Abstract

Immunocytochemical techniques were used to examine the distribution of neurons immunoreactive (-ir) for nitric oxide synthase (nNOS), somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), calbindin (CB) and calretinin (CR), in the inferotemporal gyrus (Brodmann's area 21) of the human neocortex. Neurons that colocalized either nNOS or SOM with PV, CB or CR were also identified by double-labeling techniques. Furthermore, glutamate receptor subunit profiles (GluR1, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1) were also determined for these cells. The number and distribution of cells containing nNOS, SOM, NPY, PV, CB or CR differed for each antigen. In addition, distinct subpopulations of neurons displayed different degrees of colocalization of these antigens depending on which antigens were compared. Moreover, cells that contained nNOS, SOM, NPY, PV, CB or CR expressed different receptor subunit profiles. These results show that specific subpopulations of neurochemically identified nonpyramidal cells may be activated via different receptor subtypes. As these different subpopulations of cells project to specific regions of pyramidal cells, facilitation of subsets of these cells via different receptor subunits may activate different inhibitory circuits. Thus, various distinct, but overlapping, inhibitory circuits may act in concert in the modulation of normal cortical function, plasticity and disease.

Introduction

Approximately 15–25% of all cortical neurons are of the nonpyramidal type. Most of these cells contain GABA, are distinguished by their morphologies, project to specific post-synaptic targets, and express a number of different neurotransmitters, neuropeptides and calcium-binding proteins [for reviews, see (Houser et al., 1984; Hendry, 1987; DeFelipe, 1993, 1997; Somogyi et al., 1998)]. Furthermore, these cells have been shown to express different receptors, including both GABA and glutamate receptor subunits. While many studies have focused on the receptor profiles of pyramidal cells (Huntley et al., 1994, 1997; Muñoz et al., 1999; González-Albo and DeFelipe, 2000), few studies have focused on receptor subunit expression in nonpyramidal cells (Kharazia et al., 1996; Standaert et al., 1996; Vissavajjhala et al., 1996; Kondo et al., 1997, 2000). In addition, what is known about receptor subunit expression in nonpyramidal neurons is derived from studies of primary sensory areas of the monkey, cat and rat [for reviews, see (Jones, 1993; Jones et al., 1994; Somogyi et al., 1998)]. As primary sensory areas differ markedly from non-primary areas at the molecular (Lewis et al., 1981; Nelson et al., 1987; Rakic et al., 1988; Huntley et al., 1994, 1997; Muñoz et al., 1999), cellular (Hendry and Jones, 1983; Vercelli and Innocenti, 1993; Elston and Rosa, 1997, 1998, 2000; Peters et al., 1997; Elston et al., 1999a,b, 2001; Elston, 2000; Jacobs et al., 2001) and systems (Kritzer et al., 1992; Lund et al., 1993; Hof and Morrison, 1995; DeFelipe et al., 1999; Kondo et al., 1999; Lewis et al., 2001) levels, and aspects of cortical circuitry differ markedly between homologous cortical areas in different species (Nimchinsky et al., 1999; Hof et al., 2000; Preuss, 2000; Elston et al., 2001), patterns of immunoreactivity revealed in a particular cortical area cannot be applied to all areas. Similarly, patterns revealed in one species cannot be extrapolated to all species.

Consequently, relatively little is known about the neurochemical content and receptor subunit profiles of human neocortical neurons. Cortical tissue removed during the course of neurosurgery for the treatment of patients with intractable epilepsy represents an excellent opportunity for research on the human brain because resected tissue can be immersed immediately in the fixative and post-mortem factors do not apply. As the temporal lobe is the major cortical region affected in patients suffering partial epileptic seizures (Wieser et al., 1993), and changes in inhibitory nonpyramidal cell circuitry may underlie the development and maintenance of seizure activity (DeFelipe, 1999), it is fundamental that aspects of normal circuitry be determined to better understand the disease. In the present study, we determined the morphology and distribution of neurons that contain nitric oxide synthase (nNOS), somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), calbindin (CB) or calretinin (CR) in the human temporal lobe neocortex obtained following surgery. Moreover, we determined the degree of colocalization of some of these antigens in the different cells. In addition, we quantified the expression of the glutamate receptor subunits GluR1, GluR2/3, GluR2/4, GluR5/ 6/7 and NMDAR1 in these neurochemically characterized subtypes of nonpyramidal neurons. The different morphological and neurochemical types of nonpyramidal cells examined were found to be characterized by different glutamate receptor subunit profiles.

Materials and Methods

Patients and Ethics

Human tissue was obtained following surgical removal from three male patients (25–30 years old) suffering pharmaco-resistant right temporal lobe epilepsy (partial complex with secondary generalized tonic–clonic seizures). Surgical treatment required anterior temporal cortical resection, including the amygdala, the anterior portion of the hippocampus (1–3 cm) and adjacent cortex. All portions of neocortex used in the present study were from the right anterolateral middle temporal gyri [Brodmann's area 21 (Brodmann, 1907)]. In all cases, the epileptic focus was localized by video-EEG recording from bilateral foramen ovale electrodes. Furthermore, the epileptogenic regions were identified by subdural recordings with a 20-electrode grid (lateral neocortex) and a four-electrode strip (uncus and parahippocampal gyrus) at the time of surgery. Only tissue that showed no abnormal spiking, and was characterized by normal ECoG activity, was used in the present study. Histopathological examination of tissue used in the present study revealed no abnormalities. Although we cannot rule out the possibility that the tissue used may have been indirectly influenced by epileptogenic activity, we are reasonably confident that the results presented here represent the normal condition. Informed consent was obtained from all patients before surgery.

Tissue Preparation

Resected tissue was immediately immersed in cold 4% paraformaldehyde (Merck, Darmstadt, Germany; UN2213) in 0.1 M phosphate buffer, pH 7.4 (PB), for 2–3 h, and then cut into small blocks and post-fixed 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 30% glycerol (Panreac, Barcelona, Spain; 141339) and 30% ethylene glycol (Panreac, 141316) in PB. The blocks were cut at 100 μm on a Vibratome and the sections pre-treated with a solution of 50% ethanol and 1% hydrogen peroxide (Merck, 107210) 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 fluorescent tags. Adjacent sections were stained with thionin to reveal laminar boundaries.

Single Labeling for nNOS, SOM, NPY, PV, CB or CR

Sections were pre-incubated in a stock solution [3% normal goat or horse serum (Vector Lab, Burlingame, CA; S-1000 and S-2000, respectively) and 0.25% Triton X-100 (Merck, 86031000) in PB] 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 (see Table 1 for a list of primary antibodies and dilutions): rabbit anti-nNOS, rabbit anti-SOM, rabbit anti-NPY, mouse anti-PV, rabbit anti-PV, mouse anti-CB, rabbit anti-CB, mouse anti-CR and rabbit anti-CR. The sections were subsequently washed in PB, incubated in a species-specific biotinylated antibody (Vector BA1000, BA-2000) (diluted 1:200 in PB) for 1 h at room temperature, and processed by the avidin–biotin–peroxidase method (Vectastain ABC immunoperoxidase kit: Vector, PK6100). DAB (3,3′-diaminobenzidine tetrahydrochloride: Sigma, St Louis, MO; D-5905) was used as the chromogen. Sections were mounted on glass slides, dehydrated, cleared in xylene and coverslipped.

Double-labeling for nNOS, SOM, NPY, PV, CB or CR

Sections processed for double immunocytochemical staining were pre-incubated in stock solution [3% normal serum (horse or goat: Vector; S-100 and S-200, respectively), 0.25% Triton X-100 in PB] for 2 h at room temperature, and then with primary antibodies for 48 h at 4°C (see Table 1 for a list of primary antibodies and dilutions). Sections were then washed three times in PB and incubated in a solution containing biotinylated goat anti-rabbit IgG or horse anti-mouse IgG (1:200 in PB) (BA-1000 and BA-2000, respectively: Vector). The sections were then incubated in a mixture of Cy5-conjugated goat anti-mouse IgG (1:200 in PB) and Cy2-conjugated streptavidin (1:1000 in PB; Amersham Life Science, Arlington Heights, IL) or Alexa Fluor 594 conjugated goat anti-rabbit and Alexa Fluor 488-conjugated streptavidin (1:2000 in PB; Molecular Probes, OR). Thereafter, the sections were mounted in 50% glycerol and PB. The following combinations were studied: nNOS with PV, CB and CR; SOM with PV, CB and CR.

Determination of Receptor Subunit Expression in Neurochemically Identified Cells

Sections were pre-incubated in stock solution for 2 h at room temperature, then with primary antibodies for 24 h at 4°C (see Table 1 for a list of primary antibodies and dilutions). Sections were then washed three times in phosphate buffer and incubated in a solution containing biotinylated goat anti-rabbit IgG or horse anti-mouse IgG (1:200 in PB) (Vector). The sections were then incubated in a mixture of Cy5-conjugated goat anti-mouse IgG (1:200 in PB) and Cy2-conjugated streptavidin (1:1000 in PB; Amersham Life Science) or Alexa Fluor 594-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated streptavidin (1:2000 in PB; Molecular Probes). Thereafter, the sections were mounted in 50% glycerol and PB. The following combinations were studied: GluR1 with PV, M-CB (CB raised in mouse) and CR; GluR2/3 with PV, M-CB and CR; GluR2/4 with nNOS, SOM, NPY, PV and CR; GluR5/6/7 with nNOS, SOM, NPY, PV and CR; NMDAR1 with PV, M-CB and CR.

Controls

Control sections were processed as per the above protocols; however, the primary antibody was replaced with normal serum. Alternatively, the secondary antibody was replaced with an inappropriate secondary antibody. No specific labeling was observed under these control conditions. In all cases, sections were processed as a single batch (for each combination of antibodies).

Microscopy, Quantification and Statistical Analyses

Colocalization of nNOS and SOM with Calcium-binding Proteins and Quantitative Analysis

Double-labeled sections for nNOS with PV, CB and CR or SOM with PV, CB and CR were studied with the aid of an Olympus BX50WI microscope equipped with a mercury fluorescence light source. Images were captured at a single focal depth in the superficial portion of the section, to which both primary antibodies had penetrated, with Micrografx Picture Publisher (Micrografx, Dallas, TX). Twenty images (×40 objective) were captured for each combination of antibodies and for each of the three cases examined in layers II and III from different sections processed. A composite image was composed by superimposing the two images captured for each fluorophore, using features of Micrografx Picture Publisher, and the number of cells that colocalized both antigens was determined.

Glutamate Receptor Profiles of Cells Containing nNOS, SOM, NPY, PV, CB and CR

Double-labeled sections were examined with a Leica TCS 4D confocal laser scanning workstation equipped with an argon–krypton mixed gas laser and a Leitz DMIRB fluorescence microscope. Fluorescent-labeled profiles were imaged through separate channels. Excitation peaks at 489 and 649 nm were used to visualize Cy2- and Cy5-labeled profiles, respectively. Excitation peaks for Alexa Fluor 594 and 488 were 585 nm and 491 nm, respectively. Images of 0.7 μm focal depth were taken of tissue processed from each of the three cases, using Scanware software (Leica, Cambridge, UK). In all instances, we initially focused beyond the depth at which data were collected to establish that the antibodies had penetrated to the level studied.

Eleven quadrants of 250 × 250 μm (×40 objective) were sampled in images containing layers II and III for each double-labeling combination, and individual case. Every immunostained somata (including fragments of cell bodies) was counted. A composite image was made by superimposing the two images captured for each fluorophore, using features of Micrografx Picture Publisher, and the number of cells that colocalized both antigens was determined.

Statistical Analyses

Statistical comparisons of the densities of labeled cells were performed by ANOVA. When the ANOVA revealed a significant difference in the data set, post hoc Tamhane tests were performed. All statistical analyses were performed with the aid of the SPSS statistical package (SPSS Inc., Chicago, IL).

Results

Specificity of Antibodies Against Calcium-binding Proteins

In some cases (PV, CR or CB), we used antibodies raised in two different species. For antibodies to both PV and CR, we saw no appreciable differences in the number and distribution of labeled cells (Fig. 1). However, in the case of CB, we found that the number of cells revealed in any given preparation depended on the antibody used: the one raised in the mouse labeled fewer cells than the one raised in the rabbit (0.71 ± 0.41 neurons per 10 000 μm2 and 1.47 ± 0.54 neurons per 10 000 μm2, respectively) (Fig. 1). Moreover, many of the additional cells labeled with the rabbit antibody were of the pyramidal type. Thus, in the following sections, CB-ir cells include those labeled with antibodies raised either the rabbit or mouse, unless otherwise specified (M-CB and R-CB for those raised in mouse and rabbit, respectively). No distinction is made for PV-ir or CR-ir neurons.

Patterns of nNOS, SOM, NPY, PV, CB and CR Expression

Labeled cells displayed a variety of forms, their sizes ranging from 8 to 25 μm in diameter. Cells labeled for nNOS and SOM were heterogeneous, with fusiform, bipolar and multipolar morphologies being represented (Fig. 2A,B). In the cases of PV-ir and M-CB-ir cells (Figs 2D and 3A), the most common morphology was multipolar with a rounded soma, whereas NPY-ir and CR-ir cells included fusiform and bipolar forms (Figs 2C and 3B, respectively). Many CB-ir cells were of small soma size, whereas many PV-ir cells were larger.

Cells labeled for nNOS, SOM, NPY, PV, CB and CR were distributed throughout layers II–VI (Fig. 4). No, or very few, nNOS-ir, SOM-ir, NPY-ir or PV-ir cells were seen in layer I. In contrast, CB-ir and CR-ir cells were present throughout layer I. nNOS-ir and NPY-ir neurons were relatively homogeneously distributed in layers II–VI, whereas SOM-ir, PV-ir, CB-ir and CR-ir neurons were more prevalent in layers II and III. The general pattern of labeling is summarized in Figure 4. The densities of cells immunopositive for the different antigens were quantified by counting the number of labeled cells within quadrants (250 × 250 μm) sampled from layers II and III. As can be seen from Figure 5, the density of cells labeled for nNOS, SOM, NPY, PV, CB and CR differed markedly for different antigens. Cells immunostained for R-CB were the most numerous (1.47 cells per 104 μm2), followed by CR-ir cells (1.14 cells per 104 μm2), M-CB (0.71 cells per 104 μm2), nNOS (0.57 cells per 104 μm2), SOM (0.40 cells per 104 μm2), PV (0.35 cells per 104 μm2) and NPY (0.17 cells per 104 μm2). An ANOVA revealed a significant difference in the densities of cells labeled for the different antigens (F6 = 1.44 × 105, P < 0.05). Post hoc Tamhane tests revealed that 19 of the 21 possible pairwise comparisons (density of labeled cells containing nNOS, SOM, NPY, PV, CB or CR) were significantly different (see Fig. 5).

nNOS-ir and SOM-ir Colocalization with Calcium-binding Proteins

Several hundred layer II/III neurons labeled for each antigen were analyzed to estimate the percentage of colocalization between different antigens in individual neurons (Table 2 and Fig. 6). As seen in Table 2, the degrees of colocalization between different antigens varied markedly according to the pairs of antigens studied. The percentages of nNOS-ir and SOM-ir neurons labeled for M-CB were moderate (40%) or high (81%), respectively. Furthermore, in the case of nNOS, we distinguished two populations of neurons: one characterized by large cell bodies (20–25 μm in diameter) that did not colocalize M-CB, and the other formed by small cell bodies (8–10 μm in diameter) that often colocalized M-CB. In contrast, nNOS-ir and SOM-ir cells did not colocalize PV or CR. The percentages of M-CB-ir cells labeled for nNOS and SOM were relatively moderate (24 and 44%, respectively).

Glutamate Receptor Subunit Expression in Neurochemically Characterized Cells

Both pyramidal and nonpyramidal cells expressed the GluR1, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1 receptor subunits. Moreover, these subunits were selectively expressed in different neurochemically characterized cells (Fig. 7). The degree of expression of the different GluR subunits was quantified in a total of 11 303 cells located within layers II and III (Tables 3 and 4). As shown in Table 3, all immunostained neurons, except nNOS-ir cells, showed high degrees of expression of the GluR5/6/7 subunits (82–96%). Expression of other GluR subunits was, however, variable. For example, a moderate number of nNOS-ir neurons expressed the GluR2/4 subunits (34%). High percentages of SOM-ir and NPY-ir neurons expressed the GluR2/4 subunits (74% and 100%, respectively). Most PV-ir and M-CB-ir neurons expressed the GluR1 subunit (93 and 88%, respectively). Furthermore, high or moderate percentages of PV-ir neurons expressed the NMDAR1 (81%) and GluR2/4 (61%) subunits, whereas fewer M-CB-ir neurons expressed the NMDAR1 subunit (39%). High proportions of CR-ir neurons expressed the GluR2/4 and GluR5/6/7 subunits (94–96%), whereas fewer cells expressed the GluR1 subunit (20%). A small population of PV-ir cells expressed the GluR2/3 subunits (11%). No M-CB-ir or CR-ir cells were found to express the GluR2/3 subunits.

We also made the reverse comparisons to determine the percentage of cells that expressed any given receptor subunit which also contained either nNOS, SOM, NPY, PV, M-CB or CR (Table 4). Forty-four percent of cells that expressed the GluR1 receptor subunit also contained M-CB. A similar percentage of cells expressing the GluR5/6/7 receptor subunits contained CR (42.5%). Forty-one percent of cells that expressed the GluR2/4 receptor subunits contained CR. Twenty-nine percent of cells that expressed the GluR1 subunit contained PV. Twenty-eight percent of cells that expressed the GluR5/6/7 subunits contained PV. Other comparisons revealed lower proportions of cells that expressed the given receptor subunit contained nNOS, SOM, NPY, PV, M-CB or CR. No cells that expressed GluR2/3 were labeled for M-CB or CR.

As seen in Table 3, some of the neurochemically identified cells necessarily coexpressed different receptor subunits. The extent of coexpression depended on the type of nonpyramidal cell and receptor subunit combinations studied. For example, ~93% of PV-ir cells expressed GluR1, 61% expressed GluR2/4, and 94% expressed GluR5/6/7. Thus, as at least 54% PV-ir cells that express GluR2/4 must also express GluR1. Moreover, as many as 55% of PV-ir cells that express GluR2/4 also express GluR5/6/7. CR-ir cells also coexpressed different receptor subunits; for example, at least 15% of CR-ir cells that expressed GluR2/4 coexpressed GluR1 (Table 3).

In some instances it was possible to identify PV-ir, CB-ir and CR-ir cells based on their morphology. Large PV-ir multipolar cells which are likely to be large basket cells (Houser et al., 1984), and CR-ir bipolar cells were easily identified: large basket cells had large fusiform or multiangular somata that gave rise to several dendrites, whereas bipolar cells had round or fusiform somata that gave rise two vertically oriented primary dendrites. Thus, in some cases we were able to determine unequivocally which receptor subunits they may express. That is not to say, however, that all of these particular cells necessarily expressed the receptor subunit. Examples were observed of PV-ir large presumptive basket cells that expressed the GluR1, GluR2/4 and NMDAR1 subunits (Fig. 8A,B). In addition, examples of CR-ir bipolar cells were seen to express the GluR2/4 subunits (Fig. 8C,D).

Discussion

By using double-labeling immunocytochemical techniques and confocal laser microscopy, we have shown that distinct subpopulations of neurons that contain nNOS, SOM, NPY, PV, CB or CR colocalize these different substances to varying degrees, depending on the antigens compared. In addition, nNOS, SOM, NPY, PV, CB or CR-ir cells express different AMPA, kainate and NMDA receptors. These results reveal that, not only do cortical nonpyramidal cells show great diversity in their morphology, chemical content and projections, but can be further classified according to which glutamate receptor subunits they express.

Neurochemical Profiles of Nonpyramidal Neurons

The present results confirm, and extend, previous findings of neurochemical profiles of cortical nonpyramidal neurons in the human cortex. In agreement with previous studies (Vincent et al., 1982; Chan-Palay et al., 1985; Kowal and Beal 1988; Hayes et al., 1991; Hornung et al., 1992; del Río and DeFelipe, 1994, 1996; Uylings and Delalle, 1997), we found that all cells that were immunoreactive for nNOS, SOM, NPY, PV and CR were of the nonpyramidal type. We also found that the density of neurons immunoreactive for any given antigen was considerably different from those of cells labeled for the other antigens. For example, the density of CR-ir neurons was 61% higher than that of nNOS-ir cells and 88% higher than that of NPY-ir cells. Thus, each of these subpopulations of cells may contribute differently to inhibitory circuitry. In many cases, it was possible to identify nonpyramidal cell types, based on the labeling. For example, we found that nonpyramidal neurons labeled for PV included chandelier cells and large basket cells, CR-ir cells included bipolar and double bouquet cells, and CB-ir cells included double bouquet cells [see also (del Río and DeFelipe, 1996, 1997a,del Río and DeFelipe, b,c)]. As previously reported (DeFelipe et al., 1990; Hayes and Lewis, 1992; Kondo et al., 1994, 1999), we also found that pyramidal cells were CB-ir, the number of which depended on the type of antibody used.

By employing double-labeling methods, we were further able to characterize the neurochemical profiles of these cell types. We found no evidence that chandelier cells colocalize nNOS or SOM. Nor did we find evidence that CR-ir bipolar cells colocalize nNOS or SOM. Small populations of M-CB-ir neurons colocalized nNOS and SOM (24% and 44%, respectively). However, most double bouquet cells colocalized both CB and CR (del Rio and DeFelipe, 1997b). Thus, as we found that CR did not colocalize with nNOS or SOM, it is probable that most double bouquet cells do not contain nNOS or SOM. Furthermore, as in a recent study on the monkey cortex (Smiley et al., 2000), we also found that large cell-bodied nNOS-ir neurons did not colocalize M-CB, whereas a subpopulation of small cell-bodied nNOS-ir cells did. Thus, cells that contain nNOS or SOM are likely to comprise different subpopulations of nonpyramidal cells.

GluR Receptor Subunit Profiles of Nonpyramidal Cells

Comparison of the present results with those of the macaque prefrontal cortex (Vickers et al., 1993) and rat somatosensory cortex (Vissavajjhala et al., 1996) reveal some consistencies as well as some marked differences in the receptor subunit profiles of neurochemically characterized cells. However, in making such comparisons it should be noted that glutamate receptors have been shown to vary in their density and laminar distribution between different sensory areas (Huntley et al., 1994; Xu et al., 1997; Muñoz et al., 1999), as has the number and distribution of neurochemically characterized cells, which express specific receptor subunits (Huntley et al., 1994, 1997). For example, in the macaque, PV-ir cells that colocalize the NMDAR1 subunit are more numerous in primary motor and sensory areas (80–90%) than in association cortex (prefrontal area 46 and inferotemporal area TE1; 45–60%), and the percentage of CB-ir cells that express this receptor subunit differs markedly between prefrontal area 46 (24%) and area TE1 (82%) (Huntley et al., 1994). Thus, the extent to which nonpyramidal neuron receptor subunit profiles of cells in the human temporal lobe (area 21) differ from those in other species can only be determined by the comparison of data taken from homologous cortical areas across species. The study of different species will also be necessary to determine whether nonpyramidal cells in humans show the greatest diversity in glutamatergic receptor subunit profiles, as previously suggested for pyramidal cells (González-Albo and DeFelipe, 2000).

Implications for Cortical Processing

Laminar Specializations

Nonpyramidal neurons in the different cortical layers are characterized by different physiological properties, and form synapses with specific connectional patterns between themselves and with pyramidal cells (Hendry et al., 1984; Condé et al., 1994; Gabbott and Bacon, 1996a,b; Kawaguchi and Kubota, 1996, 1997, 1998; DeFelipe, 1997; del Río and DeFelipe, 1997c; Meskenaite, 1997; Thomson and Deuchars, 1997; Tamás et al., 1998; DeFelipe et al., 1999; Gonchar and Burkhalter, 1999; Gupta et al., 2000). Furthermore, pyramidal cells located in different layers show unique morphological (Feldman, 1984; White, 1989; Larkman et al., 1991a,b; DeFelipe and Fariñas, 1992; Preuss et al., 1997; Elston, 2001) [see also (Elston and Rosa, 2000)], connectional (Jones, 1984) and physiological (Markram, 1997; Thomson and Deuchars, 1997) characteristics. Thus, the laminar distribution of nonpyramidal neurons that contain nNOS, SOM, NPY, PV, CB or CR, and that colocalize these different neurochemicals, may be instrumental in the modulation of activity throughout the cortical layers. That is, as nonpyramidal neurons can be distinguished by their synaptic connections with pyramidal and nonpyramidal cells, as well as their neurochemical content, cells in the different layers may be involved in the modulation of specific inhibitory ‘subcircuits’. Such specificity may also be important in determining the varying degrees to which cells in different cortical layers exhibit plastic changes following manipulation of inputs (Diamond et al., 1994; Trachtenberg et al., 2000).

Calcium Kinetics

The present results suggest a correlation between the receptor subunit composition of different populations of nonpyramidal cells and their targets. For example, cells immunopositive for CB and CR do not to express the GluR2 receptor subunit [see also (Kondo et al., 1997, 2000); but see (Vickers et al., 1993; Vissavajjhala et al., 1996)]. On the other hand, PV-ir cells that do not colocalize CR or CB may express the GluR2 receptor subunit. As the presence of the GluR2 subunit decreases the permeability of AMPA receptors to Ca2+ (Hollmann et al., 1991; Hume et al., 1991), the two subpopulations of neurons may be characterized by different Ca2+ kinetics. In the former group, which does not contain the GluR2 subunit, calcium may enter through the faster-opening AMPA receptors, whereas in the latter group Ca2+ may enter through the slower-opening NMDA receptors. As GluR2-positive and GluR2-negative cells project to different regions of pyramidal neurons they potentially modulate different aspects of pyramidal cell function. For example, inhibitory projections to the dendrites of pyramidal neurons may be important in determining integration within the dendritic arbor, whereas inputs directly to the soma and axon initial segment of pyramidal neurons may determine whether the cell fires or not [see Somogyi et al. for a review (Somogyi et al. 1998)]. Thus, it may be possible that at least two inhibitory systems act in parallel, receiving different sets of inputs, characterized by different calcium kinetics, and projecting to different regions of pyramidal cells (Fig. 9). However, this interpretation requires further investigation. As shown in the present study, different antibodies to CB label different populations of cells. Some cells labeled with the antibody to CB that was raised in the rabbit do express the GluR2/3 subunits (unpublished observations). In addition, a subpopulation of chandelier cells in infragranular layers has been shown to be immunoreactive for CB (del Río and DeFelipe, 1997a), suggesting that interneurons in the different cortical layers may have different receptor subunit profiles. Nonetheless, the results suggest that not only do the glutamate receptor subunit profiles of cells determine their inputs (Landsend et al., 1997; Rubio and Wenthold, 1997; Tóth and McBain, 1998), but that cells of different receptor subunit profiles may project to different targets.

Conclusions

The present results suggest that various neurochemically identified subpopulations of nonpyramidal cells express different AMPA, kainate and NMDA receptors. The different receptor subunit profiles of these cells may endow them with different functional characteristics that could be important in separating parallel inhibitory circuits. Such specificity in inhibitory circuitry is likely to be important in the modulation of normal cortical function, plasticity and disease.

Notes

The authors are grateful to Azucena Ortiz for technical assistance and Concha Bailón and Carmen Hernández for assistance with the confocal laser scanning microscope. This work was supported by a DGCYT PM99-0105 grant, and Comunidad de Madrid grant 08.5/0036/2000. GNE was supported by a CJ Martin Fellowship from the National Health and Medical Research Council of Australia.

Table 1

Primary antibodies

Immunoreactivity Type Dilution Source (cat. no.) 
M IgG, raised in the mouse; R IgG, raised in the rabbit. 
nNOS R IgG 1:250 Transduction Lab. (N31030/L5) 
SOM R IgG 1:2000 Peninsula Lab. (IHC8001) 
NPY R IgG 1:2000 Peninsula Lab. (IHC7180) 
PV M IgG 1:2000 Swant (235) 
PV R IgG 1:2000 Swant (PV-28) 
CB M IgG 1:2000 Swant (300) 
CB R IgG 1:2000 Swant (CB28) 
CR M IgG 1:4000 Swant (6B3) 
CR R IgG 1:2000 Swant (25392) 
GluR1 R IgG 1:50 Chemicon (AB1504) 
GluR2/3 R IgG 1:100 Chemicon (AB1506) 
GluR2/4 M IgG 1:100 Chemicon (MAB396) 
GluR5/6/7 M IgM 1:100 Chemicon (MAB379) 
NMDAR1 R IgG 1:100 Chemicon (AB1516) 
Immunoreactivity Type Dilution Source (cat. no.) 
M IgG, raised in the mouse; R IgG, raised in the rabbit. 
nNOS R IgG 1:250 Transduction Lab. (N31030/L5) 
SOM R IgG 1:2000 Peninsula Lab. (IHC8001) 
NPY R IgG 1:2000 Peninsula Lab. (IHC7180) 
PV M IgG 1:2000 Swant (235) 
PV R IgG 1:2000 Swant (PV-28) 
CB M IgG 1:2000 Swant (300) 
CB R IgG 1:2000 Swant (CB28) 
CR M IgG 1:4000 Swant (6B3) 
CR R IgG 1:2000 Swant (25392) 
GluR1 R IgG 1:50 Chemicon (AB1504) 
GluR2/3 R IgG 1:100 Chemicon (AB1506) 
GluR2/4 M IgG 1:100 Chemicon (MAB396) 
GluR5/6/7 M IgM 1:100 Chemicon (MAB379) 
NMDAR1 R IgG 1:100 Chemicon (AB1516) 
Table 2

Percentages of cells that colocalized nNOS or SOM with PV, M-CB or CR (mean ± SE)

Neurons that contain Neurons that colocalize 
 nNOS SOM PV M-CB CR 
Numbers in parentheses signify the total number of cells counted that were immunoreactive for the antigens in the far left column. 
nNOS  –  – 0 (171) 40.0 ± 4.5 (201) 0 (160) 
SOM  –  – 0 (160) 81.5 ± 3.3 (196) 0 (89) 
PV  0 (212)  0 (159) –  – – 
M-CB 24.5 ± 3.3 (465) 44.3 ± 3.3 (428) –  – – 
CR  0 (530)  0 (457) –  – – 
Neurons that contain Neurons that colocalize 
 nNOS SOM PV M-CB CR 
Numbers in parentheses signify the total number of cells counted that were immunoreactive for the antigens in the far left column. 
nNOS  –  – 0 (171) 40.0 ± 4.5 (201) 0 (160) 
SOM  –  – 0 (160) 81.5 ± 3.3 (196) 0 (89) 
PV  0 (212)  0 (159) –  – – 
M-CB 24.5 ± 3.3 (465) 44.3 ± 3.3 (428) –  – – 
CR  0 (530)  0 (457) –  – – 
Table 3

Percentages (± SEM) of cells that contained nNOS, SOM, NPY, PV, M-CB or CR that also expressed the different glutamate subunits

Neurons that contained Neurons that also expressed 
 GluR1 GluR2/3 GluR2/4 GluR5/6/7 NMDAR1 
Numbers in parentheses signify the total number of cells counted that were immunoreactive for the antigens in the far left column. 
nNOS  –  –  33.9 ± 5.8 (109) 50.8 ± 5.1 (128)  – 
SOM  –  –  73.9 ± 5.9 (94) 81.6 ± 5.5 (73)  – 
NPY  –  – 100 (40) 81.8 ± 6.8 (31)  – 
PV 93.2 ± 3.7 (71) 11.4 ± 4.0 (64)  61.4 ± 7.4 (72) 93.7 ± 2.1 (84) 81.4 ± 6.2 (68) 
M-CB 88.2 ± 3.0 (197)  0 (91)  –  – 39.2 ± 5.9 (151) 
CR 19.6 ± 3.4 (182)  0 (253)  94.5 ± 1.6 (231) 96.4 ± 1.5 (194) 75.9 ± 4.3 (262) 
Neurons that contained Neurons that also expressed 
 GluR1 GluR2/3 GluR2/4 GluR5/6/7 NMDAR1 
Numbers in parentheses signify the total number of cells counted that were immunoreactive for the antigens in the far left column. 
nNOS  –  –  33.9 ± 5.8 (109) 50.8 ± 5.1 (128)  – 
SOM  –  –  73.9 ± 5.9 (94) 81.6 ± 5.5 (73)  – 
NPY  –  – 100 (40) 81.8 ± 6.8 (31)  – 
PV 93.2 ± 3.7 (71) 11.4 ± 4.0 (64)  61.4 ± 7.4 (72) 93.7 ± 2.1 (84) 81.4 ± 6.2 (68) 
M-CB 88.2 ± 3.0 (197)  0 (91)  –  – 39.2 ± 5.9 (151) 
CR 19.6 ± 3.4 (182)  0 (253)  94.5 ± 1.6 (231) 96.4 ± 1.5 (194) 75.9 ± 4.3 (262) 
Table 4

Percentages (± SEM) of cells that expressed the different glutamate subunits and that also contained nNOS, SOM, NPY, PV, M-CB or CR

Neurons that expressed Neurons that also contained 
 nNOS SOM NPY PV M-CB CR 
Numbers in parentheses signify the total number of cells counted that were immunoreactive for the antigens in the far left column. 
GluR1  –  –  – 29.3 ± 3.5 (283) 43.8 ± 3.6 (423)  8.6 ± 1.2 (379) 
GluR2/3  –  –  – 2.9 ± 1.1 (342)  0 (373)  0 (619) 
GluR2/4  8.4 ± 1.7 (476) 12.4 ± 1.2 (499) 17.1 ± 1.8 (277) 8.8 ± 1.3 (482)  – 40.8 ± 3.2 (564) 
GluR5/6/7 12.0 ± 1.5 (544) 13.0 ± 1.6 (465) 16.7 ± 3.3 (300) 28.1 ± 4.3 (383)  – 42.5 ± 4.2 (501) 
NMDAR1  –  –  – 9.9 ± 1.2 (585) 10.8 ± 1.6 (551) 24.4 ± 2.3 (862) 
Neurons that expressed Neurons that also contained 
 nNOS SOM NPY PV M-CB CR 
Numbers in parentheses signify the total number of cells counted that were immunoreactive for the antigens in the far left column. 
GluR1  –  –  – 29.3 ± 3.5 (283) 43.8 ± 3.6 (423)  8.6 ± 1.2 (379) 
GluR2/3  –  –  – 2.9 ± 1.1 (342)  0 (373)  0 (619) 
GluR2/4  8.4 ± 1.7 (476) 12.4 ± 1.2 (499) 17.1 ± 1.8 (277) 8.8 ± 1.3 (482)  – 40.8 ± 3.2 (564) 
GluR5/6/7 12.0 ± 1.5 (544) 13.0 ± 1.6 (465) 16.7 ± 3.3 (300) 28.1 ± 4.3 (383)  – 42.5 ± 4.2 (501) 
NMDAR1  –  –  – 9.9 ± 1.2 (585) 10.8 ± 1.6 (551) 24.4 ± 2.3 (862) 
Figure 1.

Histograms showing the densities (mean ± SE) of cells immunoreactive for CB, CR and PV, per 10 000 μm2, in layers II and III. M (mouse) and R (rabbit) indicate the species in which the antibodies were raised.

Figure 1.

Histograms showing the densities (mean ± SE) of cells immunoreactive for CB, CR and PV, per 10 000 μm2, in layers II and III. M (mouse) and R (rabbit) indicate the species in which the antibodies were raised.

Figure 2.

High-power photomicrographs showing examples of nonpyramidal neurons in layer III which are immunoreactive for nNOS (A), SOM (B), NPY (C) and PV (D). Scale bar: 30 μm.

Figure 2.

High-power photomicrographs showing examples of nonpyramidal neurons in layer III which are immunoreactive for nNOS (A), SOM (B), NPY (C) and PV (D). Scale bar: 30 μm.

Figure 3.

High-power photomicrographs showing examples of nonpyramidal neurons in layer III which are immunoreactive for M-CB (A) and CR (B). Scale bar: 30 μm.

Figure 3.

High-power photomicrographs showing examples of nonpyramidal neurons in layer III which are immunoreactive for M-CB (A) and CR (B). Scale bar: 30 μm.

Figure 4.

Camera lucida drawings showing the distributions of cells immunoreactive for nNOS, SOM, NPY, PV, CB and CR in layers I–VI. Scale bar: 150 μm.

Figure 4.

Camera lucida drawings showing the distributions of cells immunoreactive for nNOS, SOM, NPY, PV, CB and CR in layers I–VI. Scale bar: 150 μm.

Figure 5.

Histograms showing the densities (mean ± SE) of somata immunostained for nNOS, SOM, NPY, PV, CB and CR per 10 000 μm2 in layers II and III. M (mouse) and R (rabbit) indicate the species in which the antibody was raised. All pairwise comparisons of the densities were significantly different, except that between PV and SOM and that between M-CB and nNOS.

Figure 5.

Histograms showing the densities (mean ± SE) of somata immunostained for nNOS, SOM, NPY, PV, CB and CR per 10 000 μm2 in layers II and III. M (mouse) and R (rabbit) indicate the species in which the antibody was raised. All pairwise comparisons of the densities were significantly different, except that between PV and SOM and that between M-CB and nNOS.

Figure 6.

Pseudo-colored confocal images of M-CB (A, G), nNOS (B, E), CR (D) and SOM (H) immunoreactivities. (C) Colocalization of M-CB with nNOS. (F) shows that many cells that contain CR do not colocalize nNOS. (I) Colocalization of M-CB with SOM. Arrows indicate double-labeled cells. The stars in (D–F) indicate a single unlabeled pyramidal cell in the different images, identifiable by the appearance of the cell body and apical dendrite. Scale bar: 50 μm.

Figure 6.

Pseudo-colored confocal images of M-CB (A, G), nNOS (B, E), CR (D) and SOM (H) immunoreactivities. (C) Colocalization of M-CB with nNOS. (F) shows that many cells that contain CR do not colocalize nNOS. (I) Colocalization of M-CB with SOM. Arrows indicate double-labeled cells. The stars in (D–F) indicate a single unlabeled pyramidal cell in the different images, identifiable by the appearance of the cell body and apical dendrite. Scale bar: 50 μm.

Figure 7.

Pseudo-color confocal images illustrating receptor subunit expression in selected examples of neurochemically identified cell types, as revealed by fluorescent double-labeling immunohistochemistry. Each row includes three images, two of which (left and center) illustrate cells studied that contain either the particular antigen (left) or receptor subunit (center). The third image in each row (right column) is the composite image showing examples of cells containing particular calcium-binding proteins that either express (C, F), or do not express (I), a particular glutamate receptor subunit. (A–C) PV-ir cells and those which express GluR5/6/7. (D–F) M-CB-ir cells and those which express GluR1. (G–I) CR-ir cells and those which express GluR2/3. Arrows indicate double-labeled cells. Scale bar: 50 μm.

Figure 7.

Pseudo-color confocal images illustrating receptor subunit expression in selected examples of neurochemically identified cell types, as revealed by fluorescent double-labeling immunohistochemistry. Each row includes three images, two of which (left and center) illustrate cells studied that contain either the particular antigen (left) or receptor subunit (center). The third image in each row (right column) is the composite image showing examples of cells containing particular calcium-binding proteins that either express (C, F), or do not express (I), a particular glutamate receptor subunit. (A–C) PV-ir cells and those which express GluR5/6/7. (D–F) M-CB-ir cells and those which express GluR1. (G–I) CR-ir cells and those which express GluR2/3. Arrows indicate double-labeled cells. Scale bar: 50 μm.

Figure 8.

Pairs of fluorescence photomicrographs (A and B, C and D) showing examples of cells that contain PV or CR which also express the GluR2/4 subunits. The PV-ir cell shown in (A) and (B) (arrows) is a large basket cell. The CR-ir cell shown in (C) and (D) (arrows) is a bipolar neuron. Scale bar: 30 μm.

Figure 8.

Pairs of fluorescence photomicrographs (A and B, C and D) showing examples of cells that contain PV or CR which also express the GluR2/4 subunits. The PV-ir cell shown in (A) and (B) (arrows) is a large basket cell. The CR-ir cell shown in (C) and (D) (arrows) is a bipolar neuron. Scale bar: 30 μm.

Figure 9.

Putative expression of receptor subunits in some neurochemically identified nonpyramidal cells in layers II–III of the human temporal cortex. PV-ir cells include chandelier cells and large basket cells; CB-ir cells include double bouquet cells; and CR-ir cells include bipolar cells and double bouquet cells. Two populations of CB-ir double bouquet cells can be distinguished, those that colocalize CR and those that do not. Of particular importance is the observation that cells which express the GluR2 subunit may project to different regions of pyramidal cells, as compared with cells that do not express GluR2. PV-ir chandelier cells, which project to the initial segments of pyramidal cell axons, and PV-ir large basket cells, which project to the soma and dendrites of pyramidal cells, contain the GluR 2 subunit. Double bouquet cells and bipolar cells, which project to the dendrites of pyramidal cells, do not contain the GluR2 subunit. Thus, interneurons that project to the soma and axon initial segment of pyramidal cells may be distinguished from those that do not by their GluR subunit expression. The source of excitatory inputs to the receptor subunit profiles and the post-synaptic targets of the different interneurons may be important in determining parallel inhibitory circuits.

Figure 9.

Putative expression of receptor subunits in some neurochemically identified nonpyramidal cells in layers II–III of the human temporal cortex. PV-ir cells include chandelier cells and large basket cells; CB-ir cells include double bouquet cells; and CR-ir cells include bipolar cells and double bouquet cells. Two populations of CB-ir double bouquet cells can be distinguished, those that colocalize CR and those that do not. Of particular importance is the observation that cells which express the GluR2 subunit may project to different regions of pyramidal cells, as compared with cells that do not express GluR2. PV-ir chandelier cells, which project to the initial segments of pyramidal cell axons, and PV-ir large basket cells, which project to the soma and dendrites of pyramidal cells, contain the GluR 2 subunit. Double bouquet cells and bipolar cells, which project to the dendrites of pyramidal cells, do not contain the GluR2 subunit. Thus, interneurons that project to the soma and axon initial segment of pyramidal cells may be distinguished from those that do not by their GluR subunit expression. The source of excitatory inputs to the receptor subunit profiles and the post-synaptic targets of the different interneurons may be important in determining parallel inhibitory circuits.

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