Elsevier

Neuroscience

Volume 159, Issue 2, 17 March 2009, Pages 682-691
Neuroscience

Neuroanatomy
Expression of layer-specific markers in the adult neocortex of BCNU-Treated rat, a model of cortical dysplasia

https://doi.org/10.1016/j.neuroscience.2008.12.064Get rights and content

Abstract

The experimental model of cortical dysplasia (CD) obtained by administering carmustine (1-3-bis-chloroethyl-nitrosurea [BCNU]) in pregnant rat uterus mimics the histopathological abnormalities observed in human CD patients: altered cortical layering, and presence of heterotopia and dysmorphic/heterotopic neurons. To investigate further the cortical layering disruption and the neuronal composition of heterotopia in BCNU-exposed cortex, we analyzed the expression pattern of the transcription factors Nurr1, Er81, Ror-β, and Cux2 (respectively specific markers of layers VI, V, IV and superficial layers) in the cortical areas of BCNU-treated rats by means of in situ hybridization, and compared the findings with those observed in adult control rats. Combining in situ hybridization and immunohistochemistry we also investigated the origin of dysmorphic or heterotopic neurons. The main results of the present study are (i) the analysis of cortical layer thickness revealed that the cortical thinning in the BCNU model was prevalently restricted to the superficial layers; (ii) in cortical and periventricular heterotopia, the prevalent presence of superficial layer neurons in the internal areas, and deeper layer neurons in a more peripheral region, demonstrated a rudimentary pattern of laminar organization in nodule formation; and (iii) the Er81 signal in the dysmorphic and heterotopic pyramidal neurons located in layers I/II showed that they belong to layer V. These results shed light on the disorganization of the laminar architecture of the BCNU model by providing correlations with normal cortical layering and revealing the ontogenesis of heterotopia and heterotopic/dysmorphic neurons. They also provide strong evidence of the usefulness of layer-specific markers in investigating the neuropathology of CD, thus opening up the possibility of expanding their application to human neuropathology.

Section snippets

Animal treatment and tissue fixation

The experiments were performed in accordance with the guidelines defined by the European Communities Council Directive (86/609/EEC) and every effort was made to limit the number and suffering of animals used.

Pregnant Sprague–Dawley rats (Charles River Italia, Calco, Italy) were i.p. injected with 20 mg/kg of BCNU solution (5% sterile glucose in water at 4 mg/ml) at E (embryonic day) 15 (E1 was defined as the day of vaginal plug), at the time of peak cortical neurogenesis. On the same

Overview of the histological alterations found in BCNU-treated rats

Fig. 1 summarizes the most prominent histopathological features identified in BCNU-exposed brain. They included: (i) the disorganization of cortical layers I/II (Fig. 1A), (ii) the large nodular clusters of heterotopic neurons prevalently localized in the lowest part of the cortex and periventricular heterotopia (Fig. 1B) identified with CB immunolabeling, (iii) ectopic and inverted pyramidal neurons in cortical layers I/II (Fig. 1C) identified with SMI311 immunolabeling. Another striking

Discussion

We reported a comparative analysis of the expression of some layer-specific markers between control rats and a model of CD, namely BCNU-treated rats, with the aim of mapping the alterations in the distribution of the cortical laminae that characterize CD. Our findings concerning the details of cortical layering disruption revealed the neuronal composition of heterotopia, and our combined use of ISH and ICC demonstrated the origin of the heterotopic and dysmorphic neurons characterizing this

Conclusion

In conclusion, layer-specific markers turned out to be a useful means of examining the disorganized laminar architecture and ontology of neurons in a model of CD. Similarly they can be applied to the investigation of human neuropathology to reveal the details of cortical alterations and shed light on the interpretations of pathogenesis.

Acknowledgments

This work was supported by grants of the Italian Ministry of Health to C.F, Fondazione Banca del Monte di Lombardia, and EU grant “Functional Genomics and Neurobiology of Epilepsy” (EPICure) contract no. LSHM-CT-2006.0373315. We acknowledge the technical assistance of Ms. Sonoko Ohsawa.

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