− mice

PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 5 3 1 - 6

Neuroscience Vol. 115, No. 3, pp. 657^667, 2002 D 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

www.neuroscience-ibro.com

MORPHOLOGICAL ORGANIZATION OF SOMATOSENSORY CORTEX IN Otx13=3 MICE B. CIPELLETTI,a G. AVANZINI,a L. VITELLARO-ZUCCARELLO,b S. FRANCESCHETTI,a G. SANCINI,a T. LAVAZZA,a D. ACAMPORA,c;d A. SIMEONE,c;d R. SPREAFICOa and C. FRASSONIa a

Dipartimento Neuro¢siologia Sperimentale, Istituto Nazionale Neurologico ‘C. Besta’, via Celoria 11, 20133 Milano, Italy b

Sez. Istologia e Anatomia umana, Dip. Fisiologia e Biochimica generali, via Celoria 26, 20133 Milano, Italy c d

International Institute of Genetics and Biophysics, CNR, via Marconi 12, 80125 Naples, Italy

MRC Centre for Developmental Neurobiology, King’s College London, London SE1 9RT, UK

Abstract
for brain morphogenesis (Simeone et al., 1992a; Bu£one et al., 1993). The homeobox genes include Otx1, Otx2, Emx1 and Emx2 which are expressed in the developing rostral brain of mouse embryos (Simeone et al., 1992a,b; 1993; Boncinelli et al., 1995). It is proposed that these four genes are concerned with establishing the identity of the various embryonic brain regions in a stepwise process centered on the dorsal telencephalon (Simeone et al., 1992a; Boncinelli et al., 1995). The Otx1 expression domain is a continuous region that includes part of the telencephalon, diencephalon and mesencephalon. This gene is expressed by the precursors of deep layer neurons within the developing cerebral ventricular zone. In situ hybridization studies (Frantz et al., 1994) have shown that Otx1 mRNA is also expressed in a subpopulation of neurons within cortical layers V and VI during postnatal and adult life. More recently it has been shown that Otx1 has no obvious role in laminar speci¢cation, but has an important role in the later stages of cortical di¡erentiation. Thus, Otx1 is strongly expressed in a subset of layer V neurons and is required for the development of a normal pattern of connectivity of these neurons during development (Weimann et al., 1999). Otx1 knock-out (Otx13=3 ) mice are characterized by

Brain development and neural di¡erentiation are a complex series of temporally and spatially regulated morphogenetic events (Shimamura and Rubenstein, 1997; Rubenstein et al., 1998; Beddington and Robertson, 1998, 1999). Several families of genes coding for transcription factors involved in the regulation of these events are recognized (Lemaire and Kodjabachian, 1996; Tam et al., 1997; Tam and Steiner, 1999; Rubenstein et al., 1998) and many have been cloned including the homeo-box genes (Finkelstein and Boncinelli, 1994; Simeone et al., 1993; Brunelli et al., 1996). The observation of overlapping patterns of expression of these factors in the developing rostral CNS has led to the proposal of a regionalization model

*Corresponding author. Tel.: +39-2-2394279; fax: +39-2-70600775. E-mail address: [email protected] (C. Frassoni). Abbreviations : ABC, avidin^biotin^peroxidase complex ; CB, calbindin; CR, calretinin; GAD67, glutamic acid decarboxylase of 67 kDa; GAT-1, GABA transporter 1; GAT-3, GABA transporter 3; GFAP, glial ¢brillary acidic protein; NHS, normal horse serum; NGS, normal goat serum; NMDAR1, subunit 1 of the NMDA receptor ; P, postnatal days; pAb, polyclonal antibody; PB, phosphate bu¡er; PBS, phosphate-bu¡ered saline. 657

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recurrent epileptic seizures and a microencephalic phenotype, mainly evident as marked reduction in neocortical thickness, particularly involving the perirhinal and temporal areas (Acampora et al., 1996, 2001). Although layering is less evident than in wild-type mice (Acampora et al., 2001), analysis of the expression of layer-speci¢c molecular markers has shown that laminar identities are preserved (Weimann et al., 1999). Recently, epileptogenic mechanisms have been investigated in the Otx13=3 model by electrophysiological recordings of somatosensory cortex slices (Sancini et al., 2001). This study found signi¢cant changes in synaptic excitability, that disrupt the normal balance between excitatory and inhibitory neurotransmission. The present study used immunocytochemical methods to investigate whether these physiological ¢ndings could be related to morphological changes involving the excitatory and inhibitory network in the somatosensory cortex. Preliminary results have been presented in abstract form (Cipelletti et al., 2001).

EXPERIMENTAL PROCEDURES

Genotyping of wild-type and mutant mice Wild-type, heterozygous and homozygous o¡spring were identi¢ed by polymerase chain reaction (PCR). Tail tips were incubated in lysis bu¡er (20 mM Tris, pH 8.0, 5 mM EDTA, 400 mM NaCl, 1% sodium dodecyl sulfate, 0.6 mg/ml proteinase K) overnight at 55‡C, phenol-chloroform extracted, ethanol precipitated and redissolved in sterile water at a ¢nal concentration of 0.2^1.0 Wg/Wl. The wild-type allele was detected using the sense primer (5P-AGCAGACACATGGAAACCTTC-3P) and antisense primer (5P-CACTTGGGATTTTGCACCCTC-3P). The mutated allele was detected using the lacZ sequence sense primer (5P-GCGTTGGCATTTAACCGCC-3P) and antisense primer (5P-CAGTTTACCCGCTCGCTAC-3P). Thirty-¢ve PCR cycles (denaturation: 3 min, 94‡C; annealing: 30 s, 58‡C; elongation: 1 min, 72‡C) were performed and the ampli¢ed products of 430 and 300 base pairs (bp), were separated on 2% agarose gels by electrophoresis. Tissue preparation Experiments were performed on six male wild-type (B6D2F1) and eight male Otx13=3 mice (supplied by Acampora) housed in controlled conditions at the facility of Charles River Italia

(Calco LC, Italy). The experiments were performed in accordance with the Principles of Laboratory Animal Care (European Communities Council Directive 86/609/EEC), all e¡orts being made to minimize animal su¡ering and minimize the number used. The animals were killed at 60 postnatal days; they were ¢rst deeply anesthetized with 4% chloral hydrate (1 ml/100 g i.p.) and then rapidly perfused through the ascending aorta with 1% paraformaldehyde in 0.1 M phosphate bu¡er (PB), pH 7.2 followed by 4% paraformaldehyde in PB. The brains were immediately removed, post¢xed for 12 h in 4% paraformaldehyde in PB at 4‡C, transferred to PB, pH 7.2, and processed for histological and immunocytochemical examination. 50-Wm-thick serial coronal sections of the entire rostro-caudal extent of the somatosensory cortex were cut with a vibratome (Leica) and collected in PB, pH 7.2. Sections adjacent to those processed for immunocytochemistry were stained with Thionin (0.1% in distilled water) for cytoarchitectonic control and determination of cortical layers and thickness. Determination of thickness of cortex and cortical layers Selected Thionin-stained sections from ¢ve wild-type and ¢ve knock-out mice were used for this analysis. The thickness of the cortex and of each cortical layers were evaluated on three Thionin-stained sections from each brain taken at approximately the rostral, central and caudal levels of the somatosensory cortex, using a U20 objective and the Image Measure software (Microscience, Seattle, WA, USA). The mean total cortical thickness and mean thickness of each layer, level and group were then calculated, again with the aid of the Image Measure software. Di¡erences in thickness were tested using the Student’s t-test, with P 6 0.05 being considered statistically signi¢cant. Immunocytochemistry Selected free-£oating 50-Wm sections from wild-type and knock-out mice were processed in the same vial to standardize conditions. Immunoperoxidase and immuno£uorescent labeling procedures were used. The following antibodies were used: (a) anti-non-phosphorylated neuro¢lament (SMI311) antibody and anti-subunit 1 of NMDA receptor (NMDAR1) antibody, to clearly reveal pyramidal cells. (b) Markers of inhibitory neurons: anti-glutamic acid decarboxylase (GAD67; 67 kDa) antibody, antibodies against the calcium-binding proteins parvalbumin (PV), calbindin D-28K (CB) and calretinin (CR) (expressed in non-overlapping subpopulations of inhibitory cortical interneurons). (c) Anti-GABA transporter 1 (GAT-1) antibody to reveal inhibitory terminals and distal astrocyte processes. (d) Markers of glia: anti-glial ¢brillary acidic protein (GFAP) antibody and anti-GABA transporter 3 (GAT-3). See Table 1 for antibody sources, technical details and concentrations used.

Table 1. Sources, technical details and concentrations of antibodies used Antigen

Source

Type

Speci¢city

SMI 311 NMDAR1 PV GAD67 CR CB GAT-1

Sternberger Monoclonals Incorporated Chemicon Sternberger Monoclonals Incorporated Chemicon Swant-Swiss antibodies Swant-Swiss antibodies Chemicon

mouse IgG mAb rabbit IgG pAb mouse IgG mAb rabbit IgG pAb rabbit IgG pAb mouse IgG mAb rabbit IgG pAb

GAT-3 GFAP

Chemicon Boehringer Mannheim

rabbit IgG pAb mouse IgG mAb

non-phosphorylated NF R1 subunit of ionotropic receptors subpopulations of GABAergic interneurons GABAergic cell bodies and axon terminals subpopulations of GABAergic interneurons subpopulations of GABAergic interneurons GABAergic axon terminals, astrocytic processes astrocytic processes IF in astrocytes

Working dilutions LM

LSCM

EM

1:1 000 1:100 1:10 000 1:1 000 1:5 000 1:10 000 1:500

1:100

1:500

1:250 1:500

EM = Electron microscopy ; GAD = glutamic acid decarboxylase ; GFAP = glial ¢brillary acidic protein; IF = intermediate ¢laments; LM = light microscopy ; LSCN = laser scanning confocal microscopy ; mAb = monoclonal antibody; NF = neuro¢laments ; pAb = polyclonal antibody.

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Immunoperoxidase staining. Sections were pre-treated with 3% H2 O2 for 5 min, preincubated for 45 min in 0.1 M phosphate-bu¡ered saline (PBS), pH 7.4. containing 10% normal goat serum (NGS) for polyclonal antibodies, or normal horse serum (NHS) for monoclonal antibodies and 0.2% Triton X-100 to mask non-speci¢c adsorption sites. Sections were then incubated overnight at 4‡C in the primary antibody. After rinsing in PBS, they were incubated in biotinylated goat anti-rabbit or anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) diluted 1:200, in PBS/1% NGS or NHS. After rinsing in PBS, the sections were further incubated in avidin^biotin^peroxidase complex (ABC kit, Vector Laboratories, Burlingame, CA, USA) diluted 1:100 in PBS/1% NHS or NGS. In the ¢nal step the sections were incubated with 0.075% 3,3P-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA) in 0.05 M Tris^ HCl and 0.002% H2 O2 ; they were then mounted on gelatincoated slides, air-dried, dehydrated and coverslipped. Control sections were processed without primary antibody; no immunostaining was ever observed on control sections. The distribution of PV-immunoreactive neurons was plotted on maps using an X^Y plotter connected by linear potentiometers to the stage of a Leitz Diaplan microscope. Cortical layers were identi¢ed on adjacent Thionin-stained sections and transferred to the chart by means of a projection microscope. Immuno£uorescence staining. Selected sections were incubated for 30 min in PBS containing 1% bovine serum albumin and 0.2% Triton X-100, and then in primary anti-GAT-1 diluted in PBS containing 0.1% bovine serum albumin (48 h at 4‡C). Subsequently sections were incubated in indocarbocyanine (Cy2)conjugated goat anti rabbit (Jackson Immunoresearch Laboratories, West Grove, PA, USA) diluted 1:80. The sections then examined in a TCS NT confocal laser-scanning microscope (Leica Lasertecknik GmbH, Heidelberg, Germany) equipped with a 75-mW krypton/argon laser. Cy2 was excited at 488 nm and imaged using Leica Power Scan software. Quantitation of GAT-1 immunoreactivity in layer V The areas of GAT-1-immunoreactive structures in layer V were estimated on peroxidase-stained sections of somatosensory cortex examined in a Zeiss III photomicroscope using a U63 oil immersion objective. For the estimation, layer V images were captured using a digital video camera. Six to eight frames (8439 Wm2 each) per animal were recorded (from three knock-out and three wild-type mice) and imported into the public domain software NIH-Image. Background was subtracted using the Rolling Ball command and thresholding was performed at a constant gray level to obtain binary images on which the total area of GAT-1-immunoreactive structures was calculated in Wm2 . The densities of GAT-1-immunoreactive terminal boutons (number per 100 Wm2 of cell membrane) attached to the cell bodies of 15 knock-out (one animal) and 15 wild-type pyramidal neurons from layer V were estimated using the disector method (Sterio, 1984; Coggeshall and Lekan, 1996; Simon and Horcholle-Bossavit, 1999). To determine these densities, serial 0.4-Wm sections were cut from vibratome-cut sections that had been processed for GAT-1 immunocytochemistry and then embedded in epoxy resin. These thick section tissues had been permeabilized with Triton X-100 (0.2% in NGS, 45 min) to increase the penetration of the immunoreagents for density determination. A diamond knife (Histo, Diatome) was used to ensure consistently uniform section thickness. Counting was performed on pro¢le pairs (two to six) of the central portion of cell bodies in which the nucleolus was visible in at least two sections. Pyramidal neurons were identi¢ed by their triangular shape and presence of apical dendrites.

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GAT-1 antibody using the ABC method, post¢xed with 2.5% glutaraldehyde in PB (1 h) followed by 1% osmium tetroxide (1 h) and £at embedded in Epon-Spurr. Ultrathin sections were then cut on a Reichert ultramicrotome and examined, unstained or counterstained with lead citrate only, in a Zeiss 902 electron microscope. Statistical analyses Di¡erences in the thickness of the cortex and cortical layers were tested using the Student’s t-test, with P 6 0.05 being considered statistically signi¢cant.

RESULTS

Thickness of cortex and cortical layers Although layering was clearly evident in the cortex of knock-out mice (Fig. 2B), we found that the total thickness of the somatosensory cortex was about 25% less than in wild-type mice (Fig. 1). The thickness of the individual layers were also reduced: layers II and III by 35%, layer VI by 30% and layer V by 22%; the average thickness of layer I was the same as in both wild-type and mutated cortex. Unmistakable cytological alterations were not observed, however neurons in layer V of mutant cortex were less densely packed (Fig. 2D) than in the corresponding layer of wild-type cortex (Fig. 2C). Immunocytochemical ¢ndings SMI311 and NMDAR1 as markers of pyramidal neurons. In wild-type cortex, SMI311-immunoreactive neurons were mainly present in layer III and the lower part of the layer V (Fig. 3A). Scattered immunoreactive cells were also found in layer IV, but never in layer I. Neuropil immunolabeling was evident in all cortical layers except layer I. SMI311 immunostaining was particularly intense in cell bodies, basal and apical dendrites of pyramidal neurons and also in rounded multipolar cells (Fig. 3A).

Immunoelectron microscopy Some vibratome sections, ¢xed as for light microscopy immunocytochemistry, were permeabilized by sequential incubation in 10%, 25% and 10% aqueous ethanol solution (5 min each) following which they were immunolabeled with anti-

Fig. 1. Histogram showing thickness of somatosensory cortex and each cortical layer ( T SD) in ¢ve wild-type and ¢ve Otx13=3 mice. *Signi¢cantly di¡erent (P 6 0.05).

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Fig. 2. Photomicrographs of Thionin-stained coronal sections of somatosensory cortex. Low-power micrographs of whole cortex, from pia mater (top) to white matter (bottom), in wild-type (A) and Otx13=3 (B) mice. Note the reduced cortical thickness in Otx13=3 compared to the wild-type. Higher magni¢cation of layer V in wild-type (C) and Otx13=3 (D) mice. Note that neurons are less densely packed in knock-out. Scale bars = 100 Wm (A, B) and 50 Wm (C, D).

In Otx13=3 cortex, neuropil immunoreactivity for SMI311 was more intense than in wild-type cortex. Scattered layer II pyramidal neurons with apical dendrites arborizing in layer I were immunopositive for SMI311 in mutant but not in wild-type cortex (Fig. 3B). There were fewer immunopositive cell bodies and apical den-

drites in layers III and V than in the corresponding wildtype cortical layers (Fig. 3A, B). In wild-type cortex, most pyramidal cell bodies were NMDAR1-immunopositive (Fig. 3C). The entire neuropil of the cortex was also uniformly positive for NMDAR1.

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Organization of somatosensory cortex in Otx13=3 mice

Fig. 3. Photomicrographs showing immunostaining for SMI311 (A, B) and NMDAR1 (C, D) in coronal sections of cortex from wild-type (A, C) and Otx13=3 (B, D) mice. SMI311 immunoreactivity (A, B) is particularly evident in cell bodies (arrows) and apical dendrites (arrowheads) of neurons including pyramidal cells. Note the reduced number of immunoreactive cell bodies in layer V of Otx13=3 (B) compared to wild-type (A) and the presence of large pyramidal cells in the upper part of the cortex (arrows) of Otx13=3 (B). NMDAR1 immunoreactivity (C, D) is mainly present in the cell bodies of the layer V pyramidal neurons in wild-type cortex (C, arrows) and in intensely immunoreactive neurons in the super¢cial cortical layer of Otx13=3 cortex (D, arrows). Scale bars = 100 Wm.

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Fig. 4. Photomicrographs showing PV (A, B) and GAD67 (C, D) immunoreactivity in coronal sections of wild-type (A, C) and Otx13=3 (B, D) cortex. In wild-type cortex, PV- (A) and GAD67- (C) immunoreactive cells are distributed uniformly throughout the cortex. In Otx13=3 cortex PV (B) and GAD67 (D) labeled neurons are distributed patchily : there are areas where immunoreactive interneurons are rare or absent (asterisks) and adjacent areas where their density is comparable to that in wild-type cortex. Scale bars = 100 Wm.

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Organization of somatosensory cortex in Otx13=3 mice

In Otx13=3 cortex, occasional large pyramidal cells were NMDAR1 positive in the super¢cial layers, particularly rostrally (Fig. 3D). Fewer NMDAR1-immunoreactive neurons were present in layer V than the same layer of wild-type cortex. The distribution of pyramidal neurons labeled for NMDAR1 in Otx13=3 cortex was similar to that for SMI311 (Fig. 3B, D). Markers of inhibitory neurons. Cell bodies, proximal dendrites and neuropil were intensely positive for PV in both wild-type and knock-out cortex (Fig. 4A, B). In wild-type cortex, PV-positive neurons were present in all layers except, as expected, layer I (Figs. 4A and 5). In both wild-type and Otx13=3 mice, intensely immunoreactive punctate structures were present throughout the cortex excepting only the upper part of layer V where overall PV positivity was least intense (Fig. 4A, B). These structures, most of which were probably axon terminals, were particularly evident surrounding unlabeled pyramidal cells. In Otx13=3 mice, PV-immunoreactive cell bodies were not uniformly distributed, with areas in which immunolabeling was scanty or absent adjacent to those where the labeling was similar to that in wild-type cortex (Figs. 4B and 5). GAD67-immunoreactive cells were present in all layers in both wild-type and knock-out cortex (Fig. 4C, D). The cells thus picked out were small- to medium-sized neurons of rounded or fusiform shape. Proximal dendrites were also labeled occasionally. Large numbers of punctate structures, interpreted as terminals or ¢ber crosssections, were intensely positive for GAD67. GAD67 immunolabeling in Otx13=3 cortex was patchy and similar to that of PV immunolabeling (Fig. 4D). We found that interneurons and neuropil in wildtype cortex were immunoreactive to CR and CB in the same way as described by Hof et al. (1999). Knock-out cortex mice did not di¡er from wild-type cortex in these respects (data not shown). GAT-1. GAT-1 immunoreactivity was exclusively

663

localized in punctate structures scattered in the neuropil or surrounding cell bodies and dendrites. This was the case both in wild-type and knock-out cortex. Staining intensity varied between layers, being greatest in layers II and III and in the upper part of layer V (Fig. 6A, B). The density of labeling was higher in Otx13=3 than wildtype cortex (Fig. 6B), this was particularly evident in layer V viewed under the confocal microscope (Fig. 6C, D). However, the densities of terminals apposed to pyramidal cell bodies did not di¡er between wild-type and Otx13=3 cortex. This was demonstrated by quantitative analysis, which showed that the total area of GAT-1labeled structures in layer V of the somatosensory cortex was 40^160% higher in knock-out than wild-type, while the density of GAT-1 terminals (number/100 Wm2 of cell membrane T SD) apposed to layer V pyramidal cell bodies did not signi¢cantly di¡er between wild-type (17.7 T 3.3) and knock-out (21.2 T 7.1) cortex. The sizes of terminals in Otx13=3 cortex were closely similar in knock-out and in wild-type cortex (1.2 Wm2 T SD 0.4 in both). The labeling of GAT-1-immunoreactive structures at the ultrastructural level was similar in wild-type and knock-out cortex. Electron-dense reaction product labeled terminals and distal astrocytic processes throughout the neuropil. The labeled terminals always formed symmetric contacts with unlabeled dendrites or somata (Fig. 7). Labeled distal astrocytic processes were numerous, typically surrounded axon terminals that formed both symmetric or asymmetric synapses, and also surrounded unlabeled dendrites and somata. GAT-1 labeling was also observed in perivascular astrocytic processes (Fig. 7). Markers of glia. The distribution and intensity of GAT-3 immunoreactivity did not di¡er between wildtype and knock-out cortex. GAT-3-positive puncta were observed both in the neuropil and in close relationship with cell bodies, but the density of these structures varied, being lowest in the upper part of layer V and in layer VI (data not shown).

Fig. 5. Schematic representation of the distribution of PV-immunoreactive neurons in wild-type and Otx13=3 mice in four representative coronal sections from four di¡erent mice. Note the patchy distribution in Otx13=3 compared to wild-type.

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Fig. 6. Photomicrographs of GAT-1 immunoreactivity in coronal sections of wild-type (A) and Otx13=3 (B) cortex revealed by immunoperoxidase staining. Confocal laser scanning photomicrographs of GAT-1 immunoreactivity in layer V sections of wild-type (C) and Otx13=3 (D) cortex revealed by immuno£uorescence. Note the greater staining intensity in knock-out (B) than wild-type cortex (A). Note also the greater density of GAT-1-positive punctate structures in the neuropil of knock-out (D) than in wild-type layer V (C) while there is no apparent di¡erence in the density of terminals (arrows) apposed to the cell bodies of pyramidal neurons (picked out by asterisks in C and D). Scale bars = 100 Wm (A, B) and 10 Wm (C, D).

The distribution and intensity of GFAP immunoreactivity did not di¡er between wild-type and knock-out cortex. GFAP-positive cells and processes were mainly present, as expected, in the white matter; a few scattered GFAP-immunoreactive astrocytes were also present in the gray matter (data not shown).

DISCUSSION

This study has provided the ¢rst detailed morphological and immunocytochemical description of the somatosensory cortex of Otx13=3 mice. As also reported for the perirhinal and temporal cortices (Acampora et al., 1996,

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Organization of somatosensory cortex in Otx13=3 mice

Fig. 7. Ultrastructural localization of GAT-1 immunoreactivity in layer V of a knockout cortex. (A) Axon terminals (t) make symmetric synapses on dendrites (D); asterisks indicate astrocytic processes sparse in the neuropil; arrows indicate astrocyte processes surrounding an unlabeled axon terminal that forms an asymmetric synapse. (B) A GAT-1 axon terminal (t) makes a symmetric synaptic contact with a neuronal cell body (CB). (C) A GAT-1 terminal (t) makes a symmetric synapse with a dendrite also contacted by an unlabeled axon terminal (triangle) and is adjacent to a labeled astrocytic process (asterisk). Scale bars = 1 Wm. (A), 0.45 Wm (B) and 0.35 Wm (C).

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2001), we found that the somatosensory cortex in these mutants was markedly reduced in thickness, while its laminar organization was substantially preserved. Furthermore, as previously observed in slices of neocortex (Sancini et al., 2001), pyramidal neurons in layer V of the somatosensory cortex were less densely packed in mutant than wild-type cortex. Our immunocytochemical characterization using pyramidal cell markers con¢rmed the altered distribution of these cells in layer V, and also revealed the presence of large pyramidal neurons in the upper part of the cortex. It is possible that these cells may be part of the cohort of layer V pyramidal neurons migrates to an ectopic position and that absence of the Otx1 gene may interfere with the inside^out processes of corticogenesis (Acampora et al., 1996, 2001). However, this supposition requires testing by combined thymidine radiographic or BrdU and immunocytochemical studies, and it cannot be excluded that the large pyramidal neurons in the super¢cial layers are a subset of cells normally destined for the upper layers that have developed unusually large morphologies. These glutamatergic neurons may also contribute to the seizures facility in Otx13=3 mice (Acampora et al., 1996, 2001). We investigated the expression of the calcium-binding proteins CB, CR and PV to identify non-overlapping subclasses of GABAergic interneurons (Celio, 1986; Conde' et al., 1994; DeFelipe, 1997; Gonchar and Burkhalter, 1997; Hof et al., 1999). We found that the distribution of these proteins in the somatosensory cortex of wild-type mice was similar to that described in other rodents (Celio, 1986; Gonchar and Burkhalter, 1997; Hof et al., 1999), while the distribution and morphology of CB- and CR-positive neurons in the cortex of Otx13=3 mice were similar to those of the controls, indicating that these classes of interneurons are not a¡ected by lack of the Otx1 gene. However, PV-positive neurons were patchily distributed in Otx13=3 cortex, indicating that chandelier and basket cells ^ the neurons that express PV and provide the most important inhibitory inputs to pyramidal cells ^ are unevenly localized. This ¢nding was con¢rmed by the GAD67 data which showed GAD67-labeled cell bodies were also irregularly distributed. Whether this alteration is directly related to absence of the Otx1 gene or is a consequence of the inappropriate location of a subset of excitatory cells remains to be elucidated. PV- and GAD67-positive neurons are also found patchily distributed in the cortex of patients with intractable epilepsy associated with architectural dysplasia. These alterations were related to the pathophysiological activities underlying epileptogenesis (DeFelipe et al., 1994; Sprea¢co et al., 2000). However, while PV- or GAD67-immunoreactive puncta were markedly reduced in the dysplastic human cortex (Sprea¢co et al., 2000), their densities in the neuropil of Otx13=3 somatosensory cortex were similar to those in wild-type mouse cortex. To provide information on GABAergic transmission we investigated the expression of GAT-1 and GAT-3 in the Otx13=3 cortex. These are GABA transporters, have high a⁄nity for this neurotransmitter, and are present in pre-synaptic terminals and the glia surrounding synapses.

They regulate the concentration of extracellular GABA and hence the intensity and duration of post-synaptic inhibition potentials (Borden and Caplan, 1996; Gadea and Lopez-Colome, 2001). Studies on the cerebral cortex of various mammals (Minelli et al., 1995, 1996; Conti et al., 1998) have shown that GAT-1 immunoreactivity is both glial and neuronal, while GAT-3 immunoreactivity is restricted to astrocytes. Our results demonstrated that, while the labeling density of GAT-3 was similar in mutant and wild-type cortex, the density of GAT-1-immunoreactive structures was much higher in the former. The ultrastructural data revealed that GAT-1 immunoreactivity, as previously shown in other mammals (Minelli et al., 1995; Conti et al., 1998) was present in astrocyte pro¢les and in terminals making symmetric synapses in both Otx13=3 and control mice. The higher density of GAT-1 immunoreactivity in knock-out mice could re£ect an increase in labeled distal astrocyte processes, or labeled synaptic terminals, or both. However, the labeling pattern for the glial markers GFAP and GAT-3 did not di¡er between knock-out and control mice, indicating that the number and distribution of astrocytic processes were similar. We therefore propose that the higher density of GAT-1-positive structures in Otx13=3 mice is due to an increased number of GABAergic terminals. The fact that GAD67 immunocytochemistry did not reveal di¡erences in GABAergic synaptic terminal density may be because GAT-1 is a more sensitive marker of inhibitory terminals as noted previously (Minelli et al., 1995). Since the electrophysiological properties of layer V pyramidal neurons in knock-out mice indicate alterations in GABAergic transmission (Sancini et al., 2001) we quantitated GAT-1-immunoreactive structures in this layer. As also indicated by our qualitative immunohistochemical observations, we found that the total area of GAT-1 labeled structures was greatly increased in layer V of Otx13=3 cortex, while the density of GAT-1-labeled inhibitory terminals on pyramidal neurons did not di¡er from that in wild-type cortex. Increased GAT-1 results in a reduced persistence of GABA released into the synaptic cleft due to its increased uptake from pre-synaptic terminals and glia (Bernstein and Quick, 1999). The expected result is reduced inhibition. Interestingly, however, this change seems to selectively a¡ect interneurons whose dis-inhibition results in increased inhibition of pyramidal neurons. This is consistent with the ¢ndings of recent electrophysiological experiments on cortical slices from Otx13=3 mice which showed that abnormally enhanced GABAA - and GABAB -mediated inhibitory post-synaptic potentials (IPSPs) could be readily evoked in pyramidal neurons (Sancini et al., 2001). These high IPSPs, often followed by NMDA-mediated excitatory post-synaptic potentials, could play an important role in synchronizing abnormal excitatory events thus contributing to the highly synchronized epileptogenic discharges that characterize Otx13=3 mice. Acknowledgements/This study was supported by the Italian Ministry of Health. The authors thank M.C. Regondi for the technical support and D.C. Ward for help with the English.

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