GAP-43 mRNA detection by in situ hybridization, direct and indirect in situ RT-PCR in hippocampal and cerebellar tissue sections of adult rat brain

Tiziana Casoli, Giuseppina Di Stefano, Patrizia Fattoretti, Moreno Solazzi, Alessia Delfino, Graziella Biagini, Carlo Bertoni-Freddari

Research output: Contribution to journalArticlepeer-review

Abstract

The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that is expressed at high levels during development and axonal growth. To evaluate the cellular distribution of GAP-43 mRNA in the hippocampus and cerebellum of adult rats we applied in situ hybridization (ISH) as well as direct and indirect in situ RT-PCR using biotin as a reporter molecule. ISH resulted in a positive signal in most cerebellar granular cells and in 30% of hippocampal CA3 neurons. Direct in situ RT-PCR yielded cells with strong signals in every region investigated, with elevated background levels most likely related to incorporation of labeled nucleotides into non-specific amplicons through internal priming and DNA repair activity. Indirect in situ RT-PCR turned out to be the best approach for detecting GAP-43 mRNA positive cells. Cerebellar cells exhibiting a positive signal for GAP-43 mRNA were of the granular cell type (98%). Hippocampal neurons with a positive reaction for GAP-43 mRNA included all the neuron groups analyzed, namely CA1 (99%) and CA3 pyramidal cells (94%) and dentate gyrus granule cells (92%). Dentate gyrus granule cells have not tested positive for GAP-43 mRNA detection by molecular morphology analysis. These data show that in normal rats GAP-43 mRNA is present in different cell populations of hippocampal formation, supporting the role of this protein in the ongoing processes of synaptic plasticity.

Original languageEnglish
Pages (from-to)415-422
Number of pages8
JournalMicron
Volume34
Issue number8
DOIs
Publication statusPublished - 2003

Keywords

  • Cerebellar cells
  • GAP-43
  • Hippocampal cells
  • In situ hybridization
  • In situ RT-PCR

ASJC Scopus subject areas

  • Cell Biology
  • Materials Science(all)
  • Instrumentation

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