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Neuronal localization of the GABA transporter GAT-3 in human cerebral cortex: A procedural artifact?
Journal of Chemical Neuroanatomy 30 (2005) 45–54 www.elsevier.com/locate/jchemneu
Neuronal localization of the GABA transporter GAT-3 in human cerebral cortex: A procedural artifact? Marcello Melone, Paolo Barbaresi, Giorgia Fattorini, Fiorenzo Conti * Department of Neurosciences, Section of Physiology, Universita` Politecnica delle Marche, Via Tronto 10/A, Torrette di Ancona, I-60020 Ancona, Italy Received 31 January 2005; received in revised form 8 April 2005; accepted 8 April 2005 Available online 31 May 2005
Abstract Gamma-amino butyric acid (GABA) plasma membrane transporters (GATs) contribute to the modulation of GABA’s actions and are implicated in neuropsychiatric diseases. In this study, the localization of GAT-3, the major glial GAT, was investigated in human cortex using immunocytochemical techniques. In prefrontal and temporal cortices, GAT-3 immunoreactivity (ir) was present throughout the depth of the cortex, both in puncta and in neurons. GAT-3-positive puncta were dispersed in the neuropil or closely related to cell bodies; neuronal staining was in perikarya, especially of pyramidal cells, and proximal dendrites. Electron microscopic studies showed that GAT-3 ir was in astrocytic processes as well as in neuronal elements. All GAT-3-positive neurons co-expressed heat shock protein 70. To test the possibility that the collection procedure of human samples induced the expression of GAT-3 in neurons which normally do not express it, we analyzed rat cortical tissue resected using the same procedure and found that numerous neurons are GAT-3-positive and that they co-express heat shock protein 70. Results show that in human cortex GAT-3 is expressed in astrocytic processes and in neurons and suggest that neuronal expression is related to the procedure used for collecting human samples. # 2005 Elsevier B.V. All rights reserved. Keywords: GABA uptake; Astrocytes; Neurons; Ischemia
1. Introduction Gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the human cortex (McCormick, 1989; McCormick et al., 1993; Haugstad et al., 1992; Hornung and De Tribolet, 1995), where it plays an important role in physiological and in several pathophysiological conditions, including epilepsy (Meldrum, 1989; Treiman, 2001), schizophrenia (Lewis et al., 1999; Benes and Berretta, 2001), and brain ischemia (Schwartz-Bloom and Sah, 2001). Several studies have provided detailed information on cortical GABAergic neurons, their synaptic relationships and ontogeny (Kisvarday et al., 1990; Hornung and De Tribolet, 1994; Zecevic and Milosevic, 1997), and the expression of GABA receptors (Akbarian et al., 1995). * Corresponding author. Tel.: +39 071 220 6056; fax: +39 071 220 6052. E-mail address: [email protected] (F. Conti). 0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2005.04.002
The effects of GABA on synaptic transmission also depend on the action of the plasma membrane transporters that mediate high-affinity Na+/Cl-dependent uptake of GABA into nerve endings and/or surrounding glial processes and appear to control the deactivation kinetics of inhibitory postsynaptic currents (IPSCs) as well as GABA spillover (Cherubini and Conti, 2001). Of the four GABA transporters (GATs) that have been characterized so far in the nervous system (GAT-1, GAT-2, GAT-3, and BGT-1; Borden, 1996; Conti et al., 2004), GAT-1 and GAT-3 appear to be able to influence synaptic transmission (Minelli et al., 1995, 1996; Conti et al., 1998; Jensen et al., 2003); GAT-2 seems to be more involved in the regulation of extracellular GABA levels (Conti et al., 1999a); and BGT-1 is apparently not expressed in the CNS. Investigations of the distribution and localization of GAT-1 (Conti et al., 1998; DeFelipe and Gonzalez-Albo, 1998; Ong et al., 1998) have provided useful data for studies of the role of GATs in the cerebral cortex of epileptic (DeFelipe, 1999;
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Orozco-Suarez et al., 2000; Calcagnotto et al., 2002; Arellano et al., 2004) and schizophrenic (Volk et al., 2001, 2002) patients. Since similar information on the expression of GAT3 is not available, we used immunocytochemistry with affinity-purified antibodies to determine the distribution and cellular localization of GAT-3 in the human cerebral cortex.
2. Materials and methods 2.1. Tissue preparation Human cortical tissue came from the surgical specimens of eight patients with brain tumors whose clinical data are summarized in Table 1. Two of the eight samples have also been used in a previous study (Conti et al., 1999b). Informed consent to the surgical procedure was obtained in all cases. The cortical tissue used in this study was not located in the vicinity of the tumor: it was macroscopically normal tissue that had to be resected in order to reach deep-seated tumors or was included in ‘‘tactical lobectomies’’, and showed no signs of edema. None of the patients suffered from pre- or postoperative seizures. Three additional samples of cortical tissue came from the autoptic specimens (PMI: 20 h; #200227) of a 20-year-old man without a record of neuropsychiatric disorders who died for the sequelae of a motorcycle accident. The approximate localization of all samples is shown in Fig. 1. Twelve adult albino rats (Sprague–Dawley; Charles River, Milan, Italy) weighing 180–250 g were also used in these studies. Care and handling of the animals were approved by the Animal Research Committee of Universita` Politecnica delle Marche. Seven rats were deeply anesthetized with 30% cloral hydrate; the skull and dura were opened and a large portion of one hemisphere (three rats) or both hemispheres (four rats) was resected, replicating the procedure adopted to collect the human samples. The time between the first cut of the neocortex and the immersion of the tissue in the fixative solution varied between 2 and 3 min. These samples were used either for immunocytochemistry (seven) or western blotting (four) studies (see below). The remaining five rats were used as controls and perfused transcardially, as described previously (Minelli et al., 1996; Melone et al., 2003).
Fig. 1. Diagram based on the surgeon’s estimate of the cortical resection and on magnetic resonance imaging 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. Modified from Brodmann (1909).
In both rat and human immunocytochemical studies, specimens were immediately immersed in a cold solution of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 2–3 h and then transferred to a fresh solution of 4% PFA for 24–48 h at 4 8C. Samples were cut into small blocks that were further postfixed for 24–72 h at 4 8C in the same solution. They were cut on a Vibratome in coronal and parasagittal planes into 30–35 mm thick sections which were collected in groups of 10 and stored at 4 8C in PB until processing. Of the 10 sections, one was stained for Nissl with 0.1% thionine and the remaining nine were used for immunocytochemistry. For Western blotting, rats 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). 2.2. Antibodies Affinity-purified polyclonal antibodies directed to the predicted C-terminus of rat GAT-3 (rGAT-3607–627; Borden
Table 1 Summary of clinical data Case #
Age (years)/sex
Major symptom (s)
Pathology
Drug/daily dose/duration (d)a
HBC980510 HBC980611 HBC980913 HBC981114 HBC981219 HBC990222 HBC040924 HBC040925
60/M 64/F 60/F 65/M 60/F 58/M 71/M 58/F
High intracranial pressure High intracranial pressure High intracranial pressure Absent Absent Visual field defect Visual field defect Visual field defect
Frontal meningioma Fronto-orbital meningioma Temporal fossa meningioma Temporal fossa meningioma Frontal metastasis from breast cancer Temporal fossa menigioma Temporal fossa menigioma Parasagittal meningioma
Valproate/1000 mg/30 Barbesaclone/100 mg/30 Phenobarbital/100 mg; Dexametazone/24 mg/21 Phenobarbital/100 mg/14 Phenobarbital/100 mg; Dexametazone/16 mg/14 Dexametazone/16 mg/4 Oxcarbazepine/1200 mg/2 Oxcarbazepine/900 mg/2
a
Drugs used in perisurgical prophylactic therapy.
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et al., 1992; Clark et al., 1992) and GAT-1 (rGAT-1588–599; Guastella et al., 1990) were used (GAT-3: 369-D and 374-E; GAT-1: 341-F and 346-M) as described previously (Minelli et al., 1996, for GAT-3; Conti et al., 1998 for GAT-1). Monoclonal antibodies to glial fibrillary acid protein (GFAP) and heat shock protein 70 (HSP70) were purchased from Sigma (St. Louis, MO; clones GA-5, G-3893/1014– 4800 and BRM-22, H-5147/029H4838, respectively). Specificity of GAT-3 antibodies was assessed by preadsorbing GAT-3 antibodies with 105 M rat GAT-3607–627 peptide (Minelli et al., 1996) and with 105 to 103 M human C-terminus GAT-3612–632 peptide (CDAKLKSDGTIAAITEKETHF; Borden et al., 1994), purchased from PeptidoGenic Research & Co. (Livermore, CA; lot #81204A161). 2.3. Immunocytochemical procedures For immunoperoxidase studies, free-floating sections were pretreated in 1% H2O2 for 30 min to remove endogenous peroxidase activity, rinsed in PB and preincubated in 10% normal goat serum (NGS; 1 h) containing 0.3% Triton X-100 (GAT-3 and GAT-1), or in 1% NGS containing 0.3% Triton X-100 (GFAP). Sections were then incubated in primary antiserum (2 h at room temperature and overnight at 4 8C) at dilutions of 1:300–1:500 (GAT-3); 1:200–1:500 (GAT-1) and 1:1.000 (GFAP). Sections were
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rinsed in PB and incubated in 10% NGS (GAT-3 and GAT-1 series) or 2% NGS (GFAP series), then for 1 h in biotinylated goat anti-rabbit IgG (bGAR, BA 1000/J0901; GAT-3, GAT-1 and HSP70 series; dilution 1:100) or antimouse secondary antibody (bGAM, BA 9200/K0212, Vector Laboratories, Burlinghame, CA; for GFAP; dilution 1:200), rinsed in PB, incubated in avidin–biotin peroxidase complex (ABC; Vector) for 30 min and washed in PB. They were incubated in 0.08% 3-30 -diaminobenzidine tetrahydrochloride (DAB; Carlo Erba, Milan, Italy) with 0.02% H2O2 in 0.05 M Tris buffer, pH 7.6, washed in PB, mounted on gelatin-coated slides, air dried, washed, dehydrated, and finally coverslipped. Method specificity was tested by substituting the primary antibody with PB or NGS. The same procedure was used in the rat studies, except that anti-GAT-1 antibodies were used at a dilution of 1:1000. For immunofluorescence, free-floating sections were incubated first in PBS (10 mM, NaCl 0.15 M, pH 7.8) containing 10% NGS (1 h) and then in a mixture of GAT-3 (1:500) and HSP70 (1:800) antisera (2 h at room temperature and overnight at 4 8C). Sections were washed in PBS, incubated first in a 10% NGS solution and then for 1 h in PBS containing a mixture of fluorescein isothiocyanateconjugated goat anti-rabbit affinity-purified secondary antibody (FITC; Vector; FI1000/JO114) and tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse
Fig. 2. GAT-3 ir in human cerebral cortex. (A) GAT-3 ir in layers I–III of area 21. (B) GAT-3 ir disappears following preabsorption with human GAT-3612–632. (C) GAT-3+ puncta outline unlabeled cell bodies. (D) GAT-3+ neurons. (E) GAT-1 ir is confined to puncta. (F) GAT-3 antibodies reveal a single band of 70 kDa. Cases #980613 (area 21; A, B, and D and E), 981219 (area 9; C), and 040925 (area 7; F). Bar: 100 mm for A, B and E, 10 mm for C and D.
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affinity-purified secondary antibody (TRITC; Molecular Probes, PoortGebouw, The Netherlands; T-2762/6691-1) at dilution of 1:100. Control experiments, in which one or both primary antibodies were substituted with PBS and one or both secondary antibodies (FITC/TRITC) were substituted with PBS, were performed to verify method specificity. 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 with the 488 and 543 nm lines, respectively, imaged separately (using 515/30 and 570 nm filters for FITC and TRITC, respectively) and merged using the LaserSharp Processing BioRad software (version 3.2). The fields of interest were scanned using a 60 Nikon Plan Apo oil immersion objective with a numerical aperture of 1.4 and images were acquired on a 512 pixel 512 pixel box using a confocal pinhole of 2–3. To improve signal-tonoise ratio, 10 frames of each image were averaged by Kalman filtering. 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 and one secondary antibody or with one primary and two secondary antibodies revealed no appreciable crossreactivity. For electron microscopic studies, a mild ethanol pretreatment (10, 25, and 10%; 5 min each) was used before immunocytochemistry. After completion of the immunocytochemical procedure (without Triton X-100), sections were prepared for electron microscopy. They were flat-embedded in Epon-Spurr, processes as in previous studies (Conti et al., 1999b), and examined with a Philips CM10 electron microscope. Identification of neuronal and non-neuronal elements was performed according to Peters et al. (1991). Some blocks were cut into 2 mm sections and used for quantitative analysis of immunoreactive neurons. Only neurons displaying a nucleolus were tallied. Cytoarchitectonic areas and boundaries were identified on adjacent thionine-stained sections following the descriptions of Brodmann (1909); Economo von (1928); Ong and Garey (1990), and Rajkowska and Goldman-Rakic (1995). 2.4. Western blotting The neocortex was separately homogenized with 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 mM leupeptin and pepstatin A). Cell extract preparation and protein concentration determination were performed as described in previous publications (Melone et al., 2004). Aliquots (10 mg of total protein per lane in rat
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GAT-3 studies; 40 mg in human GAT-3studies; and 30 mg in HSP70 studies) of cell extracts mixed with equal volumes of 2 electrophoresis sample buffer were subjected to SDSPAGE; separated proteins were electroblotted onto nitrocellulose filters (0.22 mm) and finally probed with GAT-3 (dilution: 1:400 in human studies; 1:500 in rat studies) or HSP70 (dilution: 1:750) antibodies. Labeled bands were visualized by the BioRad Chemidoc and Quantity One software (BioRad version 4.1.1) using the ECL western blotting detection reagents and analysis system (Amersham Bioscience, Little Chalfont, UK).
3. Results In all specimens, examination of Nissl-stained sections adjacent to those used for immunocytochemistry revealed that they were devoid of any appreciable abnormalities and consistent with previous descriptions (Ong and Garey, 1990; Rajkowska and Goldman-Rakic, 1995; Conti et al., 1998, 1999b). In sections processed for GFAP, immunoreactive cells and processes were numerous in layer I and white matter and rare in layers II–VI, in line with its distribution in normal tissue (Hansen et al., 1987; Marco et al., 1996). In all cases studied, GAT-3 ir was present throughout the depth of the cortex (Fig. 2A) and was both in puncta and neurons. GAT-3 ir was abolished when GAT-3 antibodies were substituted with NGS and when they were blocked with both rat GAT-3607–627 and human GAT-3612–632 (Fig. 2B). GAT-3 positive (+) puncta were in all cortical layers and were observed both dispersed in the neuropil and in close relation to cell bodies; in the latter case, they outlined the somata of both pyramidal and nonpyramidal cells (Fig. 2C and D). In layers II–III and V, GAT-3+ puncta were also in close association with basal and apical dendrites of pyramidal cells. Only in some cases did we detect the patches described in rat neocortex (Minelli et al., 1996). Neuronal staining was in perikarya and proximal dendrites; the intense labeling in most cases allowed to distinguish pyramidal from nonpyramidal neurons (Conti et al., 1987, 1992 for criteria): the former appeared to be the more numerous in all sections. Semithin (2 mm) sections from cases HBC990222, HBC981114, HBC980611, and HBC980510 (four to six sections/case) were used to gather quantitative data. This analysis showed that GAT-3+ neurons were 310 out of 1.426 neurons sampled (21.7%). To rule out the possibility that the tissue used in the present studies was unsuitable for immunocytochemical studies, we studied GAT-1 ir in sections adjacent to those processed for GAT-3, and observed that it exhibited a normal distribution (Conti et al., 1998) and was not localized to
Fig. 3. Ultrastructural localization of GAT-3 in human cerebral cortex. (A) GAT-3 ir in the cytoplasm of a cortical pyramidal neuron; the framed region is shown enlarged in (B). In addition, GAT-3 ir (arrow) is observed in the cytoplasm of a dendrite (C), in an axon (asterisk in D), and in axon terminals (arrowheads indicate postsynaptic densities). (I–K) GAT-3 ir in distal astrocytic processes (arrows). AT, axon terminal; AsP, distal astrocytic process; Den, dendrite; Lf, lipofuscin bodies; Nuc, nucleus; sa, spine apparatus; sp, spine. Case #981219. Bar: 5 mm for (A and D); 1 mm for (B and C); and 0.5 mm for (E–K).
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neurons (Fig. 2E). We also performed western blotting studies to investigate whether GAT-3 ir in human cortex depends on cross-reaction with unknown proteins and found that in homegenates from human cortex, GAT-3 antibodies recognized a single band of 70 kDa (Fig. 2F). Electron microscopic studies showed that the immunoreaction product indicating GAT-3 ir appeared as electrondense patches of variable size, often apposed to plasma membranes (Fig. 3). GAT-3 ir was localized to distal astrocytic processes (Fig. 3D, and I–K), which surrounded labeled (Fig. 3J) and unlabeled (Fig. 3I and K) neuropilar profiles. In some cases, GAT-3+ distal astrocytic processes were in intimate association with axon terminals forming asymmetric synapses (Fig. 3K), but they were more commonly not associated with synapses (Fig. 3D, I and J). Besides its expected astrocytic localization, GAT-3 ir was also observed in neurons and neuronal processes (Fig. 3A and C–H). In the large majority of cases, GAT-3+ neurons were pyramidal (Fig. 3A), although some nonpyramidal cells were also observed. In both cell types, the reaction product was widely distributed in the cytoplasm, forming small patches on nuclear and cytoplasmic envelopes and on the membranes of cytoplasmic organelles (Fig. 3B). GAT-3 ir was also observed in proximal and distal dendritic processes, where it was associated with microtubules, mitochondria and agranular reticulum (Fig. 3A and C). GAT3 ir was observed in axons and axon terminals (Fig. 3D–H); in axon terminals, it was on the cytoplasmic side of the plasmalemma and of synaptic membranes (Fig. 3E–H). GAT-3+ axon terminals formed exclusively asymmetric synapses (Fig. 3E–H); in most cases these axon terminals contacted unlabeled dendritic profiles and spines (Fig. 3E– H), even though they occasionally contacted labeled dendrites. These studies thus indicate that whereas in rat cortex GAT-3 is expressed exclusively in distal astrocytic processes (Minelli et al., 1996), in human cortex it is also expressed by neurons. To ascertain whether GAT-3+ neurons are a feature of the organization of human cortex or an effect of the procedure employed to collect human cortical samples (e.g., an alteration of gene expression secondary to the oxygen/ energy deprivation attending resection; Kanthan et al., 1995), we conducted further studies of human and rat cortex. Since HSP70, the major inducible heat shock protein, is robustly expressed in neurons in response to heat, heavy metals, ischemia and other stresses (Sharp et al., 1999), we investigated whether in our samples HSP70+ neurons were present and whether GAT-3+ cortical neurons coexpressed HSP70 and found that HSP70+ neurons were numerous (Fig. 4A) and that all of the 170 GAT-3+ neurons (from 58 images of cases #980510, 980519 [bioptic] and 200227 [autoptic]) were also HSP70+ (Fig. 4B and C). Next, we verified whether replication in rats of the procedure used to collect human cortical samples induced the expression of GAT-3 in neurons which do not normally express it (Minelli et al., 1996; Melone et al., 2003).
Fig. 4. Cortical neurons from human samples express HSP70 (A) and all GAT-3+ cortical neurons are also HSP70+ (B and C). Case #980510. Bar: 50 mm for (A), 10 mm for (B and C).
Accordingly, we removed about half the hemisphere, the average size of human samples, in adult rats and processed it exactly like the human resections using the same reagents in identical conditions. In all the animals we observed numerous, mostly pyramidal, GAT-3+ neurons (Fig. 5A) in all cortical layers. Western blotting from perfused (control) and resected cortices showed that in both conditions GAT-3 antibodies revealed a single band of 70 kDa (Fig. 5B). Adjacent sections processed for the visualization of GAT-1 ir (Fig. 5C) showed it to be as described in rats perfused transcardially (Minelli et al., 1995; Melone et al., 2003). Next, we used immunocytochemistry and western blotting to study whether cortical resection in adult rats induced HSP70. HSP70 ir in the cerebral cortex of control rats was localized to some dendrites and cell bodies, mostly of pyramydal neurons in layers II–III and V, in line with previous studies which suggested that this constitutive HSP70 may contribute to synaptic function (Moon et al., 2001). Both immunocytochemistry and western blotting (Fig. 5D and E) showed that HSP70 was indeed increased in the animals sujected to resections. Quantitative analysis of western blotting showed that the induction was statistically significant (P = 0.0286; Mann–Whitney). Finally, we studied whether in the samples resected from adult rats, GAT-3+ neurons were also immunoreactive for HSP70. We found that whereas in controls GAT-3 and HSP70 ir are totally segregated (Fig. 5F and G), in the resected samples all GAT-3+ neurons are also HSP70+ (Fig. 5H and I), as in human cortex.
4. Discussion The present studies showed that in several cytoarchitectonic areas of human cerebral cortex the plasma membrane
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Fig. 5. Replication in rats of the procedure used for collecting human samples induced the appearance of numerous GAT-3+ neurons (A) in the cerebral cortex, but did not change the distribution of GAT-1 in an adjacent section. (B) Western blottings on resected samples (Res) reveal a single band of 70 kDa, similar to that obtained in rats perfused transcardially (Ctr). The same procedure induces HSP70 expression (D–F) in the cerebral cortex. G–J show that whereas in controls GAT-3 and HSP70 ir are segregated (G and H), in samples of resected cortices neurons GAT-3+ neurons are also HSP70+ (I and J). Bar: (A and C) 25 mm; (D and E) 60 mm; (F–I) 10 mm.
GABA transporter GAT-3 is localized both to distal astrocytic processes and to neurons. Since in previous investigations we showed GAT-3 to be localized to distal astrocytic processes but not to cortical neurons of rat neocortex (Minelli et al., 1996), the question arose whether these observations reflected species differences in the
organization of cortical GABA uptake or methodological/ procedural biases. The total lack of ir following preincubation of the antibodies with human C-terminal peptides indicates that our antibodies recognize an epitope of the GAT-3 sequence; most importantly, the observation that GAT-3 antibodies
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recognize a single band in homogenates from human cerebral cortex indicate that immunoreactivity is highly specific and in particular it rules out a cross-reaction between the GAT-3 antibodies and unknown proteins expressed in human, but not rat, neocortex. The specificity of GAT-3 staining in the human cortex thus ascertained, we considered the possibility that neuronal expression of GAT-3 could be related to the collection procedure of cortical tissue. In particular, we surmised that the energy and oxygen deprivation associated with the surgical procedure could trigger expression of GAT-3 in neurons that normally do not express it. This hypothesis rested on three considerations: (i) cortical resection as performed in the present study lead to an acute ischemic situation in the resected brain and is often used as a model of acute focal ischemia (Kanthan et al., 1995); (ii) in many instances fixation by immersion yields perikaryal staining in cases in which fixation by perfusion does not (e.g., Tamura et al., 2000); and (iii) in the rat neocortex, transient middle cerebral artery occlusion (MCAO) triggers expression of GAT-3 in neurons that are immunoreactive for HSP70, the major inducible heat-shock protein (Melone et al., 2003). Li et al. (2004) recently observed that gene expression in postmortem human brains is correlated with tissue pH: individuals who suffered prolonged agonal state have lower pH in the brain, whereas those who died rapidly had normal pH. Our sample is too limited in number to verify whether such a correlation holds also for GAT-3 neuronal expression; it is worth noting however that we observed GAT-3 ir in neurons also in the cerebral cortex of an individual who died from a car accident. Thus, even if we cannot rule out the possibility that tissue acidity triggered GAT-3 neuronal expression, the evidence in our hands suggests that this does not seem to be the case. To test the hypothesis that energy and oxygen deprivation associated with the surgical procedure trigger neuronal expression of GAT-3, we first studied the expression of HSP70 and then investigated GAT3/HSP70 coexpression and found that the density of HSP70+ neurons paralleled that of GAT-3+ neurons, and, most importantly, that all GAT-3+ neurons coexpressed HSP70. These studies show that expression of GAT-3 by neurons that usually do not express the protein occurs in cells subjected to metabolic stress and suggest that neuronal expression of GAT-3 in cortical neurons may depend on the collection procedure employed. The presence of GAT-3+ neurons in one autoptic case we had the opportunity to study strongly supports this view. Since, for obvious reasons, the positive control cannot be performed, we replicated in rats the surgical procedure used for human cortical resection and found that it does induces both HSP70 and the appearance of numerous GAT-3+ neurons in the neocortex of rats, where GAT-3 ir is normally confined to distal astrocytic processes (Minelli et al., 1996). The observation that GAT-3 can be visualized in neurons whose gene expression has been altered (as indicated by HSP70 positivity) does not necessarily imply that GAT-3 visualized in those conditions
originates directly from the translation of the GAT-3 gene. Indeed, the presence of untranscribed mRNA in CNS neurons has been documented (e.g., Steward and Schuman, 2003) and GAT-3 mRNA has been reported in some central neurons (Clark et al., 1992; Durkin et al., 1995); and protein synthesis may persist for some minutes while the tissue is in fixative. This latter scenario would be in line with the short time lapse between the beginning of resection and the immersion in the fixative solution. Overall, our studies show that in human cerebral cortex GAT-3 is expressed in distal astrocytic processes, as in rats, as well as in cortical neurons. They also suggest that GAT-3 expression in human cortical neurons is connected with the collection procedure. The implications of these results for the validity of human cortical studies and for our understanding of the localization and regulation of GATs in the cerebral cortex can be summarized as follows: 1. From the methodological point of view, the present findings indicate that if other antigens are as susceptible as GAT-3 to factors connected with the procedures employed in human cortical studies, ‘‘species differences’’ in localization studies should be addressed with great caution. 2. The observations that GAT-3 is expressed in astrocytes and that its neuronal expression is in all likelihood due to nonphysiological causes suggest that its localization in the human cortex is similar to the one demonstrated in rats; furthermore, given that GAT-1 is similarly expressed in rats and humans (Minelli et al., 1995; Conti et al., 1998), these data indicate that the localization of the GABA transporters in the cerebral cortex may be similar in different species. 3. The finding that in both human and rat cortex GAT-3 expression is altered by procedural variables (cortical resection) and by transient MCAO (Melone et al., 2003), whereas in the very same experimental situations (and in adjacent sections) GAT-1 expression is unchanged seems to point at significant functional differences between the two major cortical GATs. In particular, it appears that the expression of the hard-wired, synaptic GAT-1 is less susceptible than that of GAT-3 to changes in the composition of the extracellular milieu, possibly pointing to an important contribution of the mechanisms regulating GAT-3 expression in the adaptive capabilities of neurons (Conti et al., 2004).
Acknowledgments We are grateful to A. Ducati, M. Scerrati and A. Tagliabracci for providing the surgical and autoptic material, to N.C. Brecha for the generous gift of GAT-3 antibodies, to J. DeFelipe for helpful comments on an earlier version of this manuscript, and to L. Bragina and F. Quagliano for
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technical assistance. This work was supported by grants from MIUR (COFIN 01) to F.C.
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Sharp, F.R., Massa, S.M., Swanson, R.A., 1999. Heat shock protein protection. Trends Neurosci. 22, 97–99. Steward, O., Schuman, E.M., 2003. Compartmentalized synthesis and degradation of proteins in neurons. Neuron 40, 347–359. Tamura, T., Said, S., Lu, W.W., Neufeld, D., 2000. Specificity of TUNEL method depends on duration of fixation. Biotech. Histochem. 75, 197– 200. Treiman, D.M., 2001. GABAergic mechanisms in epilepsy. Epilepsia 42 (Suppl. 3), 8–12. Volk, D.W., Mark, M.S., Austin, C., Pierri, J.N., Sampson, A.R., Lewis, D.A., 2001. GABA transporter-1 mRNA in the prefrontal cortex in schizophrenia: decreased expression in a subset of neurons. Am. J. Psychiatr. 158, 256–265. Volk, D.W., Pierri, J.N., Fritschy, J.-M., Auh, S., Sampson, A.R., Lewis, D.A., 2002. Reciprocal alterations in pre- and postsynaptic inhibitory markers at chandelier cell inputs to pyramidal neurons in schizophrenia. Cereb. Cortex 12, 1063–1067. Zecevic, N., Milosevic, A., 1997. Initial development of g-aminobutyiric acid immunoreactivity in the human cerebral cortex. J. Comp. Neurol. 380, 495–506.
Neuronal localization of the GABA transporter GAT-3 in human cerebral cortex: A procedural artifact? Marcello Melone, Paolo Barbaresi, Giorgia Fattorini, Fiorenzo Conti * Department of Neurosciences, Section of Physiology, Universita` Politecnica delle Marche, Via Tronto 10/A, Torrette di Ancona, I-60020 Ancona, Italy Received 31 January 2005; received in revised form 8 April 2005; accepted 8 April 2005 Available online 31 May 2005
Abstract Gamma-amino butyric acid (GABA) plasma membrane transporters (GATs) contribute to the modulation of GABA’s actions and are implicated in neuropsychiatric diseases. In this study, the localization of GAT-3, the major glial GAT, was investigated in human cortex using immunocytochemical techniques. In prefrontal and temporal cortices, GAT-3 immunoreactivity (ir) was present throughout the depth of the cortex, both in puncta and in neurons. GAT-3-positive puncta were dispersed in the neuropil or closely related to cell bodies; neuronal staining was in perikarya, especially of pyramidal cells, and proximal dendrites. Electron microscopic studies showed that GAT-3 ir was in astrocytic processes as well as in neuronal elements. All GAT-3-positive neurons co-expressed heat shock protein 70. To test the possibility that the collection procedure of human samples induced the expression of GAT-3 in neurons which normally do not express it, we analyzed rat cortical tissue resected using the same procedure and found that numerous neurons are GAT-3-positive and that they co-express heat shock protein 70. Results show that in human cortex GAT-3 is expressed in astrocytic processes and in neurons and suggest that neuronal expression is related to the procedure used for collecting human samples. # 2005 Elsevier B.V. All rights reserved. Keywords: GABA uptake; Astrocytes; Neurons; Ischemia
1. Introduction Gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the human cortex (McCormick, 1989; McCormick et al., 1993; Haugstad et al., 1992; Hornung and De Tribolet, 1995), where it plays an important role in physiological and in several pathophysiological conditions, including epilepsy (Meldrum, 1989; Treiman, 2001), schizophrenia (Lewis et al., 1999; Benes and Berretta, 2001), and brain ischemia (Schwartz-Bloom and Sah, 2001). Several studies have provided detailed information on cortical GABAergic neurons, their synaptic relationships and ontogeny (Kisvarday et al., 1990; Hornung and De Tribolet, 1994; Zecevic and Milosevic, 1997), and the expression of GABA receptors (Akbarian et al., 1995). * Corresponding author. Tel.: +39 071 220 6056; fax: +39 071 220 6052. E-mail address: [email protected] (F. Conti). 0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2005.04.002
The effects of GABA on synaptic transmission also depend on the action of the plasma membrane transporters that mediate high-affinity Na+/Cl-dependent uptake of GABA into nerve endings and/or surrounding glial processes and appear to control the deactivation kinetics of inhibitory postsynaptic currents (IPSCs) as well as GABA spillover (Cherubini and Conti, 2001). Of the four GABA transporters (GATs) that have been characterized so far in the nervous system (GAT-1, GAT-2, GAT-3, and BGT-1; Borden, 1996; Conti et al., 2004), GAT-1 and GAT-3 appear to be able to influence synaptic transmission (Minelli et al., 1995, 1996; Conti et al., 1998; Jensen et al., 2003); GAT-2 seems to be more involved in the regulation of extracellular GABA levels (Conti et al., 1999a); and BGT-1 is apparently not expressed in the CNS. Investigations of the distribution and localization of GAT-1 (Conti et al., 1998; DeFelipe and Gonzalez-Albo, 1998; Ong et al., 1998) have provided useful data for studies of the role of GATs in the cerebral cortex of epileptic (DeFelipe, 1999;
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Orozco-Suarez et al., 2000; Calcagnotto et al., 2002; Arellano et al., 2004) and schizophrenic (Volk et al., 2001, 2002) patients. Since similar information on the expression of GAT3 is not available, we used immunocytochemistry with affinity-purified antibodies to determine the distribution and cellular localization of GAT-3 in the human cerebral cortex.
2. Materials and methods 2.1. Tissue preparation Human cortical tissue came from the surgical specimens of eight patients with brain tumors whose clinical data are summarized in Table 1. Two of the eight samples have also been used in a previous study (Conti et al., 1999b). Informed consent to the surgical procedure was obtained in all cases. The cortical tissue used in this study was not located in the vicinity of the tumor: it was macroscopically normal tissue that had to be resected in order to reach deep-seated tumors or was included in ‘‘tactical lobectomies’’, and showed no signs of edema. None of the patients suffered from pre- or postoperative seizures. Three additional samples of cortical tissue came from the autoptic specimens (PMI: 20 h; #200227) of a 20-year-old man without a record of neuropsychiatric disorders who died for the sequelae of a motorcycle accident. The approximate localization of all samples is shown in Fig. 1. Twelve adult albino rats (Sprague–Dawley; Charles River, Milan, Italy) weighing 180–250 g were also used in these studies. Care and handling of the animals were approved by the Animal Research Committee of Universita` Politecnica delle Marche. Seven rats were deeply anesthetized with 30% cloral hydrate; the skull and dura were opened and a large portion of one hemisphere (three rats) or both hemispheres (four rats) was resected, replicating the procedure adopted to collect the human samples. The time between the first cut of the neocortex and the immersion of the tissue in the fixative solution varied between 2 and 3 min. These samples were used either for immunocytochemistry (seven) or western blotting (four) studies (see below). The remaining five rats were used as controls and perfused transcardially, as described previously (Minelli et al., 1996; Melone et al., 2003).
Fig. 1. Diagram based on the surgeon’s estimate of the cortical resection and on magnetic resonance imaging 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. Modified from Brodmann (1909).
In both rat and human immunocytochemical studies, specimens were immediately immersed in a cold solution of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 2–3 h and then transferred to a fresh solution of 4% PFA for 24–48 h at 4 8C. Samples were cut into small blocks that were further postfixed for 24–72 h at 4 8C in the same solution. They were cut on a Vibratome in coronal and parasagittal planes into 30–35 mm thick sections which were collected in groups of 10 and stored at 4 8C in PB until processing. Of the 10 sections, one was stained for Nissl with 0.1% thionine and the remaining nine were used for immunocytochemistry. For Western blotting, rats 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). 2.2. Antibodies Affinity-purified polyclonal antibodies directed to the predicted C-terminus of rat GAT-3 (rGAT-3607–627; Borden
Table 1 Summary of clinical data Case #
Age (years)/sex
Major symptom (s)
Pathology
Drug/daily dose/duration (d)a
HBC980510 HBC980611 HBC980913 HBC981114 HBC981219 HBC990222 HBC040924 HBC040925
60/M 64/F 60/F 65/M 60/F 58/M 71/M 58/F
High intracranial pressure High intracranial pressure High intracranial pressure Absent Absent Visual field defect Visual field defect Visual field defect
Frontal meningioma Fronto-orbital meningioma Temporal fossa meningioma Temporal fossa meningioma Frontal metastasis from breast cancer Temporal fossa menigioma Temporal fossa menigioma Parasagittal meningioma
Valproate/1000 mg/30 Barbesaclone/100 mg/30 Phenobarbital/100 mg; Dexametazone/24 mg/21 Phenobarbital/100 mg/14 Phenobarbital/100 mg; Dexametazone/16 mg/14 Dexametazone/16 mg/4 Oxcarbazepine/1200 mg/2 Oxcarbazepine/900 mg/2
a
Drugs used in perisurgical prophylactic therapy.
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et al., 1992; Clark et al., 1992) and GAT-1 (rGAT-1588–599; Guastella et al., 1990) were used (GAT-3: 369-D and 374-E; GAT-1: 341-F and 346-M) as described previously (Minelli et al., 1996, for GAT-3; Conti et al., 1998 for GAT-1). Monoclonal antibodies to glial fibrillary acid protein (GFAP) and heat shock protein 70 (HSP70) were purchased from Sigma (St. Louis, MO; clones GA-5, G-3893/1014– 4800 and BRM-22, H-5147/029H4838, respectively). Specificity of GAT-3 antibodies was assessed by preadsorbing GAT-3 antibodies with 105 M rat GAT-3607–627 peptide (Minelli et al., 1996) and with 105 to 103 M human C-terminus GAT-3612–632 peptide (CDAKLKSDGTIAAITEKETHF; Borden et al., 1994), purchased from PeptidoGenic Research & Co. (Livermore, CA; lot #81204A161). 2.3. Immunocytochemical procedures For immunoperoxidase studies, free-floating sections were pretreated in 1% H2O2 for 30 min to remove endogenous peroxidase activity, rinsed in PB and preincubated in 10% normal goat serum (NGS; 1 h) containing 0.3% Triton X-100 (GAT-3 and GAT-1), or in 1% NGS containing 0.3% Triton X-100 (GFAP). Sections were then incubated in primary antiserum (2 h at room temperature and overnight at 4 8C) at dilutions of 1:300–1:500 (GAT-3); 1:200–1:500 (GAT-1) and 1:1.000 (GFAP). Sections were
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rinsed in PB and incubated in 10% NGS (GAT-3 and GAT-1 series) or 2% NGS (GFAP series), then for 1 h in biotinylated goat anti-rabbit IgG (bGAR, BA 1000/J0901; GAT-3, GAT-1 and HSP70 series; dilution 1:100) or antimouse secondary antibody (bGAM, BA 9200/K0212, Vector Laboratories, Burlinghame, CA; for GFAP; dilution 1:200), rinsed in PB, incubated in avidin–biotin peroxidase complex (ABC; Vector) for 30 min and washed in PB. They were incubated in 0.08% 3-30 -diaminobenzidine tetrahydrochloride (DAB; Carlo Erba, Milan, Italy) with 0.02% H2O2 in 0.05 M Tris buffer, pH 7.6, washed in PB, mounted on gelatin-coated slides, air dried, washed, dehydrated, and finally coverslipped. Method specificity was tested by substituting the primary antibody with PB or NGS. The same procedure was used in the rat studies, except that anti-GAT-1 antibodies were used at a dilution of 1:1000. For immunofluorescence, free-floating sections were incubated first in PBS (10 mM, NaCl 0.15 M, pH 7.8) containing 10% NGS (1 h) and then in a mixture of GAT-3 (1:500) and HSP70 (1:800) antisera (2 h at room temperature and overnight at 4 8C). Sections were washed in PBS, incubated first in a 10% NGS solution and then for 1 h in PBS containing a mixture of fluorescein isothiocyanateconjugated goat anti-rabbit affinity-purified secondary antibody (FITC; Vector; FI1000/JO114) and tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse
Fig. 2. GAT-3 ir in human cerebral cortex. (A) GAT-3 ir in layers I–III of area 21. (B) GAT-3 ir disappears following preabsorption with human GAT-3612–632. (C) GAT-3+ puncta outline unlabeled cell bodies. (D) GAT-3+ neurons. (E) GAT-1 ir is confined to puncta. (F) GAT-3 antibodies reveal a single band of 70 kDa. Cases #980613 (area 21; A, B, and D and E), 981219 (area 9; C), and 040925 (area 7; F). Bar: 100 mm for A, B and E, 10 mm for C and D.
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affinity-purified secondary antibody (TRITC; Molecular Probes, PoortGebouw, The Netherlands; T-2762/6691-1) at dilution of 1:100. Control experiments, in which one or both primary antibodies were substituted with PBS and one or both secondary antibodies (FITC/TRITC) were substituted with PBS, were performed to verify method specificity. 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 with the 488 and 543 nm lines, respectively, imaged separately (using 515/30 and 570 nm filters for FITC and TRITC, respectively) and merged using the LaserSharp Processing BioRad software (version 3.2). The fields of interest were scanned using a 60 Nikon Plan Apo oil immersion objective with a numerical aperture of 1.4 and images were acquired on a 512 pixel 512 pixel box using a confocal pinhole of 2–3. To improve signal-tonoise ratio, 10 frames of each image were averaged by Kalman filtering. 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 and one secondary antibody or with one primary and two secondary antibodies revealed no appreciable crossreactivity. For electron microscopic studies, a mild ethanol pretreatment (10, 25, and 10%; 5 min each) was used before immunocytochemistry. After completion of the immunocytochemical procedure (without Triton X-100), sections were prepared for electron microscopy. They were flat-embedded in Epon-Spurr, processes as in previous studies (Conti et al., 1999b), and examined with a Philips CM10 electron microscope. Identification of neuronal and non-neuronal elements was performed according to Peters et al. (1991). Some blocks were cut into 2 mm sections and used for quantitative analysis of immunoreactive neurons. Only neurons displaying a nucleolus were tallied. Cytoarchitectonic areas and boundaries were identified on adjacent thionine-stained sections following the descriptions of Brodmann (1909); Economo von (1928); Ong and Garey (1990), and Rajkowska and Goldman-Rakic (1995). 2.4. Western blotting The neocortex was separately homogenized with 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 mM leupeptin and pepstatin A). Cell extract preparation and protein concentration determination were performed as described in previous publications (Melone et al., 2004). Aliquots (10 mg of total protein per lane in rat
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GAT-3 studies; 40 mg in human GAT-3studies; and 30 mg in HSP70 studies) of cell extracts mixed with equal volumes of 2 electrophoresis sample buffer were subjected to SDSPAGE; separated proteins were electroblotted onto nitrocellulose filters (0.22 mm) and finally probed with GAT-3 (dilution: 1:400 in human studies; 1:500 in rat studies) or HSP70 (dilution: 1:750) antibodies. Labeled bands were visualized by the BioRad Chemidoc and Quantity One software (BioRad version 4.1.1) using the ECL western blotting detection reagents and analysis system (Amersham Bioscience, Little Chalfont, UK).
3. Results In all specimens, examination of Nissl-stained sections adjacent to those used for immunocytochemistry revealed that they were devoid of any appreciable abnormalities and consistent with previous descriptions (Ong and Garey, 1990; Rajkowska and Goldman-Rakic, 1995; Conti et al., 1998, 1999b). In sections processed for GFAP, immunoreactive cells and processes were numerous in layer I and white matter and rare in layers II–VI, in line with its distribution in normal tissue (Hansen et al., 1987; Marco et al., 1996). In all cases studied, GAT-3 ir was present throughout the depth of the cortex (Fig. 2A) and was both in puncta and neurons. GAT-3 ir was abolished when GAT-3 antibodies were substituted with NGS and when they were blocked with both rat GAT-3607–627 and human GAT-3612–632 (Fig. 2B). GAT-3 positive (+) puncta were in all cortical layers and were observed both dispersed in the neuropil and in close relation to cell bodies; in the latter case, they outlined the somata of both pyramidal and nonpyramidal cells (Fig. 2C and D). In layers II–III and V, GAT-3+ puncta were also in close association with basal and apical dendrites of pyramidal cells. Only in some cases did we detect the patches described in rat neocortex (Minelli et al., 1996). Neuronal staining was in perikarya and proximal dendrites; the intense labeling in most cases allowed to distinguish pyramidal from nonpyramidal neurons (Conti et al., 1987, 1992 for criteria): the former appeared to be the more numerous in all sections. Semithin (2 mm) sections from cases HBC990222, HBC981114, HBC980611, and HBC980510 (four to six sections/case) were used to gather quantitative data. This analysis showed that GAT-3+ neurons were 310 out of 1.426 neurons sampled (21.7%). To rule out the possibility that the tissue used in the present studies was unsuitable for immunocytochemical studies, we studied GAT-1 ir in sections adjacent to those processed for GAT-3, and observed that it exhibited a normal distribution (Conti et al., 1998) and was not localized to
Fig. 3. Ultrastructural localization of GAT-3 in human cerebral cortex. (A) GAT-3 ir in the cytoplasm of a cortical pyramidal neuron; the framed region is shown enlarged in (B). In addition, GAT-3 ir (arrow) is observed in the cytoplasm of a dendrite (C), in an axon (asterisk in D), and in axon terminals (arrowheads indicate postsynaptic densities). (I–K) GAT-3 ir in distal astrocytic processes (arrows). AT, axon terminal; AsP, distal astrocytic process; Den, dendrite; Lf, lipofuscin bodies; Nuc, nucleus; sa, spine apparatus; sp, spine. Case #981219. Bar: 5 mm for (A and D); 1 mm for (B and C); and 0.5 mm for (E–K).
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neurons (Fig. 2E). We also performed western blotting studies to investigate whether GAT-3 ir in human cortex depends on cross-reaction with unknown proteins and found that in homegenates from human cortex, GAT-3 antibodies recognized a single band of 70 kDa (Fig. 2F). Electron microscopic studies showed that the immunoreaction product indicating GAT-3 ir appeared as electrondense patches of variable size, often apposed to plasma membranes (Fig. 3). GAT-3 ir was localized to distal astrocytic processes (Fig. 3D, and I–K), which surrounded labeled (Fig. 3J) and unlabeled (Fig. 3I and K) neuropilar profiles. In some cases, GAT-3+ distal astrocytic processes were in intimate association with axon terminals forming asymmetric synapses (Fig. 3K), but they were more commonly not associated with synapses (Fig. 3D, I and J). Besides its expected astrocytic localization, GAT-3 ir was also observed in neurons and neuronal processes (Fig. 3A and C–H). In the large majority of cases, GAT-3+ neurons were pyramidal (Fig. 3A), although some nonpyramidal cells were also observed. In both cell types, the reaction product was widely distributed in the cytoplasm, forming small patches on nuclear and cytoplasmic envelopes and on the membranes of cytoplasmic organelles (Fig. 3B). GAT-3 ir was also observed in proximal and distal dendritic processes, where it was associated with microtubules, mitochondria and agranular reticulum (Fig. 3A and C). GAT3 ir was observed in axons and axon terminals (Fig. 3D–H); in axon terminals, it was on the cytoplasmic side of the plasmalemma and of synaptic membranes (Fig. 3E–H). GAT-3+ axon terminals formed exclusively asymmetric synapses (Fig. 3E–H); in most cases these axon terminals contacted unlabeled dendritic profiles and spines (Fig. 3E– H), even though they occasionally contacted labeled dendrites. These studies thus indicate that whereas in rat cortex GAT-3 is expressed exclusively in distal astrocytic processes (Minelli et al., 1996), in human cortex it is also expressed by neurons. To ascertain whether GAT-3+ neurons are a feature of the organization of human cortex or an effect of the procedure employed to collect human cortical samples (e.g., an alteration of gene expression secondary to the oxygen/ energy deprivation attending resection; Kanthan et al., 1995), we conducted further studies of human and rat cortex. Since HSP70, the major inducible heat shock protein, is robustly expressed in neurons in response to heat, heavy metals, ischemia and other stresses (Sharp et al., 1999), we investigated whether in our samples HSP70+ neurons were present and whether GAT-3+ cortical neurons coexpressed HSP70 and found that HSP70+ neurons were numerous (Fig. 4A) and that all of the 170 GAT-3+ neurons (from 58 images of cases #980510, 980519 [bioptic] and 200227 [autoptic]) were also HSP70+ (Fig. 4B and C). Next, we verified whether replication in rats of the procedure used to collect human cortical samples induced the expression of GAT-3 in neurons which do not normally express it (Minelli et al., 1996; Melone et al., 2003).
Fig. 4. Cortical neurons from human samples express HSP70 (A) and all GAT-3+ cortical neurons are also HSP70+ (B and C). Case #980510. Bar: 50 mm for (A), 10 mm for (B and C).
Accordingly, we removed about half the hemisphere, the average size of human samples, in adult rats and processed it exactly like the human resections using the same reagents in identical conditions. In all the animals we observed numerous, mostly pyramidal, GAT-3+ neurons (Fig. 5A) in all cortical layers. Western blotting from perfused (control) and resected cortices showed that in both conditions GAT-3 antibodies revealed a single band of 70 kDa (Fig. 5B). Adjacent sections processed for the visualization of GAT-1 ir (Fig. 5C) showed it to be as described in rats perfused transcardially (Minelli et al., 1995; Melone et al., 2003). Next, we used immunocytochemistry and western blotting to study whether cortical resection in adult rats induced HSP70. HSP70 ir in the cerebral cortex of control rats was localized to some dendrites and cell bodies, mostly of pyramydal neurons in layers II–III and V, in line with previous studies which suggested that this constitutive HSP70 may contribute to synaptic function (Moon et al., 2001). Both immunocytochemistry and western blotting (Fig. 5D and E) showed that HSP70 was indeed increased in the animals sujected to resections. Quantitative analysis of western blotting showed that the induction was statistically significant (P = 0.0286; Mann–Whitney). Finally, we studied whether in the samples resected from adult rats, GAT-3+ neurons were also immunoreactive for HSP70. We found that whereas in controls GAT-3 and HSP70 ir are totally segregated (Fig. 5F and G), in the resected samples all GAT-3+ neurons are also HSP70+ (Fig. 5H and I), as in human cortex.
4. Discussion The present studies showed that in several cytoarchitectonic areas of human cerebral cortex the plasma membrane
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Fig. 5. Replication in rats of the procedure used for collecting human samples induced the appearance of numerous GAT-3+ neurons (A) in the cerebral cortex, but did not change the distribution of GAT-1 in an adjacent section. (B) Western blottings on resected samples (Res) reveal a single band of 70 kDa, similar to that obtained in rats perfused transcardially (Ctr). The same procedure induces HSP70 expression (D–F) in the cerebral cortex. G–J show that whereas in controls GAT-3 and HSP70 ir are segregated (G and H), in samples of resected cortices neurons GAT-3+ neurons are also HSP70+ (I and J). Bar: (A and C) 25 mm; (D and E) 60 mm; (F–I) 10 mm.
GABA transporter GAT-3 is localized both to distal astrocytic processes and to neurons. Since in previous investigations we showed GAT-3 to be localized to distal astrocytic processes but not to cortical neurons of rat neocortex (Minelli et al., 1996), the question arose whether these observations reflected species differences in the
organization of cortical GABA uptake or methodological/ procedural biases. The total lack of ir following preincubation of the antibodies with human C-terminal peptides indicates that our antibodies recognize an epitope of the GAT-3 sequence; most importantly, the observation that GAT-3 antibodies
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recognize a single band in homogenates from human cerebral cortex indicate that immunoreactivity is highly specific and in particular it rules out a cross-reaction between the GAT-3 antibodies and unknown proteins expressed in human, but not rat, neocortex. The specificity of GAT-3 staining in the human cortex thus ascertained, we considered the possibility that neuronal expression of GAT-3 could be related to the collection procedure of cortical tissue. In particular, we surmised that the energy and oxygen deprivation associated with the surgical procedure could trigger expression of GAT-3 in neurons that normally do not express it. This hypothesis rested on three considerations: (i) cortical resection as performed in the present study lead to an acute ischemic situation in the resected brain and is often used as a model of acute focal ischemia (Kanthan et al., 1995); (ii) in many instances fixation by immersion yields perikaryal staining in cases in which fixation by perfusion does not (e.g., Tamura et al., 2000); and (iii) in the rat neocortex, transient middle cerebral artery occlusion (MCAO) triggers expression of GAT-3 in neurons that are immunoreactive for HSP70, the major inducible heat-shock protein (Melone et al., 2003). Li et al. (2004) recently observed that gene expression in postmortem human brains is correlated with tissue pH: individuals who suffered prolonged agonal state have lower pH in the brain, whereas those who died rapidly had normal pH. Our sample is too limited in number to verify whether such a correlation holds also for GAT-3 neuronal expression; it is worth noting however that we observed GAT-3 ir in neurons also in the cerebral cortex of an individual who died from a car accident. Thus, even if we cannot rule out the possibility that tissue acidity triggered GAT-3 neuronal expression, the evidence in our hands suggests that this does not seem to be the case. To test the hypothesis that energy and oxygen deprivation associated with the surgical procedure trigger neuronal expression of GAT-3, we first studied the expression of HSP70 and then investigated GAT3/HSP70 coexpression and found that the density of HSP70+ neurons paralleled that of GAT-3+ neurons, and, most importantly, that all GAT-3+ neurons coexpressed HSP70. These studies show that expression of GAT-3 by neurons that usually do not express the protein occurs in cells subjected to metabolic stress and suggest that neuronal expression of GAT-3 in cortical neurons may depend on the collection procedure employed. The presence of GAT-3+ neurons in one autoptic case we had the opportunity to study strongly supports this view. Since, for obvious reasons, the positive control cannot be performed, we replicated in rats the surgical procedure used for human cortical resection and found that it does induces both HSP70 and the appearance of numerous GAT-3+ neurons in the neocortex of rats, where GAT-3 ir is normally confined to distal astrocytic processes (Minelli et al., 1996). The observation that GAT-3 can be visualized in neurons whose gene expression has been altered (as indicated by HSP70 positivity) does not necessarily imply that GAT-3 visualized in those conditions
originates directly from the translation of the GAT-3 gene. Indeed, the presence of untranscribed mRNA in CNS neurons has been documented (e.g., Steward and Schuman, 2003) and GAT-3 mRNA has been reported in some central neurons (Clark et al., 1992; Durkin et al., 1995); and protein synthesis may persist for some minutes while the tissue is in fixative. This latter scenario would be in line with the short time lapse between the beginning of resection and the immersion in the fixative solution. Overall, our studies show that in human cerebral cortex GAT-3 is expressed in distal astrocytic processes, as in rats, as well as in cortical neurons. They also suggest that GAT-3 expression in human cortical neurons is connected with the collection procedure. The implications of these results for the validity of human cortical studies and for our understanding of the localization and regulation of GATs in the cerebral cortex can be summarized as follows: 1. From the methodological point of view, the present findings indicate that if other antigens are as susceptible as GAT-3 to factors connected with the procedures employed in human cortical studies, ‘‘species differences’’ in localization studies should be addressed with great caution. 2. The observations that GAT-3 is expressed in astrocytes and that its neuronal expression is in all likelihood due to nonphysiological causes suggest that its localization in the human cortex is similar to the one demonstrated in rats; furthermore, given that GAT-1 is similarly expressed in rats and humans (Minelli et al., 1995; Conti et al., 1998), these data indicate that the localization of the GABA transporters in the cerebral cortex may be similar in different species. 3. The finding that in both human and rat cortex GAT-3 expression is altered by procedural variables (cortical resection) and by transient MCAO (Melone et al., 2003), whereas in the very same experimental situations (and in adjacent sections) GAT-1 expression is unchanged seems to point at significant functional differences between the two major cortical GATs. In particular, it appears that the expression of the hard-wired, synaptic GAT-1 is less susceptible than that of GAT-3 to changes in the composition of the extracellular milieu, possibly pointing to an important contribution of the mechanisms regulating GAT-3 expression in the adaptive capabilities of neurons (Conti et al., 2004).
Acknowledgments We are grateful to A. Ducati, M. Scerrati and A. Tagliabracci for providing the surgical and autoptic material, to N.C. Brecha for the generous gift of GAT-3 antibodies, to J. DeFelipe for helpful comments on an earlier version of this manuscript, and to L. Bragina and F. Quagliano for
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technical assistance. This work was supported by grants from MIUR (COFIN 01) to F.C.
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