title: 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.
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.
authors.affiliation: Neurobiology of Aging Center, N. Masera INRCA Research Department, Via Birarelli 8, Ancona 60121, Italy. t.casoli@inrca.it
authors.forename: Tiziana
authors.initials: T
authors.lastname: Casoli
authors.forename: Giuseppina Di
authors.initials: GD
authors.lastname: Stefano
authors.forename: Patrizia
authors.initials: P
authors.lastname: Fattoretti
authors.forename: Moreno
authors.initials: M
authors.lastname: Solazzi
authors.forename: Alessia
authors.initials: A
authors.lastname: Delfino
authors.forename: Graziella
authors.initials: G
authors.lastname: Biagini
authors.forename: Carlo
authors.initials: C
authors.lastname: Bertoni-Freddari
mesh_terms: D000818:Animals; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D005260:Female; D005557:Formaldehyde; D019922:GAP-43 Protein / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D017403:In Situ Hybridization / Q000379:methods*; D009474:Neurons / Q000378:metabolism; D011108:Polymers; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation
chemical_list: D019922:GAP-43 Protein; D011108:Polymers; D012333:RNA, Messenger; D005557:Formaldehyde; D019286:Endopeptidase K; C003043:paraform