Localization of the Na+-coupled neutral amino acid transporter 2 in the cerebral cortex

Neuroscience 140 (2006) 281–292

LOCALIZATION OF THE Naⴙ-COUPLED NEUTRAL AMINO ACID TRANSPORTER 2 IN THE CEREBRAL CORTEX M. MELONE,a H. VAROQUI,b J. D. ERICKSONb AND F. CONTIa*

and GABA transmitter pools through the glutamate– glutamine cycle. The strong expression of Naⴙ-coupled neutral amino acid transporter 2 in the somato-dendritic compartment and in non-neuronal elements that are integral parts of the blood– brain and brain– cerebrospinal fluid barrier suggests that Naⴙ-coupled neutral amino acid transporter 2 plays a role in regulating the levels of Gln and other amino acids in the metabolic compartment of cortical neurons. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Neuroscience (Section of Physiology), Università Politecnica delle Marche, Via Tronto 10/A, Torrette di Ancona, I-60020 Ancona, Italy b Neuroscience Center, LA State University Health Sciences Center, New Orleans, LA 70112, USA

Abstract—We studied the distribution and cellular localization of Naⴙ-coupled neutral amino acid transporter 2, a member of the system A family of amino acid transporters, in the rat and human cerebral cortex using immunocytochemical methods. Naⴙ-coupled neutral amino acid transporter 2-positive neurons were pyramidal and non-pyramidal, and Naⴙcoupled neutral amino acid transporter 2/GABA double-labeling studies revealed that Naⴙ-coupled neutral amino acid transporter 2 was highly expressed by GABAergic neurons. Double-labeling studies with the synaptophysin indicated that rare axon terminals express Naⴙ-coupled neutral amino acid transporter 2. Naⴙ-coupled neutral amino acid transporter 2-immunoreactivity was also found in astrocytes, leptomeninges, ependymal cells and choroid plexus. Electron microscopy showed robust Naⴙ-coupled neutral amino acid transporter 2-immunoreactivity in the somato-dendritic compartment of neurons and in glial processes, but, as in the case of double-labeling studies, failed to reveal Naⴙ-coupled neutral amino acid transporter 2-immunoreactivity in terminals. To rule out the possibility that the absence of Naⴙcoupled neutral amino acid transporter 1-[Melone M, Quagliano F, Barbaresi P, Varoqui H, Erickson JD, Conti F (2004) Localization of the glutamine transporter SNAT1 in rat cerebral cortex and neighboring structures, with a note on its localization in human cortex. Cereb Cortex 14:562–574] and Naⴙ-coupled neutral amino acid transporter 2-positive terminals was due to insufficient antigen detection, we evaluated Naⴙ-coupled neutral amino acid transporter 1/synaptophysin and Naⴙ-coupled neutral amino acid transporter 2/synaptophysin coexpression using non-standard immunocytochemical procedures and found that Naⴙ-coupled neutral amino acid transporter 1 and Naⴙ-coupled neutral amino acid transporter 2ⴙ terminals were rare in all conditions. These findings indicate that Naⴙ-coupled neutral amino acid transporter 1 and Naⴙ-coupled neutral amino acid transporter 2 are virtually absent in cortical terminals, and suggest that they do not contribute significantly to replenishing the Glu

Key words: glutamine– glutamate cycle, glutamate, neutral amino acids, rat, human.

Glutamine (Gln) contributes to the synthesis of the neurotransmitter pools of glutamate (Glu) and GABA, the major excitatory and inhibitory neurotransmitters in the cerebral cortex (Conti and Weinberg, 1999; Cherubini and Conti, 2001), through the “Glu–Gln” cycle, in which Glu released from axon terminals is taken up by astrocytes and converted to Gln, which is then extruded from astrocytes and finally taken up by axon terminals (Hertz et al., 1983; Hertz and Schousboe, 1988; Erecinska and Silver, 1990; Danbolt, 2001). Functional and general mapping studies suggest that Gln efflux from astrocytes is mediated by the system N transporter Na⫹-coupled neutral amino acid transporter (SNAT) 3 (or SN1; Chaudhry et al., 1999), and that two members of the family of Na⫹-coupled neutral amino acid transporters, SNAT1 (also known as GlnT, SAT1, ATA1, or SA2; see Mackenzie and Erickson, 2004 for the nomenclature) and SNAT2 (SAT2, ATA2, or SA1), mediate the influx of Gln into neurons (Varoqui et al., 2000; Albers et al., 2001; Chaudhry et al., 2002b; Yao et al., 2000; Sugawara et al., 2000; Reimer et al., 2000). Whereas this notion is supported by the demonstration that SNAT3 is indeed localized to perisynaptic astrocytes (Boulland et al., 2002; Chaudhry et al., 2002a), the recent observation that, in the cerebral cortex, SNAT1 is localized to the somatodendritic compartment of glutamatergic and GABAergic neurons (as well as to astrocytes and non-neuronal cells), but not to axon terminals (Mackenzie et al., 2003; Melone et al., 2004), suggests that this transporter is hardly suited to sustain the Gln influx that contributes to the transmitter pools of Glu and GABA (see also Rae et al., 2003). SNAT2 is a predicted 504 amino acid protein of 55 kDa and 11 putative transmembrane domains that shares 55% homology with SNAT1. Functionally, SNAT2 exhibits voltage- and Na⫹-dependent, pH-sensitive transport, and carries several neutral amino acids, including Gln and Ala (the efficiency of Ala transport being approximately four times

*Corresponding author. Tel: ⫹39-071-220-6056; fax: ⫹39-071-220-6052. E-mail address: [email protected] (F. Conti). Abbreviations: BBB, blood– brain barrier; FITC, fluorescein isothiocyanate; GA, glutaraldehyde; GFAP, glial fibrillary acidic protein; Gln, glutamine; Glu, glutamate; GST, glutathione S-transferase; ir, immunoreactivity; NeuN, neuronal nuclei; NGS, normal goat serum; PB, phosphate buffer; PBS, phosphate-buffered saline; PFA, paraformaldehyde; RT, room temperature; SI, first somatic sensory cortex; SNATs, Na⫹-coupled neutral amino acid transporters; Syn, synaptophysin; TRITC, tetramethylrhodamine isothiocyanate.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.02.042

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Table 1. Summary of clinical data Case

Cytoarch. area

Age (y/sex)

Major symptom

Pathology

Drug/daily dose/duration (days)a

HBC980510 HBC980611 HBC981219

46 10 9

60/M 64/F 60/F

High intracranial pressure High intracranial pressure None

Frontal meningioma Fronto-orbital meningioma Frontal metastasis from breast cancer

HBC990222 HBC040925

21 7

58/M 58/F

Visual field defect Visual field defect

Temporal fossa meningioma Parasagittal meningioma

Valproate/1000 mg/30 Barbesaclone/100 mg/30 Phenobarbital/100 mg/30 Dexametazone/16 mg/14 Dexametazone/16 mg/4 Oxcarbazepine/900 mg/2

a

Drugs used in perisurgical prophylactic therapy.

greater than that of Gln; Yao et al., 2000). Since Ala can be converted to Glu (Yu et al., 1983; Kihara and Kubo, 1989; Peng et al., 1991; Schousboe et al., 2003), SNAT2 could contribute to replenishing the Glu and GABA transmitter pools by transporting both Gln and Ala. These features make SNAT2 an alternative candidate for supplying Glu and GABA precursors to presynaptic terminals. Early studies showed widespread expression of SNAT2 in several brain regions, with a preferential localization to glutamatergic neurons (Reimer et al., 2000; Yao et al., 2000), and a recent general mapping study demonstrated SNAT2 in glutamatergic neurons, some axons, and several non-neuronal elements but, at least in the hippocampus and inferior colliculus, not in GABAergic neurons or axon terminals (Gonzalez-Gonzalez et al., 2005). Given the fundamental role played by glutamatergic and GABAergic transmission in the cerebral cortex in both physiological and pathophysiological conditions (Conti and Hicks, 1996; Martin and Olsen, 2000), the scarce available information on the expression of SNAT2 in the rat cerebral cortex, and the lack of data on its expression in the human cortex, we performed an extensive immunocytochemical analysis of its distribution in the neocortex of rats (with particular emphasis on its localization in axon terminals) and humans.

EXPERIMENTAL PROCEDURES Tissue preparation Rat tissue. Twenty-one adult albino 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 Committee of Università Politecnica delle Marche. Experiments conformed to the guidelines of the Society for Neuroscience. All efforts were made to minimize animal suffering and the number of animals used. For standard immunocytochemistry, rats were anesthetized with chloral hydrate (12%; i.p.) and perfused through the ascending aorta with a flush of physiological saline solution followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4 (standard fixation; n⫽9) or 4% PFA and 0.5% glutaraldehyde (GA) in PB for light microscopy (n⫽2), or by 4% PFA, 0.5% GA and 0.3% picric acid in PB for electron microscopy (n⫽2). Brains were removed and postfixed for 2 h at 4 °C in the same fixative used for perfusion before being cut with a Vibratome. Tissue preparation for non-standard immunocytochemistry was performed as described previously (Melone et al., 2005b). Animals were anesthetized and perfused with saline followed by 1% PFA (weak fixation; n⫽2) or with saline containing 50 ␮M

MG132 (EMD Biosciences, San Diego, CA, USA) and a cocktail of protease inhibitors (1 ␮M pepstatin A; 20 ␮M leupeptin; aprotinin 0.05 trypsin unit/ml and 3.6 ␮M phenylmethylsulphonylfluoride (PMSF; Sigma, Gillingham, UK) followed by 4% PFA (antiproteolytic treatment; n⫽2). Weakly-fixed brains were quickly removed after perfusion and immediately cut; antiproteolytictreated brains were removed and postfixed in 4% PFA for 2 h at 4 °C before cutting. For Western blotting, rats (n⫽4) were anesthetized with chloral hydrate (12%; i.p.); brains were rapidly harvested and the neocortex was separately homogenized in a glass–Teflon homogenizer in 6 vol of ice-cold buffer (4 mM Tris, pH 7.4; 0.32 M sucrose; 1 mM EDTA; 0.23 mM dithiothreitol; and 1 ␮M leupeptin and pepstatin A). Cell extract and crude membrane preparation and determination of protein concentrations were as described previously (Melone et al., 2001). Human tissue. Human cortical tissue came from surgical specimens of five patients (see Table 1 for anatomical localization and relevant clinical data) and has already been used in other studies (Melone et al., 2004, 2005a). 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 sign 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. For immunoblotting, cell extracts of human neocortex were prepared as described previously (Varoqui et al., 2000).

Antibodies Polyclonal antibodies directed to the predicted NH2-terminal portion of SNAT2 (1– 65) and SNAT1 (1– 63) were used. Production and characterization of SNAT2 and SNAT1 antibodies have been reported previously (Varoqui et al., 2000; Yao et al., 2000; see also Melone et al., 2004). In this study we assessed the specificity of SNAT2 antibodies by Western blotting of cell extracts and crude membranes prepared from rat cerebral cortex and of cell extracts of human cortex; and the method specificity by preadsorbing SNAT2 antibodies with 10⫺3, 10⫺4 or 10⫺5 M glutathione Stransferase (GST)-SNAT2 fusion protein and with 10⫺3, 10⫺4 or 10⫺5 M GST-SNAT1 fusion protein. Monoclonal antibodies to neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) and synaptophysin (Syn) were purchased from Chemicon (Temecula, CA, USA; MAB377) and Sigma (St. Louis, MO, USA; GFAP: GA5, G-3893; Syn: clone SVP-38, S-5768), respectively; the monoclonal antibodies to

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Table 2. Primary and secondary antibodies Primary antibody

Dilution

Secondary antibody

Dilution

SNAT2 (rat) SNAT2 (human) SNAT2/NeuN SNAT2/GFAP SNAT2/GABA (rat) SNAT2/GABA (human) SNAT1f SNAT2f SNAT1/Synf SNAT2/Synf

1:1250 1:700 1:1250/1:200 1:1250/1:800 1:1250/1:10000 1:700/1:500 1:1000 1:1250 1:1000/1:1000 1:1250/1:1000

bGARa bGAR FITC-GARb/TRITC-GAMc FITC-GARb/TRITC-GAMc FITC-GARb/TRITC-GAMc TRITC-GARd/FITC-GAMe FITC-GARb FITC-GARb FITC-GARb/TRITC-GAMc FITC-GARb/TRITC-GAMc

1:100 1:100 1:150/1:150 1:150/1:150 1:150/1:150 1:150/1:150 1:150 1:150 1:150/1:150 1:150/1:150

a

Biotinylated goat anti-rabbit (Vector. BA-1000/NO731). FITC-conjugated goat anti-rabbit (Vector. FI1000/J0114). c TRITC-conjugated goat anti-mouse (Molecular Probes, PoortGebouw, The Netherlands, T-2762/6691-1). d TRITC-conjugated goat anti-rabbit (Molecular Probes, T-2769/83A1-1). e FITC-conjugated goat anti-mouse (Molecular Probes, F-2761/6592-1). f Dilutions used for single- and double-labeling studies in standard, pepsin-pretreated, weak-fixed and antiproteolytic-treated material. b

GABA were kindly provided by Dr. C. Matute (Matute and Streit, 1986).

Immunocytochemical procedures Rat brains were cut serially in the coronal plane with a Vibratome into 30/40-␮m-thick sections, which were stored in phosphatebuffered saline (PBS) at 4 °C until processing. Weakly-fixed brains were cut at 50 ␮m and immediately processed for immunocytochemistry. Human cortical blocks were cut in the coronal and parasagittal planes into 40-␮m-thick sections, collected into groups of five and stored at 4 °C in PBS until processing. Of these five sections, one was stained with 0.1% Thionine and the remaining ones were used for immunocytochemistry. SNAT2 immunoperoxidase studies. For light microscopy, free-floating sections from 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 [RT] and overnight at 4 °C) in a solution of blocking buffer (2% normal goat serum [NGS], 2% bovine serum albumin [BSA], 0.01% Tween 20; Melone et al., 2004) containing SNAT2 antibodies (see Table 2 for dilutions). The following day, sections were rinsed three times in PBS and incubated in a solution of blocking buffer containing biotinylated secondary antibodies (Table 2; 1 h at RT). Sections were subsequently rinsed in PBS, incubated in avidin– biotin peroxidase complex (ABC Elite PK6100, Vector, Burlingame, CA, USA), washed several times in PBS, and incubated in 3,3=diaminobenzidine tetrahydrochloride (DAB; 0.05% in 0.05 M Tris 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 rats fixed with 4% PFA, 0.5% GA and 0.3% picric acid were pretreated with 1% sodium borohydride for 30 min to quench non-specific binding, rinsed several times with PBS, treated with H2O2 (1% in PBS; 30 min) to remove endogenous peroxidase, rinsed with PBS and processed as described above except 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 (Sigma, 8F119) -coated coverslips. Small blocks of tissue containing layers I–III, 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 EM 208 electron microscope coupled to a MegaViewII high-resolution CCD camera (Soft Imaging System; Munster, Germany). Identification of neuronal and non-neuronal elements was performed according to Peters et al. (1991). Colocalization studies: SNAT2/NeuN, SNAT2/GFAP, and SNAT2/GABA. SNAT2/NeuN and SNAT2/GFAP colocalization studies were performed on sections fixed in 4% PFA, whereas SNAT2/GABA double-labeling studies were performed on sections from rat brains fixed with 4% PFA and 0.5% GA, and on human cortical sections fixed with 4% PFA. Details of the antibodies used in these studies are listed in Table 2. For SNAT2/NeuN and SNAT2/GFAP studies, sections were incubated in a solution of blocking buffer containing a mixture of SNAT2 and either NeuN or GFAP primary antibodies; for SNAT2/GABA studies, free-floating sections were pretreated with 1% sodium borohydride for 30 min to quench the effects of GA on immunostaining, washed, and incubated in 10% NGS and 0.1% Triton X-100 in 0.01 M PB (1 h), and then in a solution containing a mixture of SNAT1 and GABA primary antibodies (in 1% NGS in PB; 2 h at RT and overnight at 4 °C). Sections were washed and then incubated for 1 h in a solution of blocking buffer with a mixture of affinity-purified fluorescein isothiocyanate (FITC; Vector; FI1000/J0114)- or tetramethylrhodamine isothiocyanate (TRITC; Molecular Probes, PoortGebouw, The Netherlands, T-2762/6691–1) -conjugated secondary antibodies for SNAT2/NeuN and SNAT2/GFAP colocalization, or for 20 min in 10% NGS and then for 1 h in a solution containing a mixture of FITC- and TRITC-conjugated secondary antibodies for SNAT2/GABA studies. Sections were finally washed, mounted, air-dried and coverslipped using Vectashield mounting medium (H-1000; Vector). Control experiments with single-labeled sections and sections incubated with two primary antibodies and one secondary antibody, or with one primary and two secondary antibodies, revealed no appreciable FITC/TRITC bleed-through or antibody cross-reactivity. Colocalization studies: SNAT1/Syn and SNAT2/Syn. For these studies we used sections from 4% PFA-fixed brains (for standard immunocytochemistry and pepsin pretreatment), weakly-fixed and antiproteolytic-treated material (for non-standard immunocytochemistry). Pepsin pretreatment of 4% PFA sections prior to immunoprocessing was performed according to Watanabe et al. (1998); sections were treated for 10 min at 37 °C with pepsin (1–2 mg/ml; DAKO, Carpintera, CA, USA) in 0.2 N HCl, and then washed in PBS. To minimize possible variations in immunostaining, sections prepared under different experimental conditions were processed in parallel. For single-labeling studies, sections

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were incubated in a solution of blocking buffer containing SNAT1 or SNAT2 primary antibody (see Table 2 for dilutions; 2 h at RT and overnight at 4 °C); washed in PB and then incubated in a solution of blocking buffer containing FITC-conjugated secondary antibodies. For double-labeling studies, sections were incubated in a solution of blocking buffer containing a mixture of SNAT1/Syn and SNAT2/Syn primary antibodies (see Table 2 for dilutions). Next, they were rinsed in PB and incubated directly for 1 h in a solution of blocking buffer with a mixture of affinity-purified FITCor TRITC-conjugated secondary antibodies. Sections were washed with PB, mounted, and coverslipped with Vectashield mounting medium. Controls were performed by omitting the primary antibodies. Sections incubated with the two primary antibodies and one secondary antibody, or with one primary antibody and two secondary antibodies, exhibited no appreciable crossreactivity.

Light microscopy and data collection All data were obtained from a region of the 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 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). Immunofluorescent sections 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 and imaged separately and merged using the LaserSharp Processing BioRad software (version 3.2). For SNAT2/NeuN, SNAT2/GFAP, and SNAT2/GABA colocalization rat studies, microscopic fields from SI cortex and underlying white matter (four sections/pair of antigens/hemisphere; two 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 ␮m2) using a confocal pinhole of 1.5–2. To improve the signal/noise ratio, 10 frames/image were averaged by Kalman filtering. SNAT2-, GABA- and NeuN-positive (⫹) cells were counted; merged images of SNAT2/GABA, SNAT2/ NeuN, and SNAT2/GFAP were then used to identify double-labeled cells and profiles. For SNAT2/GABA and SNAT2/GFAP human colocalization studies, confocal microscopy was performed as described for rat tissue. For SNAT1/Syn and SNAT2/Syn studies, random cortical fields from SI (layers II–III; 14 –16 from two sections/animal; two animals/experimental condition) were acquired as 512⫻512 pixel images using a 60⫻ objective with a confocal pinhole of 1.0; microscopic fields were zoomed to provide a pixel size of 180 nm; to improve the signal/noise ratio, 12 frames/image were averaged by Kalman filtering. To assess the degree of SNAT1 and SNAT2/ Syn colocalization, red (Syn) and green (SNAT1 and SNAT2) images were imported into Image J version 1.32i (NIH, Bethesda, MD, USA). As in previous studies (Melone et al., 2004), we applied a threshold for processing the Syn images that yielded the greatest number of individual puncta without causing their fusion. Syn⫹ puncta were automatically counted by Image J; for SNAT1 and SNAT2 images, a threshold was applied to subtract the background noise (Melone et al., 2004, 2005b). Each pair of processed images was then merged and yellow puncta were counted manually.

Western blotting Aliquots (12.5 and 10 ␮g of total protein per lane for rat and human studies, respectively) of cell extracts and crude membranes mixed with equal volumes of 2⫻ electrophoresis sample buffer were

Fig. 1. Antibody characterization and method specificity. The SNAT2 antibody recognizes a single band of ⬃55 kDa in both cellular extracts (CE) and crude synaptic membranes (CM) of rat cerebral cortex (A). SNAT2 ir in SI cerebral cortex (B) is abolished by preincubation with 10⫺3 M GST-SNAT2 fusion protein (C) but not with 10⫺3 M GSTSNAT1 fusion protein (D). Scale bar⫽100 ␮m (B–D).

subjected to SDS-PAGE; separated proteins were electroblotted onto nitrocellulose filters (0.22 ␮m) and finally probed with SNAT2 antibodies (dilution: 1:1250). Labeled bands were visualized with the BioRad Chemidoc and Quantity One software (BioRad version 4.1.1) using the SuperSignal West Pico (Rockford, IL, USA) chemiluminescent substrate. The same procedure was used for the human cortical samples (Melone et al., 2004).

RESULTS The specificity of SNAT2 antibody was demonstrated by Yao et al. (2000) and Armano et al. (2002), and confirmed in the present material by Western blotting of cell extracts and crude membranes from rat neocortex, which showed that SNAT2 antibodies recognized a single band of ⬃55 kDa (Fig. 1A). Method specificity was assessed by showing that SNAT2 immunoreactivity (ir) (Fig. 1B) was totally abolished by preincubation of SNAT2 antibodies with 10⫺3 M GST-SNAT2 fusion protein (Fig. 1C), but unaffected by preadsorption with 10⫺3 M GST-SNAT1 fusion protein (Fig. 1D). In line with previous northern blotting and immunocytochemical studies (Yao et al., 2000; Gonzalez-Gonzalez et al., 2005), SNAT2 ir was intense in olfactory bulb, hippocampus and cerebellum and moderate in thalamus, cerebral cortex, brainstem and striatum. Distribution and localization of SNAT2 ir in rat neocortex Light and confocal microscopy. In SI cortex, SNAT2 ir was mostly in perikarya and proximal processes (Fig. 2A), although immunoreactive profiles and punctate structures were also present in the neuropil (Fig. 2C). Morphologically heterogeneous, non-neuronal cells of variable size (diameter: ⬃5–10 ␮m) in the white matter underlying the cortex and in the corpus callosum (Fig. 2D) were also stained. Strong SNAT2 ir was detected in leptomeninges, ependymal cells of the lateral ventricle and choroid plexus (Fig. 2E–G).

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Fig. 2. Distribution of SNAT2 ir in SI cortex and neighboring structures. Laminar distribution of SNAT2⫹ cells and processes (A); roman numerals indicate cortical layers in an adjacent Nissl-stained section (B). Some SNAT2⫹ puncta are also present in the cortical neuropil (C), and small irregular cells are frequently detected in the white matter underlying the cortex and in corpus callosum (D). SNAT2 immunostaining is also observed in leptomeninges (E), ependymal cells (F) and choroid plexus (G). Scale bars⫽100 ␮m (A, B); 5 ␮m (C); 10 ␮m (D–G).

The nature of SNAT2⫹ cells was addressed by performing double-labeling studies with SNAT2 and NeuN, a protein expressed only by neurons (Mullen et al., 1992). These studies revealed that out of 2004 SNAT2⫹ cortical cells (16 microscopic fields/layer; two animals), 1986 (99.1%) were NeuN⫹ (Fig. 3A–C), whereas only 18 (0.9%) were NeuN-negative (⫺). NeuN⫺ cells were smaller (diameter: ⬃5–10 ␮m) than NeuN⫹ cells and were mostly in layers I and II (Fig. 3D–F). Small, SNAT2⫹ cells were easily detectable in the white matter underlying the cortex; colocalization studies showed that all of them were NeuN⫺. SNAT2⫹/NeuN⫺ cells were investigated with SNAT2/ GFAP colocalization studies. Analysis of layers I–II (24 microscopic fields; two rats) and underlying white matter (24 microscopic fields; two rats) showed that a considerable proportion of small SNAT2⫹ cells (Fig. 3G–L) were GFAP⫹: of 80 SNAT2⫹ cells, 60 (75%) were GFAP⫹, showing that in both cortical parenchyma and white matter this group of cells was made up of astrocytes. Some SNAT2⫹ neuropilar processes were also GFAP⫹ (Fig. 3G–L). SNAT2⫹ neurons were found in all cortical layers, but they were more numerous and intensely stained in layers II–III and V (Fig. 2A and B); most were pyramidal neurons (see Conti et al., 1987, 1992, for criteria) (Fig. 4), although SNAT2⫹ non-pyramidal neurons were also observed (Fig. 4B and C). Since most GABAergic neurons in the cortex are non-pyramidal, the latter observation raises the possi-

bility that SNAT2 is expressed by GABAergic neurons. Confocal microscopic studies of SNAT2/GABA doublestained sections revealed that out of 2430 SNAT2 neurons (16 microscopic fields/layer; two rats), 338 (14.6%) expressed GABA (Fig. 4D–F). We also calculated that 338 of the 416 GABA⫹ neurons sampled were SNAT2⫹ (86.6%). To assess the localization of SNAT2 in axon terminals, we evaluated whether the small SNAT2⫹ punctate structures observed in the cortical parenchyma were axon terminals using double-labeling studies with antibodies to SNAT2 and Syn, a marker of presynaptic axon terminals (Jahn et al., 1985; Wiedenmann and Franke, 1985). Analysis of 192 microscopic fields from all cortical layers (4 fields/layer; two rats) showed that of 317,965 Syn⫹ puncta, only 916 (0.2%) coexpressed SNAT2 (Fig. 5), suggesting that SNAT2 is rarely, if not exceptionally, localized to axon terminals. Electron microscopy. Electron microscopic studies showed that electrondense reaction product was conspicuous and frequent in perikarya (Fig. 6A) and dendritic profiles (Fig. 6B–D). SNAT2⫹ axon terminals were extremely rare: only three in all the material examined (Fig. 6B), in line with the results of Syn/SNAT2 colocalization studies. Moreover, in stained postsynaptic elements SNAT2 ir was always distant from synapses (Fig. 6D). SNAT2⫹ distal astrocytic processes were also observed (Fig. 6E) and intense SNAT2 ir was present in end-feet profiles adjacent to the endothelial basal lamina (Fig. 6F).

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Fig. 3. Neuronal and astrocytic expression of SNAT2. Most SNAT2⫹ cells in the cortical parenchyma are NeuN⫹ (A–C), but some NeuN⫺ cells are also present (arrow in D and F). SNAT2 ir is also colocalized with GFAP in some small-sized cells in upper layers (arrow in G–I) and in some processes (arrowheads in G–I). In the white matter underlying the cortex, SNAT2 is frequently colocalized with GFAP⫹ astrocytes (arrows in J–L) and processes (arrowhead in J–L). Scale bar⫽10 ␮m.

SNAT2 ir in human neocortex In the four human samples used for immunolocalization studies, Nissl- and GFAP-stained sections adjacent to those used for SNAT2 immunocytochemistry were free of any appreciable abnormality (see Conti et al., 1998, 1999; Melone et al., 2005a). Western blottings of cellular extracts prepared from area 7 showed that in human cortex SNAT2 antibodies also recognized a single band of ⬃55 kDa (Fig. 7B). In cytoarchitectonic areas 9, 10, 21 and 46 (Brodmann, 1909; von Economo, 1928; Ong and Garey, 1990; Rajkowska and Goldman-Rakic, 1995), SNAT2 ir was mostly in neuronal cell bodies (Fig. 7A, C and D) and in proximal processes. SNAT2⫹ neurons were present throughout the cortical layers, and pyramidal (Fig. 7C) and non-pyramidal (Fig. 7D) neurons were easily distinguished. Colocalization studies with SNAT2 and GABA or GFAP antibodies

showed that numerous SNAT2⫹ non-pyramidal neurons coexpressed GABA (Fig. 7E–G) and that some SNAT2⫹ elements were also GFAP⫹ (Fig. 7H–J). Effects of improved antigen detection methods on the visualization of SNAT1 and SNAT2 ir in axon terminals The data presented above, together with those from previous studies of SNAT1 localization (Mackenzie et al., 2003; Melone et al., 2004), show that in the cerebral cortex both SNAT2 and SNAT1 are localized in the somatodendritic compartment of glutamatergic and GABAergic cells but not in axon terminals, where a large amount of compelling evidence (see Discussion) indicates that they should be located to efficiently provide Gln (and/or Ala) to presynaptic terminals. The failure to visualize SNAT1 or SNAT2 may depend on the presence of other transporters

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studies using antibodies to SNAT1 and SNAT2 and Syn. To detect precisely the colocalization of the two antigens, all confocal microscopy data were obtained from the surface of immunostained sections (Melone et al., 2005b). Quantitative analysis (14 randomly selected fields of layers II–III from two sections/two animals/experimental condition) revealed that the proportion of SNAT1⫹/Syn⫹ puncta was 0.3%⫾0.06% in standard material (Fig. 8A= and J), 0.6%⫾0.1% in pepsin-treated material (Fig. 8B= and J), 1.1%⫾0.2% in weakly fixed material (Fig. 8C= and J), and 1.0%⫾0.1% in antiproteolytic-treated material (Fig. 8D= and J). Similarly, the proportion of SNAT2⫹/Syn⫹ puncta was 0.8%⫾0.1% in standard material (Fig. 8E= and K), 0.8%⫾0.1% in pepsin-treated material (Fig. 8F= and K), 1.3%⫾0.2 in weakly-fixed material (Fig. 8G= and K), and 0.8%⫾0.1% in antiproteolytic-treated material (Fig. 8H= and K). Overall, these studies show that, even though improved antigen sensitivity yielded a slightly increased proportion of SNAT1⫹ and SNAT2⫹ axon terminals, this localization is quite rare.

DISCUSSION

Fig. 4. SNAT2⫹ neurons in SI cortex are mostly pyramidal (A), although some are non-pyramidal (arrowhead in B; C); numerous SNAT2⫹ non-pyramidal neurons are also GABA⫹ (D–F). A, layer V; B, layer IV; C, layer VI. Scale bars⫽10 ␮m (A–F).

or on insufficient antigen detection. To investigate the possible influence of fixation and procedural variables on the detection of SNAT1 and SNAT2 ir in axon terminals, we investigated SNAT1 and SNAT2 immunostaining using non-standard immunocytochemical procedures, i.e. pepsin treatment, weak fixation, and antiproteolytic treatment, which increase antigen detection of synaptic proteins in the intact brain (Watanabe et al., 1998; Valtschanoff et al., 2000; Melone et al., 2005b). In pepsin-treated material, both SNAT1 and SNAT2 ir were reduced in the dendritic compartment, even though in some cases the intensity of SNAT1 cellular staining was increased (Fig. 8B); neuropilar staining, which includes positive puncta, was moderately increased (Fig. 8B and F). In weakly fixed material, the intensity of somato-dendritic labeling was slightly increased (Fig. 8C and G). In sections from tissue treated with the antiproteolytic agents, SNAT1 and SNAT2 ir appeared increased in both the dendritic and neuropilar compartments (Fig. 8D and H). To assess the expression of SNAT1 and SNAT2 in axon terminals with standard and non-standard immunocytochemical procedures, we performed double-labeling

The major results of the present study are: i) in the cerebral cortex SNAT2 is predominantly localized to the somatodendritic compartment of both glutamatergic and GABAergic neurons and virtually absent in axon terminals, in line with the in vitro studies of Armano et al. (2002) and the results from in vivo studies in other brain regions (Gonzalez-Gonzalez et al., 2005). SNAT2 is also expressed in cortical astrocytes and other non-neuronal cells; ii) the scarce expression of SNAT1 and SNAT2 in axon terminals in intact brain is unlikely to be determined by insufficient antigen detection as shown by studies performed using non-standard immunocytochemical procedures. Consistently with previous in vitro and in vivo studies (Yao et al., 2000; Armano et al., 2002; Gonzalez-Gonzalez et al., 2005), we found intense SNAT2 ir in glutamatergic pyramidal cortical neurons. We also noted that a sizeable proportion of non-pyramidal neurons express SNAT2⫹, and that these neurons are GABAergic. In an immmunocytochemical study of the distribution of SNAT2 ir in the entire rat brain, Gonzalez-Gonzalez et al. (2005) failed to detect SNAT2 in GABAergic neurons. At least two factors can account for this discrepancy. First, we used a polyclonal antiserum made in rabbit and directed against the N-terminal hydrophilic portion 1– 65 of rat SNAT2 (Yao et al., 2000), whereas Gonzalez-Gonzalez et al. (2005) used polyclonal antisera made in guinea-pig directed against 48 residues of the N-amino terminus of rat SNAT2. Secondly, we pretreated sections for SNAT2/GABA colocalization studies with sodium borohydride, which improves immunodetection in GA–PFA fixed-tissue by quenching GAinduced fluorescence (Tagliaferro et al., 1997; Casella et al., 2004). Besides cortical neurons, SNAT2 is also expressed by astrocytes and several non-neuronal cells. Similarly to our previous SNAT1 studies, and in line with the recent obser-

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Fig. 5. SNAT2/Syn double-labeling studies show highly segregated immunostaining of the two antigens (A–C); Syn⫹ puncta rarely coexpress SNAT2 (D–F; arrowhead in enlarged framed region, G–I). Scale bars⫽10 ␮m (A–F); 2 ␮m (G–I).

vation of SNAT2 ir in astrocytic somata of periaqueductal gray matter and cerebellum (Gonzalez-Gonzalez et al., 2005), we detected SNAT2⫹ astrocytes in the neocortex and underlying white matter; in particular, dense reaction

product detected in distal astrocytic processes and endfeet profiles demonstrates a widespread, albeit not intense expression of SNAT2 in cortical astrocytes. Thus, the present data and those obtained on the localization of

Fig. 6. Pre-embedding electron microscopic studies of SNAT2. Electron-dense reaction product is preferentially localized to the cytoplasm of neurons (A) and dendrites (B–D). D shows a SNAT2⫹ dendrite making an asymmetric synapse (arrowheads) with an unlabeled axon terminal; note that electron-dense material in the dendrite is far from the synaptic contact. The arrow in B indicates one of the few SNAT2⫹ axon terminals found in this study. E and F show SNAT2 ir in a distal astrocytic process (E) and in an astrocytic end-feet profile adjacent to the endothelial basal lamina (F). Asp, astrocytic process; Axt, axon terminal; End, endothelial basal lamina. Scale bars⫽5 ␮m (A), 0.5 ␮m (B–F).

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Fig. 7. SNAT2 ir in human neocortex. (A) Distribution of SNAT2 ir in the prefrontal cortex (HBC 980510; area 10). (B) SNAT2 antibody recognizes a single band of ⬃55 kDa in cellular extracts of human cortex (HBC 040925, area 7). SNAT2⫹ neurons in human cortex are both pyramidal (C) and non-pyramidal (D), and some of the latter coexpress GABA (E–G). Astrocytic cell bodies (arrowhead in H–J) and processes (arrow in H–J) both coexpress GFAP. Scale bars⫽100 ␮m (A); 10 ␮m (C–J).

SNAT1 in the neocortex (Melone et al., 2004) suggest that astrocytes do not mediate only SNAT3 Gln efflux, but contribute to Gln and Ala influx by SNAT1 and SNAT2. The expression of SNAT2 in glial end-feet profiles adjacent to the endothelial basal lamina of cortical blood vessels is in line with the notion that system A activity would contribute to Gln transport at the blood– brain barrier (BBB; Betz and Goldstein, 1978; Sanchez del Pino et al., 1995). SNAT2 is strongly expressed in non-neuronal cells, including astrocytic end-feet profiles adjacent to the endothelial basal lamina of cortical blood vessels, leptomeningeal, ependymal and choroid plexus cells, many of these cells are integral parts of the BBB and brain– cerebrospinal fluid (CSF) barrier, whose fundamental function is to regulate the composition of the extracellular neuronal milieu (e.g. Harandi et al., 1986; Brightman, 1989; Peters et al., 1991; Del Bigio, 1995; Vorbrodt and Dobrogowska, 2003; Franchi-Gazzola et al., 2004; Tanaka et al., 2005). Thus, the present findings and those reported in our previous SNAT1 study (Melone et al., 2004) suggest that non-neuronal cell SNAT1 and SNAT2 may contribute to neuronal homeostasis by regulating the influx of Gln and other neutral amino acids into neurons. The Glu–Gln cycle is held to be a crucial pathway for the replenishment of the Glu neurotransmitter pool in glutamatergic and GABAergic nerve terminals (Reubi, 1980; Bradford et al., 1983; Hertz, 1979; Hertz et al., 1983; Hertz and Schousboe, 1988; Erecinska and Silver, 1990; Conti and Minelli, 1994; Danbolt, 2001), and Ala can be converted to Glu (Yu et al., 1983; Kihara et al., 1989; Peng et

al., 1991). In line with this role of Gln (and Ala), the expression of SNAT1 and SNAT2 would be expected to be high in axon terminals, even though it has been argued that expression of SNAT1 and/or SNAT2 at axon terminals may not be a mandatory requirement for Glu/GABA recycling (Mackenzie and Erickson, 2004). Nevertheless, the strong correlation so far demonstrated between the expression of synaptically active molecules and their need (Chaudhry et al., 1995; Sheng, 2003; see also Chaudhry et al., 2002a) suggests that in order to support neurotransmitter release, Gln uptake is more likely to occur at axon terminals. Studies performed in many brain regions (Mackenzie et al., 2003; Gonzalez-Gonzalez et al., 2005) and the cerebral cortex (Melone et al., 2004; and present results) have so far failed to confirm this prediction, indicating that in the intact brain these transporters are mainly confined to non-synaptic sites. It is well known that fixation and other procedural variables can influence the detection of synaptic protein in the intact brain (Watanabe et al., 1998; Valtschanoff et al., 2000; Chen et al., 2004; Melone et al., 2005b), and several studies have shown that non-standard procedures can enhance the detection and localization of proteins (e.g. NMDA receptors subunits; Watanabe et al., 1998; Melone et al., 2005b; GLT-1; Chen et al., 2004; Melone and Conti, unpublished observations). In this study, we applied three different non-standard procedures (pepsin treatment, weak fixation and antiproteolytic treatment) to enhance SNAT1 and SNAT2 detection and noted that in all conditions their expression in axon terminals was relatively stable and consistently low (⬍1.5% of all Syn⫹

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Fig. 8. Effects of improved antigen detection on SNAT1 and SNAT2/Syn colocalization. The first column shows SNAT1 (A), SNAT1/Syn (A=), SNAT2 (E), and SNAT2/Syn (E=) in conventionally prepared material; the second (B–F=), third (C–G=), and fourth (D–H=) columns show the same sequence in material pretreated with pepsin, weakly fixed, or pretreated with antiproteolytics, respectively. In A=–D= and E=–H= arrowheads indicate puncta exhibiting SNAT1⫹/Syn⫹ or SNAT2⫹/Syn⫹ colocalization, respectively. Histograms show the percentage of Syn⫹ puncta expressing SNAT1 (J) or SNAT2 (K) in standard (std), pepsin-treated (pep), weakly-fixed (wf), and antiproteolytic-treated (ap) material. Scale bars⫽10 ␮m for A–D and E–H; 2 ␮m for A=–D= and E=–H=.

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terminals). These results seem to exclude a role for insufficient antigen detection, and to indicate that, at least in the cerebral cortex, SNAT1 and SNAT2 are not crucial for the replenishment of the Glu and GABA neurotransmitter pools. In this respect, Rae et al. (2003) have recently suggested that the activity of the neuronal system A might not have a prominent role in neurotransmitter cycling, and that its activity mainly mediates the transfer of Gln and Ala into a large turnover amino acid pool located in neuronal perikarya. The most likely explanation for our findings is therefore that other Gln transporters, either existing orphan transporters or ones yet to be identified, mediate Gln influx in the axon terminals of cortical neurons, thus sustaining glutamatergic transmission. Acknowledgments—We are grateful to A. Ducati for providing the surgical samples, C. Matute for generously providing the GABA antibodies, and Luca Antognini, Michele Bellesi, and Fiorinta Quagliano for technical assistance. This work was supported by grants from MIUR (COFIN 03) and the Stanley Medical Research Institute to F.C.

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(Accepted 1 February 2006) (Available online 17 April 2006)