Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution <em>In Situ</em> Hybridization

Abstract

AbstractmRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a singlemolecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains. Video LinkThe video component of this article can be found at

A novel pathway regulates social hierarchy via lncRNA AtLAS and postsynaptic synapsin IIb

“…FISH on mouse brain slice (10 μm) was carried out according to the previously reported protocol. 78 Plasmids and viruses GluA1, GluA2 and GluA4 plasmids were kindly provided by Dr Yu Tian Wang (University of British Columbia, Vancouver, British Columbia, Canada). Adeno-associated viruses (AAVs) for Syn2b, Syn2a, AtLAS and short hairpin RNA for AtLAS, CELF4 were purchased from Obio technology (Shanghai, China).…”

Protein synthetic machinery and mRNA in regenerating tips of spinal cord axons in lamprey

“…Evidence that local axonal synthesis of the transcription factor ATF4 is required for retrograde spread of the pathogenic effects of Aβ 1‐42 amyloid peptide was obtained by inhibiting local translation with Atf4 siRNA, with subsequent reduction in retrograde neuronal loss (Baleriola et al, ). With a high‐resolution ISH technique, these authors could demonstrate Atf4 mRNA in axons of adult mice and human brain (Baleriola et al, ).…”

“…EM in monkey and adult rats suggested the presence of ribosomal rosettes only in the initial segment but not in the shaft of CNS axons (Steward and Ribak, ), and CNS axons within the optic nerve contained much lower levels of translational components than did sciatic nerve (Verma et al, ). More recently, using high‐resolution ISH techniques capable of detecting single mRNA molecules, Atf4 mRNA has been reported in adult mouse and human brain (Baleriola et al, , ). In addition, using genetically engineered mice in which the ribosomal protein L10a was tagged with enhanced green fluorescent protein (eGFP), immuno‐EM for eGFP showed enhanced subaxolemmal staining at nodes of Ranvier in corticospinal tract axons (Walker et al, ).…”

Kisspeptin Neuron-Specific and Self-Sustained Calcium Oscillation in the Hypothalamic Arcuate Nucleus of Neonatal Mice: Regulatory Factors of its Synchronization

“…Probes from ACD were used to detect mRNA levels: Mm-Kiss1 (500141), Mm-Kiss1R (408001), Mm-Tac2-C2 (446391-C2, encoding NKB), Mm-Tacr3-C3 (481671-C3, encoding NK3R), Mm-Pdyn-C3 (318771-C3, encoding Dyn), and Mm-Oprk1-C2 (316111-C2, encoding Dyn receptor, KOR). In certain occasions, fluorescent in situ hybridization (FISH) was followed by immunohistochemistry [33] to amplify GCaMP signals using α-green fluorescent protein (GFP) antibody (Invitrogen A6455). After performing FISH and before 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) staining, the sections were briefly washed with 0.1% TBST.…”