BRIEF REPORT |
Correspondence to: Tiziana Casoli, Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8, Ancona AN 60121, Italy. E-mail: t.casoli@inrca.it
![]() |
Summary |
---|
![]() ![]() ![]() ![]() |
---|
The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that plays a key role in guiding the growth of axons and in modulating the formation of new synapses. To identify the cells that synthesize GAP-43 mRNA, we applied direct in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) in cerebellum and hippocampus of adult rat brain. In situ RT-PCR revealed GAP-43 mRNA in cerebellar granule cells, in Purkinje cells and in some interneurons of the molecular layer. Previous in situ hybridization studies had demonstrated a dense label throughout the granular layer of the cerebellar cortex but no labeling of other cerebellar neurons. Hippocampal cells showing distinct GAP-43 mRNA signal after in situ RT-PCR were CA1 and CA3 pyramidal neurons, CA4 hilar cells, and dentate gyrus granule cells, whereas in situ hybridization studies had detected GAP-43 mRNA only in CA3 and CA1 pyramidal neurons. Our data indicate that GAP-43 mRNA is widely distributed, suggesting that many cell types are potentially involved in synaptic plasticity events. (J Histochem Cytochem 49:11951196, 2001)
Key Words: GAP-43, in situ RT-PCR, cerebellar cells, hippocampal cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() |
---|
THE GROWTH-ASSOCIATED PROTEIN GAP-43 is a neuron-specific phosphoprotein whose expression has been used as an indicator of axonal growth (
Animals were sacrificed according to the guidelines of Italian Ministry of Health regarding the use of laboratory animals. Briefly, adult female Wistar rats were anesthetized and perfused intracardially with 4% paraformaldehyde. Brain samples were sectioned at 10 µm on a cryostat and sections were thaw-mounted on silane-coated slides tested for in situ PCR amplification. The hippocampus was permeabilized with 1 µg/ml and cerebellum with 2 µg/ml proteinase K for 15 min at 37C. After proteinase K inactivation at 95C for 2 min, sections were treated with RNase-free DNase I overnight at 37C and rinsed in DEPC-treated water. The RT reaction was carried out using the first-strand cDNA synthesis kit (Roche; Basel, Switzerland). Direct in situ PCR of cDNA was performed on the GeneAmp In Situ PCR System 1000 (PerkinElmer; Norwalk, CT). The following conditions were used: 1 x reaction buffer, 2.5 mM MgCl2, 200 µM of each deoxynucleotide, 50 µM Bio-11-dUTP, 1 µM of each primer (GAP-43 sense 5'-ATG CTG TGC TGT ATG AGA AGA ACC-3'; GAP-43 antisense 5'-GGC AAC GTG GAA AGC CGT TTC TTA AAG T-3'), 0.2 U/µl Taq DNA polymerase. The hot-start technique was performed. Twenty cycles were run using the following protocol: denaturation at 94C for 45 sec, annealing at 69C for 1 min, and extension at 72C for 1 min. Negative controls were performed by omission of primers. After amplification, sections were washed in 2 x SSC at 50C for 5 min, blocked in 5% BSA, and exposed to avidin DN (10 µg/ml). Sections were then incubated in biotinalkaline phosphatase (10 µg/ml) and transferred to 0.1 M Tris-HCl, pH 9.5. Visualization of the amplified biotin-labeled cDNA was performed with NBT/BCIP chromogen. Finally, sections were counterstained with methyl green, dehydrated, cleared in xylene, and coverslipped.
Fig 1A shows in situ RT-PCR of GAP-43 mRNA in rat cerebellum. Positive labeling can be observed in the granular layer (gl), in Purkinje cells (Pc), and in some interneurons of the molecular layer (ml). Fig 1B shows the negative control performed by omission of primers, demonstrating a prevalence of pale staining, due to methyl green counterstain, over the dark specific labeling. Fig 2A2C show the cellular distribution of GAP-43 mRNA in hippocampal granule cells of dentate gyrus and in pyramidal neurons of layers CA1 and CA3. Dentate gyrus granule cells (gc) are stained, as well as some CA4 hilar neurons (h). CA1 and CA3 pyramidal cells are positive, whereas neuropil areas are faintly colored. Fig 2D2F show the negative controls of dentate gyrus, CA1, and CA3, respectively. Previous ISH studies revealed a dense label throughout the granular layer of the cerebellar cortex, high levels of GAP-43 mRNA in CA3 cells of hippocampus and low levels in CA1 cells, but no labeling of other neurons (
|
Received for publication November 28, 2000; accepted February 16, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() |
---|
Benowitz LI, Routtenberg A (1997) GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurol Sci 20:84-91[Medline]
Kruger L, Bendotti C, Rivolta R, Samanin R (1993) Distribution of GAP-43 mRNA in the adult rat brain. J Comp Neurol 333:417-434[Medline]