Developmental Brain Research 142 (2003) 7–18www.elsevier.com/ locate/devbrainres

Research report

P ostnatal development of high-affinity plasma membrane GABAtransporters GAT-2 and GAT-3 in the rat cerebral cortex

*Andrea Minelli, Paolo Barbaresi, Fiorenzo Conti`Istituto di Fisiologia Umana, Universita di Ancona,Via Tronto 10/A, Torrette di Ancona, I-60020 Ancona, Italy

Accepted 9 January 2003

Abstract

We investigated the developmental profile of plasma membraneg-aminobutyric acid (GABA) transporters (GATs) GAT-2 and GAT-3expression by immunocytochemistry with affinity-purified polyclonal antibodies in the rat neocortex. At all developmental agesinvestigated, GAT-2 ir was prominent in the arachnoid and in the trabeculae of the subarachnoid space, whereas it was weak within thecortical parenchyma; the adult pattern was reached during the third week of postnatal life. GAT-3 ir was present at birth and increasedrapidly in the first week, when numerous positive cells were present throughout the cortical layers; at P10, GAT-3-positive cells becameless numerous and GAT-3 ir switched to the adult pattern, which was expressed at P20. Confocal and electron microscopic investigationsshowed that GAT-3 positive cells were both neurons and astrocytes. The present evidence indicates that early in development GAT-3 isabundantly expressed in the cerebral cortex, where its expression appears to correlate with developmental variations in GABA levels, andsuggests that it accounts for the largest fraction of GABA transport observed in the neonatal cerebral cortex. 2003 Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Cerebral cortex and limbic system

Keywords: Astrocyte; GABA; GABA uptake; Neuron

11 . Introduction GAT-2, GAT-3, and BGT-1 [9–11,13,26,30,34,44,51]have been identified. Of these, GAT-1, GAT-2, and GAT-3

The maturation ofg-aminobutyric acid (GABA)-me- are expressed in the adult cerebral cortex: GAT-1 isdiated intercellular communication in the cerebral cortex localized to axon terminals forming symmetric synapsesand its functional role have been the focus of numerous and to some astrocytic processes [15,19,40], GAT-2 isinvestigations, but many aspects are still elusive [3,45]. predominantly extraparenchymal but it is also expressed in

1 2GABA is taken up by high-affinity, Na /Cl -dependentplasma membrane transporters that contribute to shaping

1We adopt the nomenclature of Guastella et al. [26] and Borden [8] andthe inactivation phase of inhibitory postsynaptic responsesrefer to cDNA clones from rat (r) brain. An analogous, though not

and to regulating GABA’s spillover to neighboring identical, nomenclature has been introduced by Liu et al. [34], whosynapses [12]. To date, four distinct high-affinity plasma identified cDNA clones from mouse (m) brain that encode four GATs andmembrane GABA transporters (GATs), named GAT-1, named them GAT1-GAT4. Whereas mGAT1 is the homolog of rGAT-1,

mGAT2, mGAT3 and mGAT4 appear to be the homologs of dog BGT-1,rGAT-2 and rGAT-3, in this order. In addition, rGAT-3 is identical to arat clone termed GAT-B by Clark et al. [13]. Whether BGT-1 (for

*Corresponding author. Tel.:139-071-220-6056; fax:139-071-220- Betaine/GABA Transporter) [59] or its mouse or human homologs6052. contribute to GABA transport in the brain remains to be determined (see

E-mail address: f.conti@popcsi.unian.it(F. Conti). [8]).

0165-3806/03/$ – see front matter 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0165-3806(03)00007-5

8 A. Minelli et al. / Developmental Brain Research 142 (2003) 7–18

neurons and astrocytes [16], and GAT-3 is found exclu- 2 .2. Immunocytochemistrysively in distal astrocytic processes [41].

Understanding the developmental profile of GATs could 2 .2.1. Antibodiescontribute to determine the factors that regulate the de- Affinity-purified rabbit polyclonal antibodiesvelopment of GABA-mediated signalling in the cerebral (Chemicon, Temecula, CA) directed to the predicted C-neocortex and the process of cortical maturation itself termini of rat GAT-2 (rGAT-2 ) [11] and GAT-3594–602

[2,3,31,32,35,45]. Yan et al. [58] studied the maturation of (rGAT-3 ) [11,13] were used for this study. Pro-607–627

GAT-1 immunoreactivity (ir) and showed that it is low at duction and characterization of these antisera have beenearly postnatal ages, increases steadily in the first 2–3 reported previously [16,27,41]. For colocalization studies,postnatal weeks and reaches the adult pattern at P30–P45. mouse monoclonal antibodies against GABA [37], glialThese findings indicate that GAT-1 is unlikely to take up fibrillary acidic protein (GFAP; 814369; Boehringer,GABA present in the neocortex during the first postnatal Mannheim GmbH, Germany) and synaptophysin (cloneweek (see Discussion). Although general mapping studies SVP-38; S-5768, Sigma, St. Louis, MO) were used.of the development of GATs mRNA [22] or proteins[28,29] have been published, they give little information 2 .2.2. Procedureon the maturation of GAT-2 and GAT-3 in the cerebral For immunoperoxidase studies, free-floating sectionsneocortex. We have therefore investigated the postnatal were pretreated with 1% H O (30 min), rinsed several2 2

development of GAT-2 and GAT-3 ir in the rat cerebral times in PBS, preincubated for 1 h in normal goat serumcortex. (NGS, 10% in PBS) with 0.3% Triton X-100, and then

incubated overnight at 48C in primary antibody. Differentworking dilutions (ranging from 1:500 to 1:2000) weretested for each antibody; the best signal-to-noise ratio was

2 . Materials and methods obtained at 1:1000 for both GAT-2 and GAT-3. The nextday, sections were rinsed three times in PBS, incubated in

Sprague–Dawley albino rats of both sexes (Charles NGS (10%; 20 min) and then in biotinylated goat anti-River, Milan, Italy) at different postnatal ages were used in rabbit IgG (BA1000; Vector, Burlingame, CA; 1:100 inthe present study. The age groups studied were: P0 PBS, 1 h at room temperature). Sections were subsequently(postnatal day 0, within 24 h of birth), P2, P5, P10, P15, rinsed in PBS and incubated for 30 min in avidin–biotinP20, P30 and P60 (adult stage). Four animals for each time peroxidase complex (ABC Elite PK6100, Vector; 1:200 inpoint were used for immunoperoxidase studies. In addition, PBS), washed several times in PBS and incubated in 3,39

rats were sacrified at P2 and P5 and used for ultrastructural diaminobenzidine tetrahydrochloride (DAB; 0.08% in 0.05(three animals) and colocalization studies (five animals). M Tris with 0.02% H O ). Sections processed with the2 2

Care and handling of animals were approved by the Ethical same immunocytochemical protocol but using buffersCommittee for Animal Research of the University of other than PBS (0.1 M Tris buffer, pH 7.4 or 0.1 M PB,Ancona. pH 7.4) showed no appreciable differences in signal

detection.For electron microscopy, sections were first pretreated

2 .1. Tissue preparation with 1% sodium borohydryde for 30 min to quench non-specific binding due to aldehyde residues and then exposed

Rats were anesthetized with 30% chloral hydrate and to a mild ethanol treatment (10%, 25%, 10%; 5 min each)perfused transcardially with 0.1 M phosphate buffered before the immunocytochemical procedure. GAT-3 anti-saline (PBS; pH 7.4) followed by 4% paraformaldehyde body was used at dilution of 1:1000 and Triton X-100 was(PFA) in 0.1 M phosphate buffer (PB; pH 7.4). For not used. After completion of the immunocytochemicalelectron microscopy, rats were perfused with 3% PFA with procedure, sections were washed in PB, incubated in 2.5%1.5% glutaraldehyde and 20% picric acid. For double- glutaraldehyde (30 min), washed in PB and postfixed for 1labeling experiments, animals were perfused with 4% PFA h in 1% OsO . They were flat-embedded in Epon-Spurr,4

with 0.5% glutaraldehyde and 20% picric acid. Brains processed as described previously [16], and examined withwere postfixed for 8–12 h at 48C in the same fixative used a Philips CM10 electron microscope (Eindhoven, Thefor perfusion, and cut with a vibratome in the coronal or Netherlands). Identification of labeled and unlabeled pro-parasagittal plane into 30–50 mm-thick sections that were files was based on established morphological criteria [47].collected serially in PBS and stored at 48C until process- To verify whether the strong fixation protocol used toing. Every fifth section was immediately mounted on obtain material suitable for electron microscopic analysesgelatin-coated slides, air dried and stained with cresyl could affect the intensity and/or specificity of GAT-3violet. Sections for both ultrastructural and colocalization staining, some sections were mounted after the DABstudies were always processed within 24 h from cutting. reaction and examined at the light microscope. In these

A. Minelli et al. / Developmental Brain Research 142 (2003) 7–18 9

sections, the level of intensity and the distribution pattern Plan Apo oil-immersion objective (numerical aperture: 1.4)of GAT-3 ir were consistently similar to those observed in and images were acquired on a 5123512 pixels box usingsections used for optical immunoperoxidase studies, which a confocal aperture of 2 mm. To improve signal-to-noisewere taken from tissue that received a milder fixation. ratio, 20 frames were averaged by Kalman filtering.

For immunofluorescence, free-floating sections were Control experiments confirmed that there was no signifi-pretreated with 1% sodium borohydryde for 30 min, cant FITC/TRITC fluorophore bleed-through on the non-incubated in 20% NGS in PB with 0.2% Triton X-100 for corresponding channel or cross-talk when the images were1 h and then overnight at 48C in a solution of NGS (1% in acquired separately.PB) containing a mixture of one of the GATs antisera Data and representative photomicrographs were col-(used at the same working dilution as in the single-labeling lected from the frontal and parietal cortices.experiments) and, alternatively, GABA (dilution 1:12 000),GFAP (1:1000) or synaptophysin (1:1000) antibodies. Thenext day, sections were washed in PB, incubated in NGS 3 . Results(20%, 15 min) and then for 1 h in a mixture of theappropriate affinity-purified fluorescein isothiocyanate 3 .1. Developmental profiles of GAT-2 and GAT-3 in the(FITC)- or tetramethylrhodamine isothiocyanate (TRITC)- neocortexconjugated secondary antibodies (1:100). Sections weresubsequently washed in PB, mounted, air-dried and cover-3 .1.1. GAT-2slipped using Vectashield mounting medium (H-1000; At all stages, intense GAT-2 ir was detected in theVector). Sections were examined using a BioRad Mi- arachnoid and along the thin arachnoid trabeculae in thecroradiance confocal laser-scanning microscope equipped subarachnoid space (Fig. 1A–E).with an Argon gas and He/Ne mixed gas lasers. FITC and At P0–P5, faint GAT-2 ir was in the cortical paren-TRITC were excited with the 488 nm and the 543 nm chyma, without an apparent laminar pattern (Fig. 1A,B).lines, respectively, imaged sequentially and then merged GAT-2 ir was only in puncta of various sizes distributed inusing the LaserSharp Processing software from BioRad. the neuropil (Fig. 2B) and around the profile of bloodThe fields of interest were scanned using a360 Nikon vessels (Figs. 1A,B and 2A). In some cases, the entire

Fig. 1. Developmental profile of GAT-2 ir in the cerebral cortex. (A–E) GAT-2 ir in the developing cerebral cortex at P0 (A), P5 (B), P15 (C), P30 (D)and P60 (E). At all postnatal ages, GAT-2 ir is prominent in the arachnoid. Note the robust labeling around blood vessels during the first postnatal week.The adult pattern is reached at P15. Roman numerals, cortical layers; CP, cortical plate. Scale bar5100 mm.

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thus in all likelihood axon terminals, and that GAT-2positive structures surrounding blood vessels were GFAPpositive, and thus astrocytic (Fig. 3B). Neither fibers norcell bodies were labeled at P0–P5. From P10 onward,GAT-2 ir (Fig. 1C–E) was similar to that of the adult (16)and immunolabeling was detected in puncta and in few cellbodies (Fig. 2C–E). Intense GAT-2 ir was characteristical-ly observed as a thin rim of labeling around blood vessels(Fig. 2C). Few cells resembling astrocytes were present insuperficial layers, mostly in layer I (Fig. 2D), and faintlystained cells resembling neurons were occasionally foundin layers V–VI (Fig. 2E).

3 .1.2. GAT-3

3 .1.2.1. P0. GAT-3 ir was localized to puncta and cellbodies throughout the cortex. Cortical plate (CP) andupper layer I displayed the highest levels, layers V and VIthe lowest (Fig. 4A). In the superficial layers, GAT-3 irwas in numerous puncta of different sizes scattered in theneuropil and in processes (Fig. 4A) running towards layerI. A rim of intense staining was observed in corre-spondence with the glia limitans (Fig. 5A) and labeled

Fig. 2. Maturation of GAT-2 ir in the developing cerebral cortex. CP atpuncta were often around blood vessels. Numerous lightly-P2 (A) and P5 (B); layers I–II at P10 (C); layer I at P15 (D); layer V atstained GAT-3 positive cells were observed throughout theP20 (E). See text for details. Scale bars515 mm.cortex: they were more numerous in deep layers and hadirregular shape and processes radiating in all directions

caliber of blood vessels was outlined by GAT-2 ir (Fig. (astrocyte-like cells). In the upper half of CP, GAT-32A). Colocalization studies showed that some GAT-2 positive cells showing a different morphology were occa-positive puncta were synaptophysin positive (Fig. 3A), and sionally observed: they had a fusiform body, an ascending

apical dendrite that formed a terminal bouquet in layer I,and a thin process resembling the axon emerging from theopposite pole (Fig. 5A).

3 .1.2.2. P2–P5. GAT-3 ir was stronger than at birth anddisplayed a bilaminar pattern, with numerous, intenselystained, puncta in the superficial layers and numerouslabeled cells in deep layers (Fig. 4B,C). Colocalizationstudies showed that some GAT-3 positive puncta coexpres-sed the presynaptic marker synaptophysin (Fig. 6A), andthat, in regions where GFAP ir was present (i.e., layer Iand Vib; 4, 5, 52), several GAT-3 positive puncta and/orprocesses coexpressed GFAP (Fig. 6B). At P2 (and evenmore so at P5), GAT-3 ir was highest in the lower portionof CP/ layer IV (Fig. 4B,C), where labeled puncta were inthe neuropil and outlined unstained cells. Outer layer I andthe glia limitans were intensely labeled (Figs. 4B,C and5E) and numerous GAT-3-positive fibers ran verticallyFig. 3. Localization of GAT-2 ir in the developing cerebral cortex. (A)across CP (Figs. 4B,C and 5E). In all layers, blood vesselsNeuropilar GAT-2 positive processes (green) coexpressing (arrows)

synaptophysin ir (red). (B) GAT-2 positive processes (green) outlining were surrounded by GAT-3 positive puncta (Fig. 5F,G).blood vessels in most cases coexpress GFAP ir (red). In single-labeledAstrocyte-like cells were more numerous and more in-sections, synaptophysin and GFAP ir were as previously described tensely stained than at P0 (Fig. 4B,C and 5B) and were[4,5,21,52]. Single plane confocal images from CP (A) and layer VI (B) at

frequently observed in relationship with blood vessels (Fig.P5. Green and red images were acquired sequentially and subsequently5F,G); few cells with a neuronal morphology were alsomerged (see Experimental procedures for details). Bars: 5 mm in A; 20

mm in B. observed (Fig. 5C,D). We counted GAT-3-positive cells

A. Minelli et al. / Developmental Brain Research 142 (2003) 7–18 11

Fig. 4. Developmental profile of GAT-3 ir in the cerebral cortex. (A–H) GAT-3 ir in the developing cerebral cortex at P0 (A), P2 (B), P5 (C), P10 (D),P15 (E), P20 (F), P30 (G) and P60 (H). GAT-3 ir is present at birth and increases rapidly in the first week, when numerous positive cells are presentthroughout all cortical layers. At P10, GAT-3-positive cells become less numerous and GAT-3 ir switches to the adult pattern, which is fully expressed atP20. Roman numerals, cortical layers; CP, cortical plate. Scale bar5200 mm.

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Fig. 5. Maturation of GAT-3 ir in the developing cerebral cortex. Layer I at birth (A); layer V at P2 (B); CP (C and D), upper CP (E), and layer V (F andG) at P5; layer IV at P15 (H); and layer V at P20 (I and J). See text for details. Scale bars510 mm in A–G; 15mm in H–I; 30 mm in J.

within microscopic fields (2003250 mm) randomly cen- (471 cells; average: 7.963.6/field). Astrocyte-like cellstered either in the marginal layer and CP or in the cortical were the vast majority (93%) and were small-sized (majorsubplate (60 fields for each group; from four rats at P5); diameter: 3–9 mm; mean diameter [109 cells]: 5.3they were more numerous in the subplate region (2394 mm61.3). Few cells (203; 7%) displayed a neuronalcells; average: 39.968.9/field) than in superficial layers morphology (with ovoidal or fusiform soma and one or

A. Minelli et al. / Developmental Brain Research 142 (2003) 7–18 13

signal, followed by the supragranular layers, whereaslayers VI and Va showed low ir (Fig. 4E–H). No GAT-3-positive cells were found and a band of strong ir wasappreciable in layer Vb (Fig. 4E–H), where numerouslabeled puncta were observed around unlabeled pyramidalcells, sometimes in association with the emergence of theirapical dendrite (Fig. 5I). GAT-3 ir around blood vesselswas low or absent. From P20 on, several irregularly shapedareas of reduced GAT-3 ir were found in all layers (Fig.4H and 5J), giving the cortex the typical and specificpatchy appearance of the mature pattern (i.e., the presenceof restricted areas of tissue in which the expression ofGAT-3 is very low. These areas are observable in allsections from all animals and thus are not due to poorfixation of the tissue; 41).

3 .2. Electron microscopy and double-labeling studies ofGAT-3 positive cells

Whereas at late developmental stages GAT-3 ir waslocalized only to puncta, early in development it wasconsistently observed in perikarya, presumably reflectingGAT-3 molecules being transported to the plasma mem-brane. To determine whether these cells were astrocytes orneurons, we investigated at the electron microscope thelocalization of GAT-3 ir at the time points (P2 and P5) atwhich these cells were observed more frequently. Thesestudies showed that GAT-3 ir was indeed expressed inneurons as: (i) the nuclei of numerous GAT-3 positivecells were pale and large, occupied most of the cell body,so that in many cases the perikaryal cytoplasm formed athin ring around the nuclear envelope (Fig. 7A); inaddition, they contained three or four nucleoli (diameter:Fig. 6. Localization of GAT-3 ir processes in the developing cerebral2–3 mm) and electrondense clumps of chromatin werecortex. (A) Neuropilar GAT-3 positive punctate processes (green) coex-observed along the inner surface of the nuclear membranepressing (arrows) synaptophysin ir (red). (B) Neuropilar GAT-3 positive

processes (green) coexpressing GFAP ir (red). In single-labeled sections,(Fig. 7A); (ii) the cytoplasm contained many mitochondria,synaptophysin and GFAP ir were as previously described [4,5,21,52]. a well developed Golgi apparatus and a loose endoplasmicSingle plane confocal images from CP (A) and layer VI (B) at P5. Green

reticulum arranged in scattered and isolated cisternae (Fig.and red images were acquired sequentially and subsequently merged (see7A); (iii) in some cases they received axon terminalsExperimental procedures for details). Bars: 5mm in A; 10 mm in B.forming synaptic contacts; and (iv) numerous dendrites andfew axon terminals were GAT-3 positive (Fig. 7D,E).

two polar dendrites) and larger soma size (major diameter: These features are typical of neuronal cell bodies [6,39,46].6–15 mm; mean diameter [70 cells]: 9.7 mm62.1). In some cases, GAT-3-positive neurons were intensely

labeled by the reaction product, whereas in most cases ir3 .1.2.3. P10. GAT-3 ir was highest in layer IV (Fig. 4D), was in the form of small clumps scattered in the cytoplasmwhere puncta were often around unlabeled cell bodies (Fig. (Fig. 7A). GAT-3 positive neuronal cell bodies were5H). Supragranular layers stained moderately, while in numerous (33/112; 29.46%) and mostly in supragranularlayer I GAT-3 ir was lower than at earlier stages (Fig. 4D). layers.In infragranular layers, GAT-3 ir in puncta was higher than GAT-3 ir was also found in several small cells, which inat P5 (Fig. 4D), whereas labeled astrocytic-like cell bodies many cases had lobated nuclei. They had one centriolus,were less numerous (Fig. 4D). Cells with a neuronal the endoplasmic reticulum was made by a small number ofmorphology were no longer present. narrow and elongated cisternae, the Golgi apparatus was

abundant and made by few stacks of roundish cisternae.3 .1.2.4. P15-adult. From P15 onward, the intensity and Branching processes originating from the perikarya weredistribution of GAT-3 ir was similar to that of adult rats often observed (Fig. 8A). These features are typical of(P60): layers IV and Vb exibited the highest level of astrocytes [47]. These cells were mostly in infragranular

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Fig. 7. Electron microscopy studies of GAT-3 positive cortical neurons. (A) Clumps of GAT-3 ir are scattered in the cytoplasm of a cortical neuron.Framed regions are reproduced enlarged in B and C (P5). (B and C) Higher magnification of the framed regions in A showing in detail patches of labeling.(D) An axon terminal showing a patch of GAT-3 ir near the internal surface of the plasma membrane and among vesicles (P5). (E) detail of a labeleddendrite contacted by an axon terminal forming an asymmetric synapse (arrowheads). AxT, axon terminal; Den, dendrite; Nuc, nucleus; ER, endoplasmicreticulum; G, Golgi apparatus. Scale bars, 1 mm in A; 0.5mm in B–E.

layers. In addition, GAT-3 ir was frequently observed in nificantly, whereas GAT-3 ir was present at birth, in-perivascular astrocytic processes (Fig. 8C) and in elon- creased rapidly in the first week, and reached the adultgated profiles that for their size (1–2 mm) and organization pattern at P20. In addition, these data showed that duringin bundles, the paucity of microtubules and the presence of the first postnatal week GAT-3 is also expressed bysmall scattered clumps resembling those previously inter- neurons.preted as glycogen granules appeared as radial glial fibers Previous general mapping studies showed that GAT-2(Fig. 9A,B) [24,50]. Only in rare cases labelled processes protein [29] is localized exclusively to the meninges andresembling adult distal astrocytic processes were observed. blood vessels, whereas GAT-2 mRNA is expressed pre-

To ascertain whether GAT-3 positive neurons are dominantly in the meninges and faintly and transiently inGABAergic, we performed double-labeling studies using cerebellar granule cells [22]. Our findings confirmed theGABA antibodies in sections from P2 to P5 rats. GABA- predominant localization of GAT-2 to leptomeninges andpositive neurons were uniformly distributed across layers blood vessels and indicated that some GAT-2 ir is alsoand labeled puncta were more numerous in layers I and within the neuropil, as in the adult cortex [16]. TheV–VI, in line with previous descriptions [18,20,38]. These developmental profile of GAT-2 ir is relatively consistent;studies revealed that numerous GABA-positive neurons the major changes were represented by the reduction ofcontained small clumps of GAT-3 ir scattered in their large blood vessel staining and the appearance of immuno-cytoplasm, sometimes clustered in close apposition to the reactive cells at P10, when the adult pattern is reached.inner part of the cellular profile (Fig. 10A). GAT-3 ir was Although the temporal correlation between synaptogenesisalso observed in GABA-positive fibers (Fig. 10B). These [6,7,18,33] and appearance of GAT-2 ir in cell bodiesstudies showed that none of the astrocyte-like GAT-3 could suggest an influence of GAT-2 on the developingpositive cells was immunoreactive for GABA. GABAergic system, the paucity of neuronal and astrocytic

staining seems to indicate that any effect of GAT-2 onGABA-mediated responses is minor, and that during

4 . Discussion development the major function of GAT-2 is related to theregulation of GABA levels in the cerebrospinal fluid.

The present results showed that during postnatal de- The development of mouse GAT4 (the homologue of ratvelopment GAT-2 ir was low and did not change sig- GAT-3) mRNA has been studied in a general mapping

A. Minelli et al. / Developmental Brain Research 142 (2003) 7–18 15

Fig. 8. Electron microscopy studies of cortical non-neuronal GAT-3 positive elements. (A) GAT-3 ir in a presumed astrocyte at P5; arrow points to acentriolus. Framed region is enlarged in B. (B) Higher magnification of the framed region in A showing in detail GAT-3 ir. (C) A perivascular astrocyticprocess expressing GAT-3 ir. Asp, astrocytic process; B, basal lamina; Cap, capillary; Nuc, nucleus; ER, endoplasmic reticulum; G, Golgi apparatus. Scalebars, 1 mm in A and C; 0.5 mm in B.

study [22], whereas that of mouse GAT4 protein has been This possibility is supported by demonstration thatb-reported in a communication on its developmental expres- alanine, a potent GAT-3 antagonist [11], inhibits GABAsion in the entire brain and spinal cord [28]. Since both uptake in newborn rats by 93% and that this percentagepapers gave virtually no information on the development decreases steadily as maturation proceeds [56].of GAT-3 expression in the cerebral neocortex, the present In the adult neocortex, GAT-3 is expressed exclusivelydata represent the first description of GAT-3 in the in distal astrocytic processes [41]; thus, the presence ofdeveloping neocortex. We showed that: (i) GAT-3 ir was GAT-3 positive cell bodies is a feature of the developingpresent at birth and increased rapidly in the first week; (ii) neocortex. Many GAT-3 positive cells are neurons, asin the first week numerous positive cells were present suggested by the observation that many GABA-positivethroughout the cortical layers; and (iii) at P10, GAT-3- neurons contain cytoplasmic clumps of GAT-3 ir andpositive cells became less numerous and GAT-3 ir indicated by electron microscope studies which showedswitched to the adult pattern, which was expressed at P20. that many cells with ultrastructural features typical of

Yan et al. [58] have shown that GAT-1 ir is low at early cortical neurons and some axon terminals contain GAT-3postnatal ages and that it increases steadily in the first two ir.postnatal weeks to reach the adult pattern approximately at The present studies showed that GAT-3 is highlyP30 and we have confirmed these data [42,43]. The present expressed in both astrocytes and neurons in the earlyevidence thus indicates that early in development GAT-3 is phases of postnatal development, when GAD activity isthe most abundant GAT in the neocortex and suggests that low and GABAergic synapses are few and immature. Atit accounts for the largest fraction of GABA transport these stages, however, GABA concentration is high [17],observed in the neonatal cortex (30% of adult levels) [56]. and numerous GABA-positive cells and fibers are present

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VGAT [43], GAT-3 expression correlates with develop-mental variations in GABA levels.

Early in brain development, GABA modulates cellproliferation, differentiation and motility through receptor-mediated mechanisms [2,31,32,35]. The present observa-tion that in neonatal cortex GAT-3 ir is strongly expressedin outer layers and CP, i.e. in the same layers where thea2

and b subunits of GABA receptor are localized2 / 3 A

[23,48,49], suggests that GAT-3-mediated GABA transportmay modulate GABA actions on its receptors, thus in-fluencing neuronal maturation during early cortical de-velopment [35]. Whether during development GAT-3 takesup GABA, thus modulating extracellular GABA levels andpreventing GABA-mediated toxicity [36,57], or it contri-butes to GABA release by reversing its direction ([1,54];but see [25]) remains to be determined. In line with theoverall immaturity of inhibitory synaptogenesis[3,6,7,18,45], several lines of evidence emphasise the roleof the paracrine mode of GABA action during earlydevelopment [3,45], but the exact source(s) of GABA areyet elusive. Previous studies have suggested that growthcones of immature GABA-containing neurons can releaseGABA by non-vesicular mechanisms based, at least inpart, on reverse transporter action ([53,55]; but see [25]).The present observation that many GABAergic neurons inthe neonatal cortex express GAT-3 suggests that thistransporter may mediate GABA release from neuronsduring early phases of cortical development.Fig. 9. (A) Two GAT-3 positive radial glial fibers; arrows indicate

clumps of GAT-3 ir. RF, radial glial fiber; Nuc, nucleus; Cyt, cytoplasm.Bars: 0.5mm in A; 1 mm in B.

A cknowledgements[14,20,55]. Thus, whereas GAT-1 expression displaysdevelopmental changes spatially and temporally related to This work was supported by a grant from MURSTthose of GAD [58] and of the vesicular GABA transporter (COFIN99 and COFIN01) to FC. We are grateful to Carlos

Matute for the GABA antibodies and to several colleaguesfor critical comments, discussions and suggestions.

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