Detecting Single and Multiple BDNF Transcripts by In Situ Hybridization in Neuronal Cultures and Brain Sections
The neurotrophin brain-derived neurotrophic factor (BDNF) is encoded by multiple transcripts generated by differential use of eight 5′UTR exons (exons 1–8), which are alternatively spliced to the common exon (exon 9) containing the coding sequence (CDS) and the 3′UTR region. Because the 3′UTR sequence of BDNF contains two polyadenylation sites, each transcript has either a short or a long 3′ noncoding tail, generating 22 transcripts in rodents and 32 in humans. Nonradioactive in situ hybridization techniques have allowed a detailed analysis of the expression pattern of different BDNF transcripts. These studies led to the discovery that BDNF splice variants are preferentially distributed in different subcellular compartments, including the soma (exons 1, 3, 5, 7, 8), proximal dendrites (exons 2, 4, 6), and distal dendrites (exons 2, 6), thereby creating a “spatial code” for local production of BDNF protein. More recently, generation of transgenic mice with disruption of BDNF production from single Bdnf exons has provided new insights into the role of individual Bdnf transcripts in regulating social behavior, food intake, visual plasticity, sleep, sensory information processing, and fear regulation. This chapter will provide a detailed description of methods for visualizing Bdnf transcripts, including a “classical” nonradioactive in situ hybridization (ISH) technique using digoxigenin and enzyme alkaline phosphatase (AP). In addition, it will describe more modern techniques, such as fluorescent in situ hybridization (FISH) with tyramide signal amplification and the RNAscope® Multiplex Fluorescent Assay, a FISH method that allows detection of up to four gene targets or Bdnf splice variants simultaneously.
KeywordsBDNF spatial code BDNF splice variants Brain-derived neurotrophic factor Fluorescent in situ hybridization Multiplex mRNA detection Neurotrophins
- 5.Baj G, D’Alessandro V, Musazzi L, Mallei A, Sartori CR, Sciancalepore M, Tardito D, Langone F, Popoli M, Tongiorgi E (2012) Physical exercise and antidepressants enhance BDNF targeting in hippocampal CA3 dendrites: further evidence of a spatial code for BDNF splice variants. Neuropsychopharmacology 37(7):1600–1611CrossRefGoogle Scholar
- 7.Baj G, Del Turco D, Schlaudraff J, Torelli L, Deller T, Tongiorgi E (2013) Regulation of the spatial code for BDNF mRNA isoforms in the rat hippocampus following pilocarpine-treatment: a systematic analysis using laser microdissection and quantitative real-time PCR. Hippocampus 23(5):413–423CrossRefGoogle Scholar
- 11.Maynard KR, Hill JL, Calcaterra NE, Palko ME, Kardian A, Paredes D, Sukumar M, Adler BD, Jimenez DV, Schloesser RJ, Tessarollo L, Lu B, Martinowich K (2016) Functional role of BDNF production from unique promoters in aggression and serotonin signaling. Neuropsychopharmacology 41(8):1943–1955CrossRefGoogle Scholar
- 13.Mou Z, Hyde TM, Lipska BK, Martinowich K, Wei P, Ong CJ, Hunter LA, Palaguachi GI, Morgun E, Teng R, Lai C, Condarco TA, Demidowich AP, Krause AJ, Marshall LJ, Haack K, Voruganti VS, Cole SA, Butte NF, Comuzzie AG, Nalls MA, Zonderman AB, Singleton AB, Evans MK, Martin B, Maudsley S, Tsao JW, Kleinman JE, Yanovski JA, Han JC (2015) Human obesity associated with an intronic SNP in the brain-derived neurotrophic factor locus. Cell Rep 13(6):1073–1080CrossRefGoogle Scholar
- 25.Vicario A, Colliva A, Ratti A, Davidovic L, Baj G, Gricman Ł, Colombrita C, Pallavicini A, Jones KR, Bardoni B, Tongiorgi E (2015) Dendritic targeting of short and long 3′ UTR BDNF mRNA is regulated by BDNF or NT-3 and distinct sets of RNA-binding proteins. Front Mol Neurosci 8:62Google Scholar