Leitz DMIRB fluorescence microscope. Excitation peaks at 489 and 649 nm were used to visualize Cy2- and Cy5-labeled profiles respectively. Fluorescently labeled Cy2 and Cy5 profiles were recorded through separate channels.

Control sections for GluR subunits were processed as above; however, the primary antibody was replaced with normal serum. Alternatively, the secondary antibody was replaced with an inappropriate secondary antibody. No significant staining was observed under these control conditions.

Quantitative Analysis

Quantitative analysis was performed on double-labeled sections to determine the percentage of colocalization between GluR subunits. In order to determine differences in the percentage of double-labeled cells between supragranular and infragranular layers, we grouped cells in layers II–III and compared them with those in layer VI . A total of 22 squares (11 in layers II–III and 11 in layer VI) of 250 x 250 µm (40x objective) were sampled for each case. Thus, for each double-labeling combination and individual a total area of 1.375 x 106 µm2 was analyzed (0.6875 x 106 µm2 in layers II–III and 0.6875 x 106 µm2 in layer V). Every immunostained somata (all fragments of cell bodies) in the surface of the sections were counted in the confocal images, which consisted of single optical sections with a thickness of ~0.7 µm each. Possible differences in the density of stained cells between cortical layers were analyzed using an unpaired Student's t-test. The percentages of cells that colocalized different receptor subunits were compared, between cortical layers, by hypothesis testing. We assumed a binomial model for the colocalization of a given receptor subunit with any other subunit type.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Histopathology

The standard neuropathological assessment of the removed temporal cortex and mesial temporal structures revealed a normal appearance of the temporal cortex in all cases. However, patient H57 had hippocampal sclerosis. No data were available regarding mesial structures for patients H39 and H59. In addition, adjacent sections to those used for GluR subunit immunocytochemistry were first processed immunocyto- chemically for the calcium-binding protein parvalbumin and for the GABA transporter GAT-1 in order to identify possible alterations in GABAergic circuits (Marco et al., 1996Go). The patterns of parvalbumin and GAT-1 immunostaining were normal in all cortical samples. Furthermore, all cortical samples used in the present study (right anterolateral middle temporal gyri) were from non-spiking regions. Thus, we concluded that this neocortical tissue was normal.

Immunoperoxidase Staining

As previously described in the normal monkey and human neocortex (Huntley et al., 1993Go, 1994aGo, 1997Go; Vickers et al., 1993Go, 1995Go; Hof et al., 1996Go; Kohama and Urbanski, 1997Go; Conti et al., 1999Go; Muñoz et al., 1999Go), immunocytochemistry for GluR1–4, GluR5/6/7 and NMDAR1 revealed numerous stained neurons throughout layers II to VI, a moderate number in the subjacent white matter (Figs 1, 2GoGo), and a few in layer I. Further- more, differences in the density of neurons, distribution by layers, and intensity of staining were found for the different GluR subunits. The highest density of immunoreactive neurons was found for NMDAR1 and the least for GluR1, whereas the densities of neurons immunoreactive for GluR2, GluR2/3, GluR2/4 and GluR5/6/7 were similar (Table 1Go). Sections stained for GluR2/3 (Fig. 1CGo), but not for the other GluR subunits (Figs 1A,B, 2ACGoGo), showed a clear paucity of labeling in layer IV because of the few and lightly stained cells present in this layer. When the proximal dendrites were stained, both pyramidal and non-pyramidal cells were identified (Fig. 3Go).



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Figure 1. GluR subunit immunostaining through the anterolateral middle temporal gyrus (Brodmann's area 21). Low-power photomicrographs from layers I-VI of sections immunocytochemically stained for GluR1 (A), GluR2 (B) and GluR2/3 (C). Scale bar: 200 µm.

 


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Figure 2. GluR subunit immunostaining through the anterolateral middle temporal gyrus (Brodmann's area 21). Low-power photomicrographs from layers I–VI of sections immunocytochemically stained for GluR2/4 (A), GluR5/6/7 (B) and NMDAR1 (C). Scale bar: 200 µm.

 

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Table 1 Density of GluR immunoreactive neurons
 


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Figure 3. Higher-magnification photomicrographs from layer III of Figures 1AC and 2ACGoGo respectively, showing examples of neurons immunoreactive for GluR1 (A), GluR2 (B), GluR2/3 (C), GluR2/4 (D), GluR5/6/7 (E) and NMDAR1 (F). Arrow in (A) indicates an immunostained non-pyramidal neuron. Arrows in (E) indicate immunoreactive apical dendrites. Scale bar: 50 µm.

 
The most intense staining for non-pyramidal cells was ob- served with immunocytochemistry for GluR1 (Fig. 3AGo), whereas for the other GluR subunits non-pyramidal cells were lightly stained. The intensity of staining of pyramidal cells was greater for GluR5/6/7, followed by NMDAR1 and GluR2/3, then by GluR2 and GluR2/4, and finally by GluR1, which showed the lightest stained pyramidal cells (Fig. 3Go). As shown in Table 1Go, GluR-immunoreactive neurons were more abundant in layers II–III than in layer VI. Statistical analyses revealed significant differences in the density of GluR-immunoreactive neurons in the different layers, except for GluR1 and GluR5/6/7.

Colocalization

Double-labeling immunofluorescence experiments and confocal laser microscopy was used to examined the degree of co- localization in layers II/III and layer VI between the following combinations of receptor subunits: GluR1 + GluR2 (Fig. 4A,BGo); GluR1 + GluR2/4 (not shown); GluR1 + GluR5/6/7 (Fig. 5A,BGo); GluR2 + GluR2/3 (Fig. 4C,DGo); GluR2 + NMDAR1 (Fig. 6A,BGo); GluR2/3 + GluR2/4 (Fig. 4E,FGo); GluR2/3 + GluR5/6/7 (Fig. 5C,DGo); GluR2/4 + NMDAR1 (Fig. 6C,DGo); GluR5/6/7 + NMDAR1 (Fig. 6E,FGo). In this material, the patterns of staining for individual receptor subunits was the same as that observed with immuno- peroxidase. However, the staining of proximal dendrites was less intense in comparison with sections stained by im- munoperoxidase technique (compare Fig. 3EGo with Fig. 5DGo). As described by del Río and DeFelipe (del Río and DeFelipe, 1997aGo), neurons showing specific immunofluorescence staining were easily distinguished from those with autofluorescence because immunofluorescence was visualized as homogeneous staining, whereas autofluorescence was characterized by the presence of intense fluorescence clusters of small granules (lipofuscin) within the cells. The autofluorescence granules were distributed in the cytoplasm partly or completely surrounding the nuclei of the cells, in such a way that they appeared as half-moons or doughnut-shaped structures. Furthermore, neurons containing lipofuscin granules were very few in number in the present material.



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Figure 4. Colocalization of GluR subunit immunoreactivities in layer III of the anterolateral middle temporal gyrus (Brodmann's area 21). Pairs of fluorescence photomicrographs (A,B; C,D; E,F) from the same section and microscopic field, showing examples of colocalization of GluR1 and GluR2/3 (A,C,E), with GluR2 and GluR2/4 (B,D,F). Arrows in (A) and (B) indicate neurons that are not double-labeled. Scale bar: 55 µm.

 


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Figure 5. Colocalization of GluR subunit immunoreactivities in layer III of the anterolateral middle temporal gyrus (Brodmann's area 21). Pairs of fluorescence photomicrographs (A,B; C,D) from the same section and microscopic field, showing examples of colocalization of GluR1 and GluR2/3 (A,C), with GluR5/6/7 (B,D). Arrows indicate neurons that are not double-labeled. Scale bar: 55 µm.

 


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Figure 6. Colocalization of GluR subunit immunoreactivities in layer III of the anterolateral middle temporal gyrus (Brodmann's area 21). Pairs of fluorescence photomicrographs (A,B; C,D; E,F) from the same section and microscopic field, showing examples of colocalization of NMDAR1 (A,D,E), with GluR2, GluR2/4 and GluR5/6/7 (B,D,F). Arrows in (E) indicate neurons that are not double-labeled. Scale bar: 55 µm.

 
To estimate the percentage of colocalization between the different combinations (Table 2Go), several hundred neurons immunoreactive for each GluR subunit were examined in layers II/III and VI (Table 3Go). In general, a high percentage (94–99%) of colocalization was found between AMPA subunits, except for the combinations including GluR1 (51–66%). Similarly, the percentage of neurons immunoreactive for GluR2 and GluR2/4 that colocalized NMDAR1 was 100%. The converse relationship was 79 and 88% respectively. The percentage of colocalization of GluR1 and GluR2/3 with GluR5/6/7 was 66 and 71%, whereas for the converse relationship was 49 and 66% respectively. Finally, the percentage of colocalization between NMDAR1 with GluR5/6/7 was only of 38%, whereas between GluR5/6/7 with NMDAR1 was 57%. As shown in Table 2Go, when layers were compared, there were significant differences for many of the combinations, the percentages of colocalization for all combina- tions being ~20% lower in layers II–III than in layer VI.


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Table 2 Percentages of colocalization of glutamate receptor subunits in the human temporal cortex
 

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Table 3 Number of cells examined for each combination
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The main findings of the present study were threefold. First, multiple subpopulations of neurons can be identified in the human neocortex on the basis of the colocalization of GluR1–4, GluR5/6/7 and NMDAR1 subunits. Second, for all combinations of receptor subunits there was always a degree of colocalization, the minimum percentage being 30%. Third, there were differences in the percentages of colocalization of receptor subunits between layers II–III and VI. Furthermore, some of the percentages found in the human cortex were similar to those reported in the monkey and rat cortex, whereas others differed considerably. These results emphasize the diversity of excitatory circuits in the human neocortex and suggest species differences with regard to some of these circuits.

Characteristics of the Human Cortical Samples

The neocortical tissue used in the present study was considered to be normal on the basis of electrophysiological and standard histopathological examination performed in the resected tissue. While alterations of GABAergic circuits are sometimes found in neocortical regions which apparently are normal accoding to standard histopathological techniques in certain epileptic patients, these alterations can be readily identified with immuno- cytochemistry for the calcium-binding protein parvalbumin (Marco et al., 1996Go). Thus, adjacent sections to those used for GluR subunit immunocytochemistry were first processed for parvalbumin and, in addition, for the GABA transporter GAT-1, to verify normal inhibitory circuitry in this material. Immuno- cytochemistry for GAT-1 was used to label GABAergic terminals prominently (Minelli et al., 1995Go; Ribak et al., 1996Go; DeFelipe and González-Albo, 1998Go). The patterns of parvalbumin and GAT-1 immunostaining were normal in all cortical samples, as compared to those found in the normal non-epileptic human neocortex (del Río and DeFelipe, 1997aGo; Conti et al., 1998Go; Gónzalez-Albo and DeFelipe, 1998; Melchitzky et al., 1999Go). Furthermore, the patterns of immunostaining for the various GluR subunits examined were similar to those described in previous reports in the normal human neocortex (Huntley et al., 1994aGo, 1997Go; Vickers et al., 1995Go; Hof et al., 1996Go; Conti et al., 1999Go).

Immunocytochemical Localization of GluR Subunits

Immunocytochemistry for AMPA, kainate and NMDA receptor subunits revealed the existence of numerous labeled neurons in layers II–VI of the human neocortex (Huntley et al., 1994aGo, 1997Go; Vickers et al., 1995Go; Hof et al., 1996Go; Conti et al., 1999Go). This is in line with electrophysiological studies performed in human neocortical tissue maintained in vitro that have been removed during surgery of epilepsy, showing the involvement of both NMDA and non-NMDA receptors in excitatory synaptic activities mediated by excitatory amino acids (Hwa and Avoli, 1991Go, 1992Go).

In the rat and primate neocortex (including human), neurons immunoreactive for GluR subunits include both pyramidal cells and interneurons (Huntley et al., 1993Go, 1994aGo, bGo, 1997Go; Vickers et al., 1993Go; Conti et al., 1994Go, 1999Go; Vissavajjhala et al., 1996Go; Petralia et al., 1997Go; Conti and Weinberg, 1999Go; Muñoz et al., 1999Go). However, it seems clear that not all pyramidal cells and interneurons were labeled for any of the receptor subunits tested (Conti et al., 1994Go; Huntley et al., 1997Go; Kohama and Urbanski, 1997Go). The quantitative analyses performed in the present study indicate that the greatest subpopulation of neurons was represented by those neurons immunoreactive for NMDAR1, followed by neurons immunoreactive for GluR2–4 and GluR5/6/7, and then by neurons stained for GluR1. Furthermore, with the exception of GluR1 and of GluR5/6/7 all GluR- immunoreactive neurons were significantly more abundant in layers II–III than in layer VI. The present study also suggests that supragranular and infragranular circuits in the human cortex may differ with regards to the expression of some GluR subunits. This heterogeneity of excitatory cortical circuits has been proved more directly in the monkey cortex by experiments using double-labeling immunocytochemical methods, or combining tract-tracing and immunocytochemical techniques. In these studies it has been shown that different subpopulations of GABAergic interneurons and corticocortically projecting pyramidal cells located in different layers express particular GluR subunits (Huntley et al., 1994bGo).

Degree of Colocalization

The percentage of colocalization between the AMPA subunits GluR2–4 and between these AMPA subunits and NMDAR1 was nearly 100%. However, not all NMDAR1 neurons (79–88%) colocalized GluR2–4. The percentage of colocalization of GluR1 and GluR2/3 with GluR5/6/7 was moderate (66 and 71% respectively). Percentages of colocalization were lower for the converse comparisons (49 and 66% respectively). A moderate percentage of colocalization was also found between NMDAR1 and GluR5/6/7 (38 and 57% for the converse relationship). Thus, multiple subpopulations of neurons were identified by the different coexpression of GluR subunits. Furthermore, it is likely that these subpopulations are proportionally differently repres- ented in supragranular (layers II and III) and infragranular (layer VI) layers, as the percentages of colocalization of the majority of combinations studied were significantly different in layers II–III and VI.

It is unknown whether the coexpression of two or more GluR subunits in individual neocortical neurons indicates that these subunits are colocalized at the level of each single excitatory synaptic contact that the cell receives. Alternatively, there may be a particular spatial distribution of synaptic contacts within the dendritic arborization containing different GluR subunits, as reported in the hippocampus (Siegel et al., 1995Go). Further studies using a combination of postembedding gold electron microscope immunocytochemistry for various GluR subunits (Kharazia et al., 1996Go) and intracellular injection (Buhl and Schlote, 1987Go) will be required to reveal the degree of co- localization at the level of the synapse. By combining such methods, future studies will be able to address this crucial question on the excitatory synaptic circuitry. Moreover, the spatial and combinatorial patterns of GluR subunits can thus be studied.

Species Comparisons

There are no other studies on the colocalization of GluR subunits in the human neocortex to compare with the present results. However, colocalization studies of GluR subunits have been performed in the rat and monkey cortex (Vickers et al., 1993Go; Huntley et al., 1994aGo, 1997Go; Kondo et al., 1997Go; Petralia et al., 1997Go). The degree of colocalization reported here for certain combinations of receptor subunits was similar to those reported in the monkey and rat, but other combinations differed considerably. For example, by combining nonradioactive in situ hybridization with immunocytochemistry in the somatosensory cortex of the rat, Kondo et al. (Kondo et al., 1997Go) found that the majority of neurons immunoreactive for GluR2/3 were labeled with GluR2 antisense probe (Petralia et al., 1997Go). Similarly, our results showed a high degree of colocalization (94%) using antibodies against GluR2 and GluR2/3. Moreover, the percentage of colocalization of GluR2/3 with GluR5/6/7 was practically the same in the monkey [69.9% (Vickers et al. 1993Go)] and human cortex (70.6%; present results).

However, the results reported in the rat by Kondo and co-workers (Kondo et al., 1997Go) regarding the combination of GluR2 and GluR1 in layers II/III and layer VI (10 and 4.5% respectively) differ from those reported here (41 and 52% respectively). Furthermore, there are also differences in the percentage of colocalization for some combinations in the human and monkey cortex. For example, 95–100% of neurons immunoreactive for GluR5/6/7 in a variety of cortical areas (including the temporal cortex) of the monkey are also immunoreactive for GluR2/3 and NMDAR1 (Vickers et al., 1993Go; Huntley et al., 1994aGo, 1997Go), while the percentage of co- localization for these GluR subunits was lower in the human (66 and 57% respectively). Thus, the present results indicate a greater heterogeneity of neurons with regard to their content in GluR subunits, as compared to the monkey cortex.

These differences are unlikely to be due to technical factors as the cortical tissue used in the present study was immediately immersed in the fixative and the preservation of the tissue has been shown to be comparable to that obtained with experi- mental animals (del Río and DeFelipe, 1997bGo). We conclude that cortical excitatory circuitry in the human cortex shows greater diversity of receptor subunit colocalization than in other species. Therefore, it is likely that the functional diversity is more complex in the human cortex. Finally, it remains to be determined whether this diversity is related to the ability of individual neurons in humans to integrate a larger number of inputs than those in the monkey. For example, pyramidal cell dendritic arborizations in the temporal lobe of humans (G. Elston and J. DeFelipe, unpublished observations) are larger and more spinous than those reported in the monkey (Elston et al., 1999aGo,bGo).


    Notes
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The authors are grateful to Guy Elston, George Huntley and Alberto Muñoz for their helpful comments on the manuscript, and Azucena Ortiz for technical assistence. We are indebted to Concha Bailón for assistance with the confocal laser scanning microscope. This work was supported by FIS grant 98/ 0933 and Comunidad de Madrid grant 08.5/ 0014/ 1997.

Address correspondence to Dr Javier DeFelipe, Instituto Cajal (CSIC), Avenida Dr Arce 37, 28002 Madrid, Spain. Email: defelipe{at}cajal.csic.es.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
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