Abstract

SNAT1 mediates glutamine (Gln) influx into neurons and is believed to replenish the transmitters pools of glutamate (Glu) and γ-aminobutyric acid (GABA). We investigated its distribution and cellular localization in the cerebral cortex and neighboring regions of rats and humans using light and electron microscopic immunocytochemical methods with specific antibodies. In the first somatic sensory cortex of rats and in areas 9, 10, 21 and 46 of the human cortex, numerous SNAT1-positive (+) cells were present in the cortical parenchyma and in the white matter; >95% of SNAT1+ cells were neurons, but some were astrocytes. Most SNAT1+ cells were pyramidal neurons, but numerous non-pyramidal neurons were also observed: SNAT1/GABA double-labeling studies showed that SNAT1 is expressed in all GABA+ neurons. SNAT1/synaptophysin studies showed that <0.1% of all synaptophysin+ puncta coexpressed SNAT1. SNAT1 immunoreactivity (ir) was also in leptomeninges, ependymal cells and choroid plexus. Electron microscopic studies showed that neuronal SNAT1 ir was almost exclusively observed in perikarya and dendritic profiles. SNAT1 ir was also in distal astrocytic processes, including end feet profiles, and in leptomeninges. These findings suggest that the major function of SNAT1 is not to replenish the transmitter pools of Glu and GABA.

Introduction

Besides its numerous metabolic roles in the organism (Bode, 2001), the amino acid glutamine (Gln) participates in the recycling of glutamate (Glu) and γ-aminobutyric acid (GABA) (Hertz et al., 1983; Hertz and Schousboe, 1988; Erecinska and Silver, 1990; Danbolt, 2001), the main excitatory and inhibitory neurotransmitters in the CNS (Conti and Weinberg, 1999; Cherubini and Conti, 2001).

Glu recycling is mediated by complex, indirect mechanisms, among which the so-called ‘glutamate–glutamine’ cycle appears to play an important, though not exclusive, role: following its release by axon terminals, Glu is taken up by astrocytic processes ensheathing nerve terminals, where it is converted to Gln by the action of glutamine synthetase; Gln is then released into the extracellular fluid, taken up by nerve terminals and reconverted to Glu by phosphate-activated glutaminase (Van den Berg and Garfinkel, 1971; Martinez-Hernandez et al., 1977; Bradford et al., 1978, 1983; Hamberger et al., 1979a,b; Hertz, 1979; Reubi, 1980; Hertz et al., 1983; Conti and Minelli, 1994; Rothstein et al., 1994; Chaudry et al., 1995; Westergaard et al., 1995; Danbolt, 2001). GABA recycling is operated by a direct mechanism mediated by a plasma membrane transporter named GAT-1, expressed at GABAergic terminals (Guastella et al., 1990; Minelli et al., 1995; Conti et al., 1998a) and by indirect mechanisms, most notably the decarboxylation of Glu generated from Gln (Reubi, 1980; Bradford et al., 1983).

Recent studies have shed light on the molecular bases of glutamine trafficking between astrocytes and neurons (Bode, 2001; Bröer and Brookes, 2001; Chaudry et al., 2002a; for the new nomenclature, see Mackenzie and Erickson, 2003). Gln efflux from astrocytes appears to be mediated by Sodium-Coupled Neutral Amino Acid Transporter 3 (SNAT3; formerly SN1), a system N amino acid transporter localized to perisynaptic astrocytes (Chaudry et al., 1999); whether a second system N transporter, SNAT5 (formerly SN2; Nakanishi et al., 2001), is also involved in Gln efflux from astrocytes is at present unknown. Gln influx into neurons is mediated by transporters belonging to the system A family; to date, three isoforms have been characterized: SNAT1 (formerly GlnT, SAT1, ATA1, or SA2; Varoqui et al., 2000; Albers et al., 2001; Chaudry et al., 2002b); SNAT2 (formerly SAT2, ATA2, or SA1; Sugawara et al., 2000a; Yao et al., 2000; Reimer et al., 2001); and SNAT4 (formerly SAT3, or ATA3; Sugawara et al., 2000b; Hatanaka et al., 2001). As SNAT4 is exclusively expressed in liver and skeletal muscle (Sugawara et al., 2000b; Hatanaka et al., 2001), only SNAT1 and SNAT2 may influence Glu and/or GABA recycling and thus synaptic function. SNAT1, a predicted 485 amino acid protein of 54 kDa and 11 putative transmembrane domains, is an Na+-dependent amino acid transporter that prefers Gln and is inhibited by α-methylaminoisobutyric acid (MeAIB; Varoqui et al., 2000). SNAT1-mediated Gln uptake is saturable (Km 489 ± 88 µM), electrogenic and pH-sensitive (Varoqui et al., 2000; Albers et al., 2001; Chaudry et al., 2002b). Based on these features and on the broader specificity and tissue distribution of SNAT2, the second system A transporter expressed in the brain (Reimer et al., 2000; Sugawara et al., 2000a; Yao et al., 2000), SNAT1 is believed to play a crucial role in the uptake of Gln in neurons that is necessary for the glutamate–glutamine cycle (Chaudry et al., 2002a).

Full appreciation of the functional role of SNAT1 is however hampered by the scarce information on its localization. In the initial cloning study, Varoqui et al. (2000) observed high levels of SNAT1 in cerebellar granule cells but not in parallel astrocyte cultures and, based on the results of a general mapping in situ hybridization analysis, suggested a preferential localization to glutamatergic neurons, even though they observed SNAT mRNA in cerebellar Purkinje neurons. The cellular localization of SNAT1 was not addressed in that investigation. Armano et al. (2002) showed that, in primary cultures of developing hippocampal neurons (another model used for the study of glutamatergic transmission), SNAT1 is present in axon growth cones and can be detected at later stages at sites of synaptic contacts. Chaudry et al. (2002b) studied the localization of SNAT1 by in situ hybridization, providing further indirect evidence that SNAT1 is also expressed by GABAergic neurons. More recently, Mackenzie et al. (2003) suggested that SNAT1 is expressed by hippocampal, cerebellar and pallidal glutamatergic and GABAergic neurons and not by astrocytes; in addition, they found SNAT1 immunoreactivity in ependymal cells but did not observe colocalization of SNAT1 and synaptophysin. Thus, the localization of SNAT1 is poorly known and particularly so in the neocortex. Here, we report the results of an extensive immunocytochemical study of the localization of SNAT1 in rat and human cerebral cortex.

Material and Methods

Tissue Preparation

Rat Tissue

Twenty-two adult abino rats (200–225 g; Sprague–Dawley; Charles River, Milan, Italy) were used in these studies. Care and handling of animals were approved by the Animal Research Committe of Università Politecnica delle Marche.

For immunocytochemistry, rats were anesthetized with chloral hydrate (12%; i.p.) and perfused through the ascending aorta with physiological saline followed by 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB; pH 7.4; n = 8) or 4% PFA and 0.5% glutaraldehyde (GA) in PB (n = 8) for light and confocal microscopy, or by 4% PFA, 0.5 % GA and 0.3% picric acid in PB (n = 2) for electron microscopy. Brains were removed and postfixed in the same fixative used for perfusion at 4°C until processed for histology.

For Western blotting, rats (n = 4) were perfused with 4 mM cold Tris–HCl (pH 7.4) containing 0.32 M sucrose, 1 mM EDTA, 0.5 mM phenylmethylsulphonyl fluoride (PMSF) and 0.5 mM N-ethylmaleimide (NEM).

Human Tissue

The human corticaltissue used for immunocytochemical studies came from surgical specimens of four patients with brain tumors whose relevant clinical data are summarized in Table 1. Two of the four samples have already been used in another study (Conti et al., 1999a). Informed consent to the surgical procedure was given in all cases. The cortical tissue used in this study was macroscopically normal tissue included in ‘tactical lobectomies’ or resected in order to reach deep-seated tumors and showed no signs of edema. None of the patients suffered from pre- or postoperative seizures.

Tissue samples were immediately immersed in cold 4% PFA solution in PB for 2–3 h and then transferred to a fresh 4% PFA solution for 24–48 h at 4°C. Samples were then washed several times with PB, cut into small blocks and stored at –20°C in a solution containing 30% glycerol, 30% ethylene glycol, 30% distilled water and 10% PB 0.4 M for several months.

For Western blotting studies, we used a frozen autoptic specimen of cortical tissue obtained from the Harvard Brain Tissue Resource Center (Belmont, MA) and already used in a recent study (C. Matute, M. Melone, A. Vallejo-Illarramendi, M. Catalano and F. Conti, submitted for publication). Written consent was obtained from the donor’s legal next of kin.

Antibodies

We used polyclonal antibodies directed to the predicted NH2-terminal portion of SNAT1 (amino acids 1–63), whose production and characterization have been reported previously (Varoqui et al., 2000). We assessed the specificity of SNAT1 antibodies in the present material by: (i) Western blottings of cell extracts and crude membranes prepared from rat cerebral cortex (Varoqui et al., 2000; Melone et al., 2001) and of crude membranes from human cortex; (ii) preadsorbing SNAT1 antibodies with 10–3, 10–4 and 10–5 M glutathione S-transferase (GST)–SNAT1 fusion protein and with 10–3 M GST–SNAT-2 fusion protein in rat tissue; (iii) preadsorbing SNAT1 antibodies with 10–3 M GST–SNAT1 and SNAT-2 fusion protein in Western blottings of rat cortical cell extracts and crude membranes and of human cortical crude membranes.

Monoclonal antibodies to neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) and synaptophysin were purchased from Chemicon (MAB377; Temecula, CA) and Sigma (GFAP, GA5, G-3893; synaptophysin, clone SVP-38, S-5768; St Louis, MO), respectively, whereas monoclonal antibodies to GABA were kindly provided by Dr C. Matute (Matute and Streit, 1986).

Immunocytochemical Procedures

Rat brains were cut with a vibratome into 30 µm-thick coronal or parasagittal sections, which were collected serially (in groups of five) in phosphate buffered saline (PBS) and stored at 4°C until processing. One coronal section from each group was stained with 0.1% thionine; the remaining sections were used for immunocytochemical studies. Human cortical blocks were cut on a Vibratome in the coronal and parasagittal planes into 35 µm-thick sections, collected into groups of five and stored at 4°C in PBS until processing. Of the five sections, one was stained with 0.1% thionine; the remainder were used for immunocytochemistry.

Immunoperoxidase Studies

For light microscopic studies, free floating sections from both rat and human brains were pretreated with H2O2 (1% in PBS, 30 min) to remove endogenous peroxidase, rinsed with PBS and then incubated (2 h at room temperature and overnight at 4°C for rat sections; 4 h at room temperature and overnight at 4°C for human sections) in a solution of blocking buffer [2% normal goat serum (NGS), 2% bovine serum albumin (BSA), 0.01% Tween 20] containing SNAT1 antibodies (1:2000). The following day, sections were rinsed three times in PBS and incubated in a solution of blocking buffer containing biotinylated goat anti-rabbit IgG for 1 h at room temperature (BA1000, 1:100; Vector, Burlingame, CA). Sections were subsequentely rinsed in PBS, incubated in avidin–biotin peroxidase complex (ABC Elite PK6100; Vector), washed several times in PBS and incubated in 3,3′-diaminobenzidine tetrahydrochloride (DAB; 0.05% in Tris 0.05M with 0.03% H2O2). Method specificity was controlled for both rat brain and human cortical sections by substituting primary antibodies with PBS or NGS.

For electron microscopic studies, free floating sections from two rat brains fixed with 4% PFA, 0.5 % GA and 0.3% picric acid were pretreated with 1% sodium borohydryde for 30 min to quench non-specific binding, rinsed several times with PBS, pretreated with H2O2 (1% in PBS; 30 min) to remove endogenous peroxidase, rinsed with PBS and processed as described above, with the exception that Tween 20 was not used. After completion of the immunocytochemical procedure, sections were washed in PB, incubated in 2.5% GA (20 min), washed in PB and postfixed for 1 h in 1% OsO4. After dehydration in ethanol and infiltration in Epon–Spurr resin, sections were flat-embedded between two Aclar (8F119; Sigma)-coated coverslips. Small blocks of tissue containing either layers I–III or layer V and white matter, selected by light-microscopic inspection, were cut out, glued to blank epoxy and sectioned with an ultramicrotome. Thin sections were stained with uranyl acetate and lead citrate and examined with a Philips CM10 electron microscope. Identification of neuronal and non-neuronal elements was performed according to Peters et al. (1991) and, for leptomeninges, following Oda and Nakanishi (1984).

Double-labeling Studies

SNAT1/GABA double-labeling studies in rats were perfomed on sections from animals perfused with 4% PFA and 0.5% GA, whereas those in humans were performed on cortical sections fixed with 4% PFA. SNAT1/NeuN, SNAT1/GFAP and SNAT1/synaptophysin colocalization studies were all performed on sections fixed in 4% PFA. Details of the antibodies used in these studies are given in Table 2. For SNAT1/GABA studies, free floating sections were incubated in 10% NGS and 0.1% Triton X100 in 0.01M PB (1 h), then in a solution containing a mixture of SNAT1 and GABA primary antibodies (in PB with 1% NGS); for SNAT1/NeuN, SNAT1/GFAP and SNAT1/synaptophysin studies, sections were incubated in a solution of blocking buffer (2% NGS, 2% BSA and 0.01% Tween 20) containing a mixture of SNAT1 and either GFAP, NeuN, or synaptophysin primary antibodies. After incubation (2 h at room temperature and overnight at 4°C) in primary antibodies, sections were washed in PB, incubated for 20 min in 10% NGS and then for 1 h in a solution containing a mixture of affinity-purified fluorescein isothiocyanate (FITC)- or tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibodies for SNAT1/GABA colocalization studies, or directly for 1 h in a solution of blocking buffer with a mixture of FITC and TRITC secondary antibodies for SNAT1/NeuN, SNAT1/GFAP and SNAT1/synaptophysin colocalization studies. Sections were subsequently washed in PB, mounted, air-dried and coverslipped using the Vectashield mounting medium (H-1000; Vector). They were examined using a BioRad (Hemel Hempstead, UK) Microradiance confocal laser scanning microscope equipped with argon and helium/neon lasers. FITC and TRITC were excited with the 488 and 543 nm lines, respectively, imaged separately (emissions were separated by 515/30 and 570 nm filters for FITC and TRITC, respectively) and merged using the LaserSharp Processing BioRad software (v. 3.2). Control experiments with single-labeled sections confirmed that there was no significant FITC/TRITC bleed-through when images were acquired separately. Sections incubated with two primary antibodies and one secondary antibody or with one primary and two secondary antibodies revealed no appreciable cross-reactivity.

Data Analysis

All data were collected from a region of rat parietal cortex characterized by the presence of a conspicuous layer IV with intermingled dysgranular regions, densely packed layers II and III and a relatively cell-free layer Va. This area corresponds to the first somatic sensory cortex (SI; Chapin and Lin, 1990).

For SNAT1/NeuN, SNAT1/GFAP and SNAT1/GABA colocalization studies in rats, microscopic fields from SI cortex and the underlying white matter (four sections for each pair of antigens/hemisphere; four rats) were scanned using a ×60 Nikon Plan Apo oil-immersion objective with a numerical aperture of 1.4; images were acquired on a 512 × 512 pixel box (1400 µm2) using a confocal pinhole of 1.5–2. To improve signal/noise ratio, 10 frames of each image were averaged by Kalman filtering. SNAT1-, GABA- and NeuN-positive cells were counted and then merged images of SNAT1/GABA, SNAT1/NeuN and SNAT1/GFAP were used to identify double-labeled cells and profiles. To assess the degree of SNAT-1/Syn colocalization, the red images (Syn) were first imported into NIH Image software and the the density slice function was used to highlight the individual labeled puncta (by establishing a threshold that yielded the greatest number of individual puncta without causing a fusion of puncta). Next, the same level of threshold for each red image determined as described above and an appropriate level of threshold for the green image to subtract background noise were applied to the merge images using the function of a the Bio-Rad LaserSharp software (v. 3.0) and yellow puncta were counted manually.

For human cortex, cytoarchitectonic areas and boundaries were identified on adjacent thionine-stained sections according to Brodmann (1909), von Economo (1928), Ong and Garey (1990) and Rajkowska and Goldman-Rakic (1995).

Western Blotting

The neocortex was separately homogenized with a glass–Teflon homogenizer in six volumes of ice-cold buffer (4mM Tris, pH 7.4; 0.32 M sucrose; 1 mM EDTA; 0.23 mM dithiothreitol; and 1 µM leupeptin and pepstatin A). Cell extract and crude membrane preparation and protein concentration determination were performed as described in previous pubblications (Varoqui et al., 2000; Melone et al., 2001). Aliquots (15 µg of total protein per lane) of cell extracts and crude membranes mixed with equal volumes of 2× electrophoresis sample buffer were subjected to SDS–PAGE; separated proteins were electroblotted onto nitrocellulose filters (0.22 µm) and finally probed with SNAT1 antibodies (dilution: 1:2000). Labeled bands were visualized by the BioRad Chemidoc and Quantity One software (BioRad v. 4.1.1) using the SuperSignal West Pico (Rockford, IL) chemiluminescent substrate. The same procedure was used for the frozen autoptic sample of human cortex.

Results

The specificity of SNAT1 antibodies was probed in our experimental conditions by Western blottings on cell extracts and crude membranes from rat neocortex. In both cases, SNAT1 antibodies recognized a single band of ∼55 kDa (Fig. 1A), that was abolished by preincubation with 10–3 M GST–SNAT1 fusion protein (Fig. 1B) but not with 10–3 M GST–SNAT2 fusion protein (Fig. 1C).

SNAT1 immunoreactivity (ir) was highest in cerebellum, thalamus, olfactory bulb and superior colliculus, followed by brainstem, cerebral cortex (including hippocampus) and striatum, in line with the results of previous blotting studies (Varoqui et al., 2000). SNAT1 ir (Fig. 1A′) was totally abolished by preincubating SNAT1 antibodies with 10–3 M GST–SNAT1 fusion protein (Fig. 1B′), but was unaffected by preadsorbtion with 10–3 M GST–SNAT2 fusion protein (Fig. 1C′).

Distribution and Cellular Localization of SNAT1 Immunoreactivity in Rat Neocortex

Light and Confocal Microscopy

In the SI cortex, SNAT1 ir was mostly in perikarya (Fig. 2A) and proximal processes, although immunoreactive profiles and punctate structures were also present in the neuropil (Fig. 2C). Lightly stained, morphologically heterogeneous, non-neuronal cells of variable size (diameter: ∼5–10 µm) were also observed in the white matter underlying the cortex and in the corpus callosum (Fig. 2D). Strong SNAT1 ir was observed in the leptomeninges (Fig. 2E), where it was detected in the arachnoid and along the thin arachnoid trabeculae of the subarachnoid space, in the cytoplasm of ependymal cells lining the walls of the third ventricle (Fig. 2F) and in the choroid plexus (Fig. 2G).

To determine the nature of SNAT1-positive (+) cells, we performed double-labeling studies with SNAT1 and NeuN, a protein expressed only by neuronal nuclei (Mullen et al., 1992); out of 1194 SNAT1+ cells (14–16 microscopic fields/layer; two animals), 1.151 (96.4%) were NeuN+ (Fig. 3A–C) and 43 (3.6%) were NeuN-negative (–). NeuN– cells were smaller (diameter: 5–10 µm) than NeuN+ neurons and were mostly in layers I and II (Fig. 3DF). SNAT1+ cells in white matter were all NeuN–.

To find out whether SNAT1+/NeuN– cells in the cortical parenchyma and white matter were astrocytes, we performed SNAT1/GFAP double-labeling. Analysis of SI cortex (30 microscopic fields/layer; two rats) and underlying white matter (38 microscopic fields; two rats) showed that a large fraction of the small (5–10 µm) SNAT1+ cells in layers I-II (Fig. 4A–C) and white matter (Fig. 4G–I), were GFAP+: out of 147 SNAT1+ cells, 134 were GFAP+ (91,1%). Some SNAT1+ neuropilar processes were also GFAP+ (Fig. 4D–F).

In the cortical parenchyma, SNAT1+ cells that for their laminar distribution and size could be considered neurons, were present in all cortical layers, but they were more numerous and intensely stained in layers II–III and Vb (Fig. 2A); most were pyramidal neurons (for criteria, see Conti et al., 1987, 1992) (Fig. 5A,B,D). However, SNAT1+ non-pyramidal neurons were also observed (Fig. 5C,D) and in many cases they were more intensely stained than pyramidal neurons, particularly in layers II–III and VI (Fig. 5C,D). Since in the neocortex most non-pyramidal neurons are GABAergic (Hendry, 1996), the observation that a fraction of SNAT1+ neurons were non-pyramidal suggests that SNAT1 and GABA are coexpressed. Confocal microscopy studies of SNAT1/GABA double-stained sections revealed that out of 3254 SNAT1 neurons (35–40 microscopic fields/layer; two rats), 565 (17.3%) expressed GABA (Fig. 6). We also evaluated the proportion of GABA+ neurons expressing SNAT1 and found that all of the 788 GABA+ neurons sampled (43–48 microscopic fields/layer; two rats), were also SNAT1+.

To verify that the small SNAT1+ punctate structures observed in the cortical parenchyma were axon terminals, we performed double-labeling studies using antibodies to SNAT1 and synaptophysin, a marker for presynaptic axon terminals (Jahn et al., 1985; Wiedenmann and Franke, 1985). Analysis of 175 microscopic fields (four/layer; four rats) showed that out of 305 195 synaptophysin+ puncta, only 220 (0.07%) coexpressed SNAT1+ (Fig. 7), thus suggesting that the large majority of SNAT1+ puncta were not axon terminals.

Electron Microscopy

SNAT1 ir was almost exclusively observed in perikarya (Fig. 8A) and dendritic profiles (Fig. 8B–H); in all cases in which SNAT1+ dendrites possessed spines, the latter were not labeled (e.g. Fig. 8G,H). SNAT1 ir was extremely rare in axon terminals: we found only four SNAT1+ axon terminals in all the material examined (Fig. 8I). In these structures, the immunoreaction product was preferentially associated with various intracellular membranes (Fig. 8A–H).

Some SNAT1+ distal astrocytic processes were also observed, both in the vicinity of synapses whose postsynaptic element was either SNAT1+ (Fig. 8D,F) or SNAT1– (Fig. 8H) and, more often, dispersed in the neuropil (Fig. 8K,L). Intense SNAT1 ir was present in end feet profiles adjacent to the endothelial basal lamina (Fig. 8J), whereas endothelial cells were always unlabeled and in the leptomeninges (Fig. 8M). Oligodendrocytes were not labeled.

Distribution and Cellular Localization of SNAT1 ir in Human Neocortex

The approximate location of the surgical samples and of the autoptic material used in these studies is reported in Figure 9A. In the four samples used for localization studies, examination of adjacent sections processed for the detection of Nissl substance and GFAP showed them to be free of any appreciable abnormality (see Conti et al., 1998a, 1999a). Western blottings on crude membranes prepared from area 9/46 (Rajkowska and Goldman-Rakic, 1995) of a frozen specimen showed that, also in human cortex, SNAT1 antibodies recognized a single band of ∼55 kDa that was abolished by preincubation with 10–3 M GST–SNAT1 fusion protein but not with 10–3 M GST–SNAT-2 fusion protein (Fig. 9B).

In cytoarchitectonic areas 9, 10, 21 and 46 (Brodmann, 1909; von Economo, 1928; Ong and Garey, 1990; Rajkowska and Goldman-Rakic, 1995), SNAT1 ir was mostly in neuronal cell bodies (Fig. 9C) and in proximal processes, although positive puncta were also observed in the neuropil. SNAT1+ neurons were present throughout the cortical layers; the intense perikaryal staining allowed us to distinguish pyramidal (Fig. 9D) from non-pyramidal (Fig. 9E) neurons. SNAT1/GABA colocalization studies (80 microscopic fields from areas 10 and 21) showed that a considerable fraction of SNAT1+ neurons expressed GABA (e.g. Fig. 9F–H). SNAT1/GFAP colocalization studies (30 microscopic fields from areas 10 and 21) revealed that, as in rats, the two antigens were colocalized in both cells and processes (Fig. 9I–K).

Discussion

Using light, confocal and electron microscopic immunocytochemical methods to study in situ the localization of SNAT1 protein in the cerebral cortex of rats and humans, we observed: (i) that besides its expected expression by pyramidal neurons, SNAT1 is highly expressed by all GABAergic neurons; (ii) that in all cortical neurons expressing it, SNAT1 is localized exclusively to the somatodendritic compartment, with a virtually complete lack of immunoreactivity in axon terminals; and (iii) a previously unnoticed localization of SNAT1 to astrocytes and other non-neuronal brain cells.

Localization of SNAT1 in the Cerebral Cortex

SNAT1 is Expressed by all GABAergic Neurons Besides Pyramidal Cells

Robust SNAT1 expression has been detected in cultures of glutamatergic neurons (Varoqui et al., 2000; Armano et al., 2002) and SNAT1 mRNA has been observed in numerous glutamatergic neurons (Varoqui et al., 2000; Chaudry et al., 2002b). The present in situ studies of SNAT1 protein expression in the cerebral cortex show that most pyramidal neurons, which use an excitatory amino acid, in all likelihood Glu, as neurotransmitter (Conti et al., 1987, 1989), are SNAT1+, thus fully supporting the notion that SNAT1 is expressed by glutamatergic neurons.

SNAT1 expression in GABAergic neurons has been proposed on the basis of in situ hybridization studies of the distribution of SNAT1 mRNA in the CNS and of glutamine- and MeAIB-induced depolarization of hippocampal interneurons (Varoqui et al., 2000; Chaudry et al., 2002b) and of the presence of SNAT1+ GABAergic neurons, e.g. Purkinje cells in the cerebellum (Mackenzie et al., 2003). Here, we have shown that SNAT1 protein is indeed expressed in all GABAergic neurons of the cerebral cortex.

In Both Glutamatergic and GABAergic Neurons, SNAT1 is Localized to the Somatodendritic Compartment

The localization of SNAT1 at axon terminals may provide a valuable clue as to the role of SNAT1 in synaptic function; however, previous studies are inconclusive: Armano et al. (2002) showed that in both developing and mature cultured hippocampal neurons SNAT1, but not SNAT2, colocalizes with the synaptic vesicle protein synaptobrevin/VAMP2, whereas in a general mapping study of paraffin-embedded sections from rat brain, Mackenzie et al. (2003) reported that in several brain areas SNAT1 ir does not colocalize with synaptophysin.

In the cerebral cortex, SNAT1 ir is prominent in neuronal perikarya but is also observed in puncta throughout the cortical mantle. We have shown here that virtually none of these puncta coexpress synaptophysin, which is expressed in 90–95% of all axon terminals (Jahn et al., 1985; Wiedenmann and Franke, 1985). More importantly, when seeking SNAT1+ axon terminals at the electron microscope, we detected only four of them out of several thousands. Overall, these studies show that in the cerebral cortex in situ axon terminals do not express SNAT1. Whether the difference between the present results and those of Armano et al. (2002) depends on the different preparations used or on regional differences remains to be determined.

Whereas cytoplasmic accumulation of SNAT1 in cortical neurons does not per se rule out the possibility of its action at axon terminals, the simultaneous observation of cytoplasmic labeling, lack of axon terminal labeling and, most importantly, intense dendritic labeling suggests that SNAT1 either does not participate in the synaptic recycling of Glu and GABA or that it does so in a way that is not predicted by the classic glutamate–glutamine cycle. Thus, one of the crucial findings of this study, i.e. the striking somatodendritic localization of SNAT1 at both glutamatergic and GABAergic neurons, raises some doubts on the ‘synaptic’ role of SNAT1, on the validity of the concept of the ‘Gln–Glu’ cycle and/or on our understanding of Gln uptake (see below).

SNAT1 is Expressed by Astrocytes and Other Non-neuronal Cells

The observation that astrocytic processes, including end feet profiles adjacent to the endothelial basal lamina, express SNAT1 is not in itself surprising given the fundamental role played by astrocytes in Glu and GABA metabolism (Bröer and Brookes, 2001; Danbolt, 2001; Chaudry et al., 2002a) and the report of the presence of SNAT2 in glial cells (Reimer et al., 2000). Rather, our results are unexpected because SNAT1 has so far been considered a ‘neuronal’ Gln transporter (Varoqui et al., 2000; Armano et al., 2002; Chaudry et al., 2002b). The present data indicate that this notion is no longer tenable and remind of previous studies of the localization of the GABA and Glu transporters GAT-1 and EAAC1, which were also considered neuronal until electron microscopic studies revealed a consistent, albeit minor, population of astrocytic processes expressing GAT-1 and EAAC1 (Minelli et al., 1995; Conti et al., 1998a,b). Overall, these observations emphasize the scope for detailed localization studies of all amino acid transporters.

SNAT1/GFAP colocalization studies showed that SNAT1 is expressed both in GFAP+ cell bodies and neuropilar processes in the cortical parenchyma and in the underlying white matter. Most SNAT1+ non-neuronal cells in the white matter are GFAP+ and some are GFAP–: whether SNAT1+/GFAP– non-neuronal cells are GFAP– astrocytes or oligodendrocytes remains to be established, even though the negative electron microscopic search for SNAT1+ oligodendrocytes favors the former possibility. SNAT1/GFAP studies also showed that SNAT1 is expressed in non-punctate neuropilar processes, suggesting that the distal astrocytic processes observed at the electron microscope belong to the astrocytic subpopulation expressing GFAP. Since distal astrocytic processes do not contain gliofilaments (Peters et al., 1991; Privat et al., 1995), these findings suggest that, by failing to reveal the SNAT1+ distal astrocytic processes that are observed at the electron microscope, GFAP/SNAT1 colocalization studies significantly underestimate the degree of SNAT1 expression by astrocytes. SNAT1 astrocytic expression thus indicates that astrocytes play a more complex role than currently believed in Glu and GABA metabolism, at least in the cerebral cortex.

A large fraction of SNAT1+ astrocytic processes was represented by end feet profiles adjacent to the endothelial basal lamina of cortical blood vessels. Although the primary pathway for transendothelial Gln fluxes involves a system N transporter (Lee et al., 1998), system A transport activity at the blood–brain barrier has been demonstrated (Betz and Goldstein, 1978; Sanchez del Pino et al., 1995) and is responsible for ∼20% of the total Gln flux.

Leptomeninges, ependyma and choroid plexus express a variety of neurotransmitters as well as their receptors and transporters (Nilsson et al., 1992; Del Bigio, 1995; Conti et al., 1999b), which participate in the secretion and regulation of cerebrospinal fluid (CSF) and/or the detection of its composition (Harandi et al., 1986). The observation of SNAT1 ir in all these cells is therefore in line with these observations and with the expression of SNAT1 in ependymal cells (Mackenzie et al., 2003) and of SNAT2 in choroid plexus epithelial cells (Reimer et al., 2000).

Functional Implications

SNAT1 is considered a neuronal Gln transporter mediating Gln flux into glutamatergic (and, perhaps, GABAergic) axon terminals. Whereas the functional characteristics — substrate profile, kinetics, thermodynamics and ionic dependence — of SNAT1 have been extensively studied, its localization pattern has never been investigated systematically. The localization pattern of SNAT1 in the cerebral cortex of rats and humans reported here shows that its expression is more complex than, and does not fully agree with, that predicted on the basis of the original functional assumption. Indeed, we found that SNAT1 is localized almost exclusively to the somatodendritic compartment of both glutamatergic and GABAergic neurons, to astrocytic processes (including end feet profiles adjacent to the endothelial basal lamina of cortical blood vessels) and to non-neuronal brain cells of the leptomeninges, ependyma and choroid plexus. Although none of these findings per se contradicts a role for SNAT1 in the replenishment of the Glu and GABA neurotransmitters pools, the overall picture emerging from these studies is hardly reconcilable with the notion that SNAT1 is responsible for this replenisment.

The present electron microscopic findings show that whereas SNAT1 ir was high in the cytoplasm and dendrites, it was never observed in axons or axon terminals; even assuming that SNAT1 diffuses or is transported to cellular sites distant from the perikarya, these findings imply that SNAT1 does not reach the axon terminals. Furthermore, whereas dendrites represented the most common immunoreactive element, we never observed a SNAT1+ spine even from a SNAT1+ dendrite. It therefore seems that SNAT1 does not reach the synapses at all. This view is in line with the recent observation that SNAT1 ir can be demonstrated in the developing cortex at embryonic day 17 (Weiss et al., 2003), when synapses are not functional (Blue and Parnavelas, 1983a,b; Hanganu et al., 2001; Minelli et al., 2003a,b) and, most importantly, with the observation that Gln uptake inhibited by MeAIB mediates the transfer of Gln into a large turnover pool, probably located in perikarya, that does not interact with the glutamate-glutamine neurotransmitter cycle, but not in the fast turnover compartment associated with glutamate–glutamine neurotransmitter cycling (Rae et al., 2003). Altogether, these findings suggest that the major function of SNAT1 is not to replenish the transmitter pools of Glu and GABA. It follows that either our notion of how Glu and GABA transmitters are generated should be revised, or hitherto unknown Gln transporters are expressed at axon terminals and take up Gln to sustain Glu and GABA neurotransmitter pools. As reported in the Introduction, Glu and Gln metabolism are generally described according to the concept of the glutamate–glutamine cycle. As recently pointed out by Danbolt (2001), this concept represents an oversimplification: indeed, Gln administered to the intact brain is metabolized to CO2 (Zielke et al., 1998), many glutamatergic neurons do not express PAG (Laake et al., 1999) and transmitter Glu may be formed in a Gln-independent manner (Hassel and Bråthe, 2000), even at axon terminals (McKenna et al., 2000). Notwithstanding these observations, many experimental data, including some from recent studies (e.g. Lieth et al., 2001; Sibson et al., 2001; Rae et al., 2003), support the notion that Gln is necessary to mantain neuronal Glu and GABA pools. Thus, whereas several facts argue for downsizing the role of the glutamate–glutamine cycle in the replenishment of the transmitter pools of Glu and GABA (Danbolt, 2001), its contribution to the generation of releasable Glu and GABA cannot be doubted. It therefore seems safe to predict that other Gln transporters are expressed at axon terminals and that they are responsible for sustaining the Glu and GABA neurotransmitters pools.

The strong expression of SNAT1 in non-neuronal cells, including end feet profiles adjacent to the endothelial basal lamina of cortical blood vessels, leptomeningeal, ependymal and choroid plexus cells, may provide a clue to its function. Astrocyte perivascular end feet embrace the abluminal surface of the endothelial cells lining brain capillaries and are an integral part of the blood–brain barrier; epithelial cells of the choroid plexus and arachnoid and ependymal cells make up the so-called brain–CSF barrier (Brightman, 1989; Peters et al., 1991; Del Bigio, 1995; Vorbrodt and Dobrogowska, 2003). The fundamental function of these barriers and thus an important — albeit not exclusive — function of the cells contributing to them is to regulate the composition of the brain extracellular space. Thus, non-neuronal SNAT1 appear to be strategically located to regulate Gln levels in the brain, a function that may be particularly important in pathological conditions, such as hepatic encephalopathy, for the role played by Gln in ammonia detoxification (Felipo and Butterworth, 2002; Jalan et al., 2003). Whether neuronal SNAT1 also regulates Gln extracellular levels, although at a local level, is unknown; based on the close relationship between Gln levels, neuronal energy metabolism and nitrogen metabolism, such a mechanism would play an important role in neuronal homeostasis.

We are grateful to A. Ducati for providing the surgical samples and to C. Matute for generously providing the GABA antibodies. This work was supported by grants from MIUR (COFIN 01) and The Stanley Medical Research Institute to F.C.

Address for correspondence: Fiorenzo Conti, Dipartimento di Neuroscienze, Sezione di Fisiologia Umana, Università Politecnica delle Marche, Via Tronto 10/A, Torrette di Ancona, I-60020 Ancona, Italy. Email: f.conti@univpm.it.

Figure 1. Antibody characterization and method specificity. Western blotting of cell extracts (CE) and crude membranes (CM) of neocortex indicate that SNAT1 antisera recognize a single band of ∼55 kDa (A). Protein detection is completely abolished by preadsorption of antibodies with 10–3 M GST–SNAT1 (B), but not with 10–3M GST–SNAT2 (C). SNAT1 ir in the SI cortex (A′) is abolished by preincubation with 10–3M GST–SNAT1 fusion protein (B′) but not with 10–3M GST–SNAT2 fusion protein (C′). Scale bar: 100 µm (A′–C′).

Figure 1. Antibody characterization and method specificity. Western blotting of cell extracts (CE) and crude membranes (CM) of neocortex indicate that SNAT1 antisera recognize a single band of ∼55 kDa (A). Protein detection is completely abolished by preadsorption of antibodies with 10–3 M GST–SNAT1 (B), but not with 10–3M GST–SNAT2 (C). SNAT1 ir in the SI cortex (A′) is abolished by preincubation with 10–3M GST–SNAT1 fusion protein (B′) but not with 10–3M GST–SNAT2 fusion protein (C′). Scale bar: 100 µm (A′–C′).

Figure 2. SNAT1 ir in SI cortex and neighboring regions. Laminar distribution of SNAT1-immunoreactive cells and processes (A); Roman numerals indicate cortical layers in an adjacent Nissl-stained section (B). Besides cortical cells, SNAT1 ir is localized to small immunoreactive puncta scattered throughout the cortical neuropil (C) and to small cells in the white matter and callosum (D). Intense SNAT1 ir is evident in the leptomeninges (E), in ependymal cells lining the third ventricle (F) and in the choroid plexus (G). Scale bars: 100 µm (A, B); 5 µm (C); 10 µm (DG).

Figure 2. SNAT1 ir in SI cortex and neighboring regions. Laminar distribution of SNAT1-immunoreactive cells and processes (A); Roman numerals indicate cortical layers in an adjacent Nissl-stained section (B). Besides cortical cells, SNAT1 ir is localized to small immunoreactive puncta scattered throughout the cortical neuropil (C) and to small cells in the white matter and callosum (D). Intense SNAT1 ir is evident in the leptomeninges (E), in ependymal cells lining the third ventricle (F) and in the choroid plexus (G). Scale bars: 100 µm (A, B); 5 µm (C); 10 µm (DG).

Figure 3. SNAT1 ir is prevalently neuronal. SNAT1/NeuN colocalization studies show that most SNAT1+ cells in the cortical parenchyma are NeuN+ (A–C), but some (arrows in D and F) are NeuN–. Scale bar: 10 µm (AF).

Figure 3. SNAT1 ir is prevalently neuronal. SNAT1/NeuN colocalization studies show that most SNAT1+ cells in the cortical parenchyma are NeuN+ (A–C), but some (arrows in D and F) are NeuN–. Scale bar: 10 µm (AF).

Figure 4. SNAT1 ir is expressed by a subpopulation of astrocytes. SNAT1/GFAP colocalization studies show that some small SNAT1+ cells (arrows in AC) and processes (arrows in DF) in the cortical parenchyma are GFAP+ and that numerous SNAT1+ cells in the white matter underlying SI coexpress GFAP (G–I). Arrowheads in A point to the leptomeninges. Scale bar: 10 µm (AI).

Figure 4. SNAT1 ir is expressed by a subpopulation of astrocytes. SNAT1/GFAP colocalization studies show that some small SNAT1+ cells (arrows in AC) and processes (arrows in DF) in the cortical parenchyma are GFAP+ and that numerous SNAT1+ cells in the white matter underlying SI coexpress GFAP (G–I). Arrowheads in A point to the leptomeninges. Scale bar: 10 µm (AI).

Figure 5. SNAT1+ neurons in the SI cortex are both pyramidal (A, B) and non-pyramidal (C, D). Scale bar: 10 µm (A–D).

Figure 5. SNAT1+ neurons in the SI cortex are both pyramidal (A, B) and non-pyramidal (C, D). Scale bar: 10 µm (A–D).

Figure 6. SNAT1 expression in GABAergic neurons. SNAT1/GABA colocalization studies show that a sizeable fraction of SNAT1+ neurons (A) express GABA and that all GABA+ neurons express SNAT1 (B, C). Scale bar: 10 µm (AC).

Figure 6. SNAT1 expression in GABAergic neurons. SNAT1/GABA colocalization studies show that a sizeable fraction of SNAT1+ neurons (A) express GABA and that all GABA+ neurons express SNAT1 (B, C). Scale bar: 10 µm (AC).

Figure 7. Axon terminals occasionally express SNAT1. SNAT1/synaptophysin colocalization studies show highly segregated immunoreactivities (A–C), although rare SNAT1/synaptophysin+ puncta are occasionally observed at high magnification (framed regions in D–F are reproduced enlarged in G–I). Scale bars: 10 µm (AF); 2 µm (GI).

Figure 7. Axon terminals occasionally express SNAT1. SNAT1/synaptophysin colocalization studies show highly segregated immunoreactivities (A–C), although rare SNAT1/synaptophysin+ puncta are occasionally observed at high magnification (framed regions in D–F are reproduced enlarged in G–I). Scale bars: 10 µm (AF); 2 µm (GI).

Figure 8. Electron microscopic studies of SNAT1 ir confirm its preferential somatodendritic localization as well as a minor astrocytic and epithelial localization. (A) SNAT1 ir (arrows) in the cytoplasm of a cortical neuron. The asterisk indicates a bouton en passage and the arrowheads the postsynaptic density. (B–H) SNAT1 ir in cortical dendrites of medium (B–D, G, H) and small (E, F) size. Note that immunoreactive dendrites form both symmetric (B, E) and asymmetric (C, D, F–H) synapses with unlabeled axon terminals and boutons en passage (asterisk) and that SNAT1+ dendrites give rise to unlabeled dendritic spines (G, H). (I) One of the rare SNAT1+ axon terminal found in this study. (J) End feet profiles adjacent to the endothelial basal lamina (b). (K, L) SNAT1+ distal astrocytic processes in layers III (K) and V (L); note other astrocytic processes (AsP) shown in D, F and H. (M) Intense SNAT1 ir in cytoplasmic processes (arrows) of an arachnoid cell. AsP, astrocytic process; AxT, axon terminal; B, basal lamina; bv, blood vessel; Den, dendrite; End, endothelial cell; G; Golgi apparatus; Nuc, nucleus; sa, spine apparatus; sp, spine. Scale bars: 5 µm (A); 1 µm (M, I); 0.5 µm (BH, J, L).

Figure 8. Electron microscopic studies of SNAT1 ir confirm its preferential somatodendritic localization as well as a minor astrocytic and epithelial localization. (A) SNAT1 ir (arrows) in the cytoplasm of a cortical neuron. The asterisk indicates a bouton en passage and the arrowheads the postsynaptic density. (B–H) SNAT1 ir in cortical dendrites of medium (B–D, G, H) and small (E, F) size. Note that immunoreactive dendrites form both symmetric (B, E) and asymmetric (C, D, F–H) synapses with unlabeled axon terminals and boutons en passage (asterisk) and that SNAT1+ dendrites give rise to unlabeled dendritic spines (G, H). (I) One of the rare SNAT1+ axon terminal found in this study. (J) End feet profiles adjacent to the endothelial basal lamina (b). (K, L) SNAT1+ distal astrocytic processes in layers III (K) and V (L); note other astrocytic processes (AsP) shown in D, F and H. (M) Intense SNAT1 ir in cytoplasmic processes (arrows) of an arachnoid cell. AsP, astrocytic process; AxT, axon terminal; B, basal lamina; bv, blood vessel; Den, dendrite; End, endothelial cell; G; Golgi apparatus; Nuc, nucleus; sa, spine apparatus; sp, spine. Scale bars: 5 µm (A); 1 µm (M, I); 0.5 µm (BH, J, L).

Figure 9. SNAT1 ir in the human neocortex. (A) Diagram based on the surgeon’s estimate of the cortical resection and on MRI findings showing the approximate location of the cortical samples used in the present study superimposed on Brodmann’s cytoarchitectonic map of the human cerebral cortex. (B) Western blotting analysis of crude membrane of human prefrontal cortex (HBC 4981; areas 9–46) indicates that SNAT1 antisera recognize a single band of about 55 kDa (lane A). Protein detection is abolished by preincubation with 10–3 M GST–SNAT1 fusion protein (lane B), but not with 10–3 M GST–SNAT2 fusion protein (lane C). (C) Laminar distribution of SNAT1 ir in prefrontal cortex (HBC 980511; area 10). (D, E) SNAT1+ neurons in human cortex are both pyramidal (D) and non-pyramidal (E). (F–H) An example of a SNAT1+ neuron that coexpresses GABA. (I–K) SNAT1+ profiles express GFAP (arrows). Scale bars: 100 µm (C); 10 µm (DK).

Figure 9. SNAT1 ir in the human neocortex. (A) Diagram based on the surgeon’s estimate of the cortical resection and on MRI findings showing the approximate location of the cortical samples used in the present study superimposed on Brodmann’s cytoarchitectonic map of the human cerebral cortex. (B) Western blotting analysis of crude membrane of human prefrontal cortex (HBC 4981; areas 9–46) indicates that SNAT1 antisera recognize a single band of about 55 kDa (lane A). Protein detection is abolished by preincubation with 10–3 M GST–SNAT1 fusion protein (lane B), but not with 10–3 M GST–SNAT2 fusion protein (lane C). (C) Laminar distribution of SNAT1 ir in prefrontal cortex (HBC 980511; area 10). (D, E) SNAT1+ neurons in human cortex are both pyramidal (D) and non-pyramidal (E). (F–H) An example of a SNAT1+ neuron that coexpresses GABA. (I–K) SNAT1+ profiles express GFAP (arrows). Scale bars: 100 µm (C); 10 µm (DK).

Table 1


 Summary of clinical data

Case Age (years/sex) Major symptom(s) Pathology Drug/daily dose/duration (days)a 
HBC980510 60/M High intracranial pressure Frontal meningioma Valproate/1000 mg/30 
HBC980611 64/F High intracranial pressure Fronto-orbital meningioma Barbesaclone/100mg/30 
HBC981219 60/F None Frontal metastasis from breast cancer Phenobarbital/100mg/30, dexamethazone 16mg/14 
HBC990222 58/M Visual field defect Temporal fossa meningioma Dexamethazone/16mg/4 
HBC4981b 42/M – None None 
Case Age (years/sex) Major symptom(s) Pathology Drug/daily dose/duration (days)a 
HBC980510 60/M High intracranial pressure Frontal meningioma Valproate/1000 mg/30 
HBC980611 64/F High intracranial pressure Fronto-orbital meningioma Barbesaclone/100mg/30 
HBC981219 60/F None Frontal metastasis from breast cancer Phenobarbital/100mg/30, dexamethazone 16mg/14 
HBC990222 58/M Visual field defect Temporal fossa meningioma Dexamethazone/16mg/4 
HBC4981b 42/M – None None 

aDrugs used in perisurgical prophylactic therapy.

bAutoptic case (Harvard Brain Tissue Resource Center, Belmont, MA; myocardial infarction).

Table 2


 Primary and secondary antibodies

Primary antibody(ies) Dilution(s) Secondary antibody(ies) Dilution(s) 
SNAT1 1:2000 bGARa 1:100 
SNAT1/NeuN 1:1500/1:200 FITC-GARb/TRITC-GAMc 1:100/1:100 
SNAT1/GFAP 1:1500/1:800 FITC-GARb/TRITC-GAMc 1:100/1:100 
SNAT1/GABA (rats) 1:1500/1:10000 FITC-GARb/TRITC-GAMc 1:100/1:100 
SNAT1/GABA (humans) 1:1500/1:500 TRITC-GARd/FITC-GAMe 1:100/1:100 
SNAT1/Syn 1:1500/1:1000 FITC-GARb/TRITC-GAMc 1:100/1:100 
Primary antibody(ies) Dilution(s) Secondary antibody(ies) Dilution(s) 
SNAT1 1:2000 bGARa 1:100 
SNAT1/NeuN 1:1500/1:200 FITC-GARb/TRITC-GAMc 1:100/1:100 
SNAT1/GFAP 1:1500/1:800 FITC-GARb/TRITC-GAMc 1:100/1:100 
SNAT1/GABA (rats) 1:1500/1:10000 FITC-GARb/TRITC-GAMc 1:100/1:100 
SNAT1/GABA (humans) 1:1500/1:500 TRITC-GARd/FITC-GAMe 1:100/1:100 
SNAT1/Syn 1:1500/1:1000 FITC-GARb/TRITC-GAMc 1:100/1:100 

aBiotinylated goat anti-rabbit (BA-1000/NO731; Vector).

bFluorescein isothiocyanate-conjugated goat anti-rabbit (FI1000/J0114; Vector).

cTetramethylrhodamine isothiocyanate-conjugated goat anti-mouse (T-2762/6691-1; Molecular Probes).

dTetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit (T-2769/83A1-1; Molecular Probes).

eFluorescein isothiocyanate-conjugated goat anti-mouse (F-2761/6592–1; Molecular Probes).

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