Expression and mRNA splicing of glycine receptor subunits and gephyrin during neuronal differentiation of P19 cells in vitro, studied by RT-PCR and immunocytochemistry

Abstract

The mouse EC cell line P19, differentiating in vitro into neural cell types under the influence of retinoic acid, represents a well established model system for neurogenesis. In this system the expression of the α (α1–α3) and β subunits of the inhibitory glycine receptor (GlyR) and of gephyrin as well as their mRNA splice variants was analyzed by RT-PCR and by immunocytochemistry. In the course of neuronal differentiation of P19 cells mRNA of GlyRβ is constitutively expressed, GlyRα1 and α2 are induced and GlyRα3 was not detected. From the three gephyrin transcripts known to be differently spliced in the C3/C4 cassette region, the C3 transcript was found at all stages while the C4 transcript was not detectable. The insert-free form was measurable in P19 cells only 3–4 days post induction by retinoic acid. In addition a GlyRβ splice variant and a fourth gephyrin transcript were detected. Primary glial cells do not contain significant amounts of GlyRα subunits while in primary neuronal cells transcripts of GlyRα2 were found as well as the mRNA of the GlyRβ subunit and of gephyrin. PC12 cells do not express glycine receptor genes but do express gephyrin. Immunocytochemistry confirmed the constitutive expression of gephyrin at the protein level, whereas GlyR antigens could only be detected in islets of the ‘P19 neurons’. In conclusion, P19 and primary neuronal cells but not PC12 cells express the transcripts of glycine receptor components, necessary to generate functional receptors.