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.