Original Paper Dev Neurosci 2013;35:516–526 DOI: 10.1159/000355392

Received: April 3, 2013 Accepted after revision: August 30, 2013 Published online: November 14, 2013

Genesis of Heterotopia in BCNU Model of Cortical Dysplasia, Detected by Means of in utero Electroporation Ramona Frida Moroni Francesca Inverardi Maria Cristina Regondi Paolo Pennacchio Roberto Spreafico Carolina Frassoni Unit of Clinical Epileptology and Experimental Neurophysiology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy

Abstract Derangements of cortical development can cause a wide spectrum of malformations, generally termed ‘cortical dysplasia’ (CD), which are frequently associated with drug-resistant epilepsy and other neurological and mental disorders. 1,3-Bis-chloroethyl-nitrosurea (BCNU)-treated rats represent a model of CD due to the presence of histological alterations similar to those observed in human CD. BCNU is an alkylating agent that, administered at embryonic day 15 (E15), causes the loss of many cells destined to cortical layers; this results in cortical thinning but also in histological alterations imputable to migration defects, such as laminar disorganization and cortical and periventricular heterotopia. In the present study we investigated the genesis of heterotopia in BCNUtreated rats by labeling cortical ventricular zone (VZ) cells with a green fluorescent protein (GFP) expression vector by means of in utero electroporation. Here, we compared the migratory pattern and subsequent distribution of the GFPlabeled cells in the developing somatosensory cortex of con-

© 2013 S. Karger AG, Basel 0378–5866/13/0356–0516$38.00/0 E-Mail [email protected] www.karger.com/dne

trol and BCNU-treated animals. To this aim, we investigated the expression of a panel of developmental marker genes which identified radial glia cells (Pax6), intermediate precursors cells (Tbr2), and postmitotic neurons destined to infragranular (Tbr1) or supragranular layers (Satb2). The VZ of BCNU-treated rats appeared disorganized since E18 and at E21 the embryos showed an altered migratory pattern: migration of superficial layers appeared delayed, with a number of migrating cells in the intermediate zone and some neurons destined to superficial layers arrested in the VZ, thus forming periventricular heterotopia. Moreover, neurons that reached their correct position did not extend their axons through the corpus callosum in the contralateral hemisphere as in the control, but toward the ipsilateral cingulated cortex. Our analysis sheds light on how a malformed cortex develops after a temporally discrete environmental insult. © 2013 S. Karger AG, Basel

Introduction

The mammalian cerebral cortex is a complex laminated structure that forms during embryonic development through the migration of neurons from proliferative regions near the cerebral ventricles. The cortical ventricular zone (VZ) and the subventricular zone (SVZ) are Carolina Frassoni Unit of Clinical Epileptology and Experimental Neurophysiology Fondazione IRCCS Istituto Neurologico Carlo Besta Via Amadeo 42, IT–20133 Milan (Italy) E-Mail frassoni @ istituto-besta.it

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Key Words Neuronal migration defects · Cortical malformations · Periventricular heterotopia · Developmental marker genes · Callosal projection alteration · Green fluorescent protein transfection

Genesis of Heterotopia in a Model of Cortical Dysplasia

neuronal generation and migration during the development of different brain areas [15–17]. Here, we compared the migratory pattern and subsequent distribution of the GFP-labeled cells in the developing prospective somatosensory cortex of control and BCNU-treated rats and we investigated the expression of a panel of developmental marker genes in order to reveal the migration defects that cause CD in this model. Materials and Methods In utero Electroporation All the experiments were performed in accordance with the guidelines defined by the European Communities Council Directive (86/609/EEC) and all efforts were made to limit the number of animals used and their suffering. The experimental protocol was approved by the Ethics Committee of the Italian Ministry of Health (NFS-02-2012). A total of 8 pregnant Sprague-Dawley rats (Charles River Italia, Calco, Italy) were intraperitoneally injected with 20 mg/kg of BCNU solution (5% sterile glucose in water at 4 mg/ml) at E15 (the day of mating was considered E0). On the same gestational day, 6 pregnant rats were injected intraperitoneally with 5% glucose alone and used as control [9]. The timed pregnant rats were anesthetized with a solution of Domitor (300 μg/kg) and Zoletil (30 mg/kg), their abdominal cavity cut open, and the uterine horns exposed. A lateral ventricle of each E16 embryo was injected with a solution of approximately 1–2 μl of 0.5 μg/μl pCAG-GFP plasmid (Addgene plasmid 11150 [18]) and 0.05% Fast Green (Sigma, St. Louis, Mo., USA) via a microinjector (PDES-01D-2; NPI electronic GmbH, Tamm, Germany) and pulled glass capillaries (Drummond, Broomall, Pa., USA). Each embryo within its uterus was placed between tweezers-type electrodes (CUY650P5; BTX Harvard Apparatus, Holliston, Mass., USA). For targeting the cortical VZ, the angle of inclination of the electrode paddles with respect to the horizontal plane of the brain was 45°. Square electric pulses (50 V for 50 ms) were passed 5 times at 950-ms intervals using an electroporator (ECM 830; BTX Harvard Apparatus). The abdomen was closed with sutures and the embryos were allowed to develop normally until E18 or E21. Fetuses were collected by cesarean section and their brains were fixed by immersion in 4% paraformaldehyde (PFA) for 3 days; 2 litters (1 control and 1 treated with BCNU) were allowed to be born and subsequently analyzed at postnatal day 7 (P7). They were transcardially perfused with 4% PFA, and their brains were removed from the skull and post-fixed in PFA for 24 h. The brains, included in agar, were then cut into 60-μm-thick serial coronal sections by means of a Vibratome VT1000S (Leica, Heidelberg, Germany) and processed for immunofluorescent staining. Immunofluorescent Staining and Confocal Microscopy Selected free-floating sections were blocked in phosphate-buffered saline (PBS) containing 10% normal horse or goat serum and 0.2% Triton-X100, and the sections were incubated overnight at 4 ° C with the following antisera diluted in 10% normal horse or goat serum in PBS: 1:5,000 anti-calbindin (CB; Swant, Bellinzona, Switzerland); 1: 300 anti-Pax6 (Covance, Princeton, N.Y., USA); 1:1,000 Tbr2 (Merck Millipore, Billerica, Mass., USA); 1:400 Tbr1  

 

 

Dev Neurosci 2013;35:516–526 DOI: 10.1159/000355392

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the proliferating regions where radial glia and progenitor cells divide and give birth to excitatory neurons; immature neurons migrate radially toward the pial surface through the intermediate zone (IZ) and settle in the cortical plate (CP) following an inside-out gradient according to birthdates, i.e. the earliest-born neurons are destined to become the future layer VI, and the last-born neurons will become layer II [for review, see 1, 2]. Concomitantly, GABAergic interneurons, generated in subcortical germinative zones (the ganglionic eminence), migrate tangentially and ultimately integrate themselves into the correct layers in the cortex [for review, see 3, 4]. The migration of newly born neurons is a precisely regulated process that is critical for the development of the brain architecture. Derangements of cortical development can cause a wide spectrum of malformations, generally termed ‘cortical dysplasias’ (CD), which are frequently associated with drug-resistant epilepsy and other neurological and mental disorders [5–7]. 1,3-Bis-chloroethyl-nitrosourea (BCNU)-treated rats represent a model of CD due to the presence of histological alterations similar to those observed in human CD. BCNU is an alkylating agent that, administered at embryonic day 15 (E15) at the peak of neurogenesis, causes the loss of many cells destined to cortical layers; this results in cortical thinning and also in histological alterations imputable to  migration defects, such as laminar disorganization and cortical and periventricular heterotopia [8, 9]. The cytological and histological alterations of this model have been widely investigated in both adult and developing animals by means of immunohistochemical markers recognizing interneurons or pyramidal neurons, by analyzing the expression pattern of an appropriate panel of cortical layer-specific genes and by using magnetic resonance imaging [9–11]. Changes in neocortical organization of BCNU-treated rats have been revealed since the embryonic stages, when the disorganization of the ventricular wall, the disruption of the radial and tangential fibers, and the defective organization of the CP and subplate can be observed [9–11]. In the present report, with the aim of studying the genesis of heterotopia in BCNU-treated rats, we investigated the development of CD by labeling cortical VZ cells with a green fluorescent protein (GFP) expression vector by means of in utero electroporation. This technique enables the introduction, locally and temporally, of a marker gene into cortical or other neural progenitor cells in developing embryos in the uterus in order to visualize the process of migration and layer formation [12–14]. In utero electroporation has been successfully applied to the study of

 

Cell Counts For quantification of cell distribution in E18 and E21 embryos, the percentage of GFP+ cells in SVZ/VZ, IZ and CP were calculated in 3 control and 3 BCNU-treated embryos for each age (three adjacent sections for each embryo). Different subregions of the cerebral cortex were identified based on cell density using DAPI (Molecular Probes) staining. Cell counting was done using Image Pro-Plus 7.0 software (Media Cybernetics, Bethesda, Md., USA) on images acquired with confocal microscope D-Eclipse C1 (Nikon). Eight Z-series images were collected at 6-μm steps for each section and all the GFP+ cells of the section were counted in three images of the stack. For quantification of GFP+ cells immunolabeled for NeuN, Tbr1 and Satb2 in the ventricular heterotopia, all GFP+ cells and the double-labeled cells in three ventricular heterotopia were counted, taking confocal microscope images with a 40× objective (three confocal levels for each heterotopia).

Results

By means of in utero electroporation we introduced a GFP-expressing plasmid into the VZ cells of the neocortex of control and BCNU-treated E16 embryos. We analyzed the GFP-expressing cells in embryos at E18 and E21 and in postnatal rats at P7. To clarify the neuronal composition of developing heterotopia, we explored the expression of a panel of developmental marker genes identifying radial glial cells (Pax6), intermediate precursors (Tbr2), and supragranular (Satb2) and infragranular (Tbr1) layers. Of 52 control electroporated embryos, 27 survived, giving an average survival rate of 52%; of 74 BCNU-treated electroporated embryos, 42 survived, giving an average survival rate of 57%. Of these, 70% (n = 19) control and 38% (n = 16) BCNU-treated embryos contained GFP+ cells correctly positioned in the prospective somatosen518

Dev Neurosci 2013;35:516–526 DOI: 10.1159/000355392

sory cortex. An addition of 11 control and 11 BCNUtreated embryos were electroporated and allowed to be born; 10 control and 7 BCNU-treated pups were born and let grow until P7. Among them 5 control and 3 BCNUtreated pups contained GFP+ cells correctly positioned. E18: Birthday of Periventricular Heterotopia At 2 days after electroporation, there was no obvious difference in the location of GFP+ cells between control and BCNU-treated embryos (fig.  1a, b). At this time, most of the electroporated cells were located in the IZ (55.36 ± 4.3% in control and 55.35 ± 4.7% in BCNUtreated embryos) and some were migrating towards the CP (11.26 ± 5.9% in control and 11.08 ± 5.4% in BCNUtreated embryos; fig. 1c, d); GFP+ radial glial cells were clearly detectable in the VZ (33.36 ± 5.1% in control and 33.55 ± 5.7% in BCNU-treated embryos; fig. 1e, g). The typical morphological transitions of migrating neurons were appreciable in both control and BCNU-treated rats. After a start as bipolar progenitors and precursors in the VZ (fig. 1e, g), cells became multipolar and branched as they entered the lower IZ (fig. 1f, h); within the IZ, neurons re-established a bipolar morphology as they migrated towards and into the CP (fig. 1c, d). As shown by calbindin immunohistochemistry, which labels the subplate and the marginal zone, the main alterations detectable in the cortical anlage of the BCNU-treated embryos were the thickening of the subplate and the thinning of the CP (fig. 1b, d) compared to the controls (fig. 1a, c). Moreover, the ventricular wall of the BCNUtreated embryos was disorganized. In control embryos the ventricular wall consisted of two different proliferating regions: an apical VZ, identified by Pax6 immunoreactivity, which labeled radial glial cells (fig. 2a) and a basal SVZ, identified by Tbr2 immunoreactivity, which labeled intermediate precursors cells (fig.  2b). GFP+/ Pax6+ radial glial cells were correctly located in the VZ of control embryos, and GFP+/Tbr2+ intermediate precursors were located in SVZ. In the BCNU-treated embryos, though Pax6 immunoreactivity (fig. 2d) showed that the VZ was similar to the control, Tbr2 immunoreactivity revealed the ventricular disarrangement (fig. 2e). Indeed, some GFP+/Tbr2+ cells were located in the VZ of the BCNU-treated animals among groups of heterotopic Tbr2+ intermediate precursors. These cells probably represent the primordium of the periventricular heterotopia, which were clearly detectable later at E21. In the control embryos, Satb2 was expressed in the CP and IZ but not in the VZ/SVZ (fig. 2c) and Tbr1 was expressed in layers VI and V but not in the VZ/SVZ (data not Moroni/Inverardi/Regondi/Pennacchio/ Spreafico/Frassoni

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(Merck Millipore); 1: 600 Satb2 (Abcam, Cambridge, UK), and 1: 400 anti-α-internexin (Chemicon International, Temecula, Calif., USA). After several rinses in PBS, the sections were incubated in Cy3-conjugated goat anti-rabbit IgG (1:600), Cy3-conjugated goat anti-mouse IgG (1:200) or DyLight549-conjugated goat anti-chicken IgG (1:600; all Jackson ImmunoResearch Laboratories, West Grove, Pa., USA). In order to check antibody specificity, some sections were processed omitting the primary antibody; no specific staining was detected. Sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, Oreg., USA), mounted with FluorSave (Calbiochem, San Diego, Calif., USA), and examined through a confocal microscope DEclipse C1 (Nikon, Tokyo, Japan) mounted on a light microscope Eclipse TE2000-E (Nikon). Confocal images were imported into Adobe Photoshop CS5 (Adobe Systems Incorporated, San Jose, Calif., USA). Montages of images were constructed after adjustment of contrast and brightness.

gration in the prospective somatosensory cortex of control (a, c, e, f) and BCNUtreated E18 embryos (b, d, g, h), as revealed by GFP (green) expression following in utero electroporation. Brain sections have been immunolabeled for calbindin (red) and counterstained with DAPI (blue). MZ = Marginal zone; SP = subplate. a, b No obvious differences in the location of GFP+ cells between control and BCNU-treated embryos are detectable – most of the GFP+ cells are located in the IZ, some are located in the VZ and some are migrating towards the CP. c, d Detail of the cortex showing bipolar migrating neurons (arrows) towards the CP and multipolar neurons in the IZ. e, g High magnification of the VZ showing GFP+ radial glial cells (arrows) with the typical bipolar morphology. f,  h High magnification of the IZ showing multipolar and branched cells (arrowheads). Scale bars: 231 μm (a, b); 58 μm (c, d); 44 μm (e–h).

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shown). Similar to that observed in control, neither Satb2 nor Tbr1 were expressed in the VZ/SVZ of E18 BCNUtreated embryos (fig. 2f).

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E21: Disarrangements of Callosal Projections and Periventricular Heterotopia Maturation We found a striking difference in the pattern of migration of GFP+ cells between the E21 BCNU-treated and age-matched control embryos. In fact, in control embryos most of the GFP+ cells reached the CP (73.2 ± 6.6%) and only few were detectable in the IZ (18.2 ± 5.6%) and in the VZ/SVZ (8.7 ± 1.8%; fig. 3a); by contrast, in BCNU-treat-

ed animals, though many cells were located in the CP (48.1 ± 5.6%), some were located in the IZ (17.4 ± 2.7%) and groups of GFP+ cells were found in the ventricular wall (34.6 ± 6.9%; fig.  3c). Moreover, GFP+ cells that reached the CP extended their axon across the corpus callosum of the control embryos (fig. 3a, b), whereas most of the GFP+ axons in the BCNU-treated rats formed heterotopic bundles that projected towards the medial cortex (fig. 3c, d). It is interesting to note that fibers projecting from and to the thalamus (labeled with α-internexin) and callosal projections represented two well-separated groups of tangential fibers in control embryos (fig. 3b),

Genesis of Heterotopia in a Model of Cortical Dysplasia

Dev Neurosci 2013;35:516–526 DOI: 10.1159/000355392

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Fig. 1. Comparison between neuronal mi-

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mediate precursors are located in the SVZ in both the con-

trol  and the BCNU-treated rats but groups of Tbr2+ cells are erroneously located in the VZ of BCNU-treated rats (asterisk in e). Examples of double-labeled cells are indicated by arrows (a, b, d, e). c, f Satb2+ cells are not present in the SVZ or in the VZ, neither in the control nor in the BCNU-treated rats. Scale bar: 58 μm.

whereas they were intermingled in BCNU-treated embryos (fig. 3d), in which thalamic fibers were dispersed in a wider region in comparison to the control. The ventricular wall of the BCNU-treated embryos was evidently disarranged and, frequently, groups of heterotopic neurons protruded into the ventricles. In order to characterize the ventricular wall, we analyzed the expression of Pax6 and Tbr2 genes (fig. 4). The Pax6 gene (fig. 4a) was specifically expressed in the VZ of the control embryos and Tbr2 was specifically expressed in the SVZ (fig. 4b). Conversely, in the BCNU-treated embryos we found Pax6+ (fig. 4c) and Tbr2+ (fig. 4d) cells scattered both through the VZ and SVZ. In the BCNU-treated embryos we frequently observed clusters of GFP+

cells protruding into the ventricles, which morphologically differed from the GFP+ radial glial cells occasionally detectable in the controls. With the aim of characterizing these cells we analyzed the expression of NeuN (marker of postmitotic neurons), Tbr1 (marker of layers VI and V) and Satb2 (expressed in the CP), none of which was expressed in the ventricular wall of the controls (fig. 5a, d, g). About 31% of the GFP+ cells localized in the heterotopic clusters expressed NeuN (fig. 5b, c) and about 65% expressed Satb2 (fig. 5h, i) whereas just 1% expressed Tbr1 (fig. 5e, f), thus demonstrating that most of the GFP+ cells in the periventricular clusters are postmitotic neurons prevalently destined to superficial cortical layers.

Fig. 2. Details of the ventricular wall of control (a–c) and BCNUtreated E18 embryos (d–f) electroporated with GFP (green) and immunolabeled (red) for Pax6 (a, d), Tbr2 (b, e), and Satb2 (c, f). Sections are counterstained with DAPI (blue). a, d Pax6+ radial glial cells are correctly located in the VZ. b, e Tbr2+ inter-

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most of the GFP+ cells are located in the upper CP and they extend their axons across the corpus callosum. b Detail of the control cortex showing callosal projection (green) running towards the corpus callosum and thalamocortical afferents immunolabeled for α-internexin

(red) running through layer VI. c In the BCNU-treated embryo, though many GFP+ cells are located in the upper CP, some are located in the IZ (asterisk) and a group protrude in the ventricle (arrowhead); callosal axons project toward the medial cortex (arrow). d Detail of the cortex of a BCNU-treated embryo showing bundles of callosal axons (green) projecting towards the medial cortex and dispersed thalamocortical afferents (red). Sections are counterstained with DAPI (blue). Scale bars: 387 μm (a, c); 198 μm (b, d).

P7 In the P7 control rats, GFP+ cells were mainly distributed in layers IV and V (fig. 6a, c) whereas in the BCNUtreated rats, in addition to the GFP+ cells correctly positioned in the cortex, there were groups of electroporated cells located under the white matter (fig. 6b, d), forming periventricular heterotopia. In order to investigate the nature of the heterotopic cells, we analyzed the expression of Tbr1 and Satb2 genes. In the control rats, Tbr1 was prevalently expressed in layer VI (fig. 6a) whereas Satb2 was expressed by a subset of neurons throughout the cortical layers, with a prominent expression in layers II–IV (fig. 6c). In the electroporated region, the BCNU-treated rats showed a cortical expression pattern of Tbr1 (fig. 6b) and Satb2 genes (fig. 6d) similar to the controls. Never-

theless, groups of Satb2+ neurons were located in the periventricular heterotopia together with the electroporated cells (fig. 6d). These cells were GFP+, Satb2+ (fig. 6f) and Tbr1– (fig. 6e), thus indicating that they were cells destined to the superficial layers that arrested their migration. Few Tbr1+ cells were located at the border of the heterotopic nodules (fig. 6e).

Genesis of Heterotopia in a Model of Cortical Dysplasia

Dev Neurosci 2013;35:516–526 DOI: 10.1159/000355392

Discussion

The BCNU-treated rats represent an injury-based model of cortical malformations mimicking the histopathological abnormalities observed in human CD patients [9, 10]. The expression analysis of some layer-spe521

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Fig. 3. Neuronal migration and callosal projection in the prospective somatosensory cortex of control (a, b) and BCNU-treated E21 embryos (c, d), as revealed by GFP (green) expression following in utero electroporation. cc = Corpus callosum. a In the control embryo,

Color version available online

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Fig. 4. Details of the ventricular wall of control (a, b) and BCNU-treated E21 embryos (c, d) electroporated with GFP (green) and immunolabeled (red) for Pax6 (a, c) and Tbr2 (b, d). In the control embryo Pax6 is expressed in the VZ (a) and Tbr2 is expressed in the SVZ (b). In the BCNU-treat-

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ed embryo both Pax6+ and Tbr2+ cells are scattered through the VZ and SVZ (c, d). The sections are counterstained with DAPI (blue). Scale bar: 59 μm.

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Dev Neurosci 2013;35:516–526 DOI: 10.1159/000355392

ronal migration. Indeed, the efficiency of cell transfection in control rats was consistent with data reported in the literature [12, 21, 22], whereas that of the BCNU-treated rats was less so, probably due to cell death induced by drug administration [11]. The overall analysis of electroporated cell distribution during cortical development revealed that the most obvious alterations of neuronal migration of the BCNU-treated cortices were detectable since E21. At this age we revealed the simultaneous presence of cells that reached their correct location in the CP and cells with a delay or an arrest of migration. This is supposed to happen because cortical neuroblast proliferation is not synchronous, but occurs through ‘proliferative units’ [23, 24], which may become the target of BCNU. It is interesting to note that in the BCNU-treated cortex some neurons, which probably were not affected by BCNU, underwent the same series of morphological transition as the control ones – bipolar in the VZ, multipolar in the IZ, and bipolar again as the neurons migrate toward and into the CP [15, 25]. This suggests that only the proliferative units affected by BCNU underwent defects in migration. The main finding of this study concerns the ontogenesis of periventricular heterotopia, an additional feature revealed in the BCNU-treated rats during adulthood and development [9–11]. Contrary to what has been previously described, in which periventricular heterotopia were detected since P1 [11], here we identified perivenMoroni/Inverardi/Regondi/Pennacchio/ Spreafico/Frassoni

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cific markers during embryonic and postnatal development has strongly suggested defects in neuronal migration and a rudimentary laminar pattern of the periventricular nodule found in BCNU-exposed cortex [11]. In the present study, we provide new evaluations about the altered neurogenesis that underlies CD development in the BCNU-treated rats by means of in utero electroporation – an in vivo approach that enables long-term analyses of neuronal migration. Depending on the timing of electroporation and the region targeted during the prenatal stages, in utero electroporation allows the targeting of specific cell populations [19]. With the aim of following neuronal migration after the insult induced by BCNU exposure, we transfected cortical radial glial cells with a plasmid encoding GFP. As the BCNU-treated rats are characterized by cortical and periventricular heterotopia mainly localized in the somatosensory cortex and prevalently consisting of cells destined to layers II–IV, we chose to target the prospective somatosensory cortex at E16, the age at which layer IV begins to be born [20]. The viability of electroporated embryos was slightly less than that expected from the literature [12, 21, 22]; this technical hitch was probably correlated to different experimental conditions (reported embryo viability varies from one group to another) or to species-specific differences (rats vs. mice). Nonetheless, the number of transfected embryos was sufficient to perform a good analysis of neu-

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Fig. 5. Periventricular heterotopia in E21 BCNU-treated embryos electroporated with GFP (green) and immunolabeled (red) for NeuN (b, c), Tbr1 (e, f) and Satb2 (h, i). None of the markers is expressed in the ventricular wall of the control embryos (a, d, g).

Most of the GFP+ cells localized in the heterotopia express NeuN (arrows in b, c), and Satb2 (arrows in h, i); just a few Tbr1+ cells are localized in the heterotopia (e, f). The sections are counterstained with DAPI (blue). Scale bar: 59 μm.

Genesis of Heterotopia in a Model of Cortical Dysplasia

Dev Neurosci 2013;35:516–526 DOI: 10.1159/000355392

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Fig. 6. Distribution of the GFP+ cells in the somatosensory cortex of control (a, c) and BCNU-treated P7 rats (b, d–f). wm = White matter. Sections are immunolabeled with Tbr1 (a, b, e) and Satb2 (c, d, f). Both in control and BCNU-treated rats, GFP+ cells are

distributed mainly in layers IV and V. Groups of GFP+ cells are

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located under the white matter in BCNU-treated rats, forming periventricular heterotopia. e, f Details of a periventricular heterotopia; the nodule is prevalently constituted of Satb2+ cells (f), while few Tbr1+ cells are located at its border (e). Double-labeled cells are indicated by arrows in f. Scale bars: 139 μm (a–d); 39 μm (e, f).

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tricular heterotopia already during embryonic development and we traced back their origin at E18. The birth of periventricular heterotopia could be correlated to the disorganization of the VZ that we observed in the BCNU-treated rats. The ventricular wall of control embryos consists of two proliferating regions: an apical VZ, where Pax6+ radial glial cells reside, and a basal SVZ, populated by Tbr2+ intermediate precursors [26, 27]. Moreover, radial glial fibers run through the VZ/SVZ until the pial surface, providing a scaffold to neuronal migration [28–30]. Conversely, at E18 we found some electroporated cells expressing Tbr2 in the VZ of the BCNU-treated embryos; these represent intermediate precursors that arrested their migration, probably because of the disarrangement of the radial glial scaffold previously revealed in this region [11]. At E21, most of the electroporated cells arrested in the VZ began to express Satb2 (marker of upper layer neurons) [31], while they were negative for Tbr1 (marker of layer VI) [32], thus proving that these cells became postmitotic neurons destined to supragranular layers. Both at E21 and at P7, periventricular heterotopia consisted mainly of Satb2+ cells, whereas Tbr1+ neurons were located at their border, thus showing a pseudolaminar structure and confirming our previous findings that demonstrated a rudimentary laminar organization of the nodular formations [10, 11]. Another feature of BCNU-treated cortex was the presence of cortical heterotopia, i.e. heterotopic neuronal clusters largely localized in infragranular layers at the intermediate rostrocaudal level of the somatosensory cortex [9, 10]. Unfortunately, we were unable to find these neuronal clusters by means of GFP in utero electroporation, probably because of the constricted area that is transfected in both the medial-lateral and rostrocaudal extensions of the brain, which probably has not covered the above-mentioned alterations. Conversely, we could identify the periventricular heterotopia because they were localized in a more anterior level in comparison to the cortical heterotopia and were located in the electroporated zone. The BCNU-treated rats are characterized by the hypoplasia of the corpus callosum, which was postulated to be caused by cell loss and the consequent reduction in the number of callosal fibers [9]. Our present work shows that the defect in callosal development is also due to the defective growth of callosal axons – they are unable to arrive at the midline, as they project to the ipsilateral prospective cingulated cortex. The corpus callosum forms through a sequence of events that includes the formation of the telencephalic midline and the generation of callosal

neurons and their axons, and their targeting [33]. In normal rats, callosal projecting neurons principally reside in the cortical layers II–III and to a lesser extent in layers V and VI [34]. Information regarding the direction of the axonal projection (if callosal or subcortical) is genetically specified within the neuron before it reaches the CP. Subsequently, axonal extension and pathfinding are regulated both by intrinsic and extrinsic guidance mechanisms that direct axons towards and through the midline, where they form the corpus callosum [33]. As we showed that cortical thinning in the BCNU model was prevalently restricted to the superficial layers, it is plausible to hypothesize that a loss of part of callosal-projecting neurons induces corpus callosum reduction. Moreover, though the callosal axons project medially, they are unable to enter the midline and they erroneously target the medial cortex, thus representing a further cause of corpus callosum thinning. A general disorganization of cortical fibers in BCNUtreated rats has been previously described [9, 11]. Here we can distinguish between fibers projecting from and to the thalamus, immunoreactive for α-internexin [35], and callosal axons, which have been transfected with the GFP vector. Both types of fibers are disarranged in BCNU-treated rats, with thalamic fibers widespread almost all over the cortical layers and callosal fibers projecting towards wrong targets. The alteration of the cortical circuitry may be one of the causes of the cortical hyperexcitability previously found in the BCNU model [8, 36].

Genesis of Heterotopia in a Model of Cortical Dysplasia

Dev Neurosci 2013;35:516–526 DOI: 10.1159/000355392

Conclusion

Targeting radial glial cells by means of in utero electroporation afforded us to trace back the origin of periventricular heterotopia and to demonstrate that this disarrangement is caused by the arrest of migration of neuronal precursor cells in the VZ and their subsequent maturation in upper layer neurons. Moreover, we could unravel cortical fiber disarrangement by distinguishing between thalamic and callosal projections, shedding light on the pathological circuitry that could contribute to the neuronal hyperexcitability. Acknowledgments

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We thank Dr. Françoise Watrin for her technical advice about in utero electroporation. This study was supported by grants from the Italian Ministry of Health to C.F.

References

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Genesis of heterotopia in BCNU model of cortical dysplasia, detected by means of in utero electroporation.

Derangements of cortical development can cause a wide spectrum of malformations, generally termed 'cortical dysplasia' (CD), which are frequently asso...
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