Advertisement

pp 1-27 | Cite as

Detecting Single and Multiple BDNF Transcripts by In Situ Hybridization in Neuronal Cultures and Brain Sections

  • Andrea Colliva
  • Kristen R. Maynard
  • Keri Martinowich
  • Enrico TongiorgiEmail author
Protocol
Part of the Neuromethods book series

Abstract

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.

Keywords

BDNF spatial code BDNF splice variants Brain-derived neurotrophic factor Fluorescent in situ hybridization Multiplex mRNA detection Neurotrophins 

References

  1. 1.
    Aid T, Kazantseva A, Piirsoo M, Palm K, Timmusk T (2007) Mouse and rat BDNF gene structure and expression revisited. J Neurosci Res 85(3):525–535Google Scholar
  2. 2.
    Pruunsild P, Kazantseva A, Aid T, Palm K, Timmusk T (2007) Dissecting the human BDNF locus: bidirectional transcription, complex splicing, and multiple promoters. Genomics 90(3):397–406Google Scholar
  3. 3.
    Timmusk T, Palm K, Metsis M, Reintam T, Paalme V, Saarma M, Persson H (1993) Multiple promoters direct tissue-specific expression of the rat BDNF gene. Neuron 10(3):475–489Google Scholar
  4. 4.
    Oliff HS, Berchtold NC, Isackson P, Cotman CW (1998) Exercise-induced regulation of brain-derived neurotrophic factor (BDNF) transcripts in the rat hippocampus. Brain Res Mol Brain Res 61(1–2):147–153Google Scholar
  5. 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):1600–1611Google Scholar
  6. 6.
    Khundakar AA, Zetterström TSC (2006) Biphasic change in BDNF gene expression following antidepressant drug treatment explained by differential transcript regulation. Brain Res 1106(1):12–20Google Scholar
  7. 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):413–423Google Scholar
  8. 8.
    Mizuno K, Dempster E, Mill J, Giese KP (2012) Long-lasting regulation of hippocampal Bdnf gene transcription after contextual fear conditioning. Genes Brain Behav 11(6):651–659Google Scholar
  9. 9.
    Neeley EW, Berger R, Koenig JI, Leonard S (2011) Prenatal stress differentially alters brain-derived neurotrophic factor expression and signaling across rat strains. Neuroscience 187:24–35Google Scholar
  10. 10.
    Sakata K, Martinowich K, Woo NH, Schloesser RJ, Jimenez DV, Ji Y, Shen L, Lu B (2013) Role of activity-dependent BDNF expression in hippocampal-prefrontal cortical regulation of behavioral perseverance. Proc Natl Acad Sci U S A 110(37):15103–15108Google Scholar
  11. 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):1943–1955Google Scholar
  12. 12.
    Hill JL, Hardy NF, Jimenez DV, Maynard KR, Kardian AS, Pollock CJ, Schloesser RJ, Martinowich K (2016) Loss of promoter IV-driven BDNF expression impacts oscillatory activity during sleep, sensory information processing and fear regulation. Transl Psychiatry 6(8):e873Google Scholar
  13. 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):1073–1080Google Scholar
  14. 14.
    Han JC, Liu QR, Jones M, Levinn RL, Menzie CM, Jefferson-George KS, Adler-Wailes DC, Sanford EL, Lacbawan FL, Uhl GR, Rennert OM, Yanovski JA (2008) Brain-derived neurotrophic factor and obesity in WAGR syndrome. N Engl J Med 359(9):918–927Google Scholar
  15. 15.
    Gao M, Maynard KR, Chokshi V, Song L, Jacobs C, Wang H, Tran T, Martinowich K, Lee HK (2014) Rebound potentiation of inhibition in juvenile visual cortex requires vision-induced BDNF expression. J Neurosci 34(32):10770–10779Google Scholar
  16. 16.
    Tongiorgi E, Righi M, Cattaneo A (1997) Activity-dependent dendritic targeting of BDNF and TrkB mRNAs in hippocampal neurons. J Neurosci 17(24):9492–9505Google Scholar
  17. 17.
    Tongiorgi E, Righi M, Cattaneo A (1998) A non-radioactive in situ hybridization method that does not require RNAse-free conditions. J Neurosci Methods 85(2):129–139Google Scholar
  18. 18.
    Simonato M, Bregola G, Armellin M, Del Piccolo P, Rodi D, Zucchini S, Tongiorgi E (2002) Dendritic targeting of mRNAs for plasticity genes in experimental models of temporal lobe epilepsy. Epilepsia 43(Suppl 5):153–158Google Scholar
  19. 19.
    Tongiorgi E, Armellin M, Giulianini PG, Bregola G, Zucchini S, Paradiso B, Steward O, Cattaneo A, Simonato M (2004) Brain-derived neurotrophic factor mRNA and protein are targeted to discrete dendritic laminas by events that trigger epileptogenesis. J Neurosci 24(30):6842–6852Google Scholar
  20. 20.
    Chiaruttini C, Sonego M, Baj G, Simonato M, Tongiorgi E (2008) BDNF mRNA splice variants display activity-dependent targeting to distinct hippocampal laminae. Mol Cell Neurosci 37(1):11–19Google Scholar
  21. 21.
    Chiaruttini C, Vicario A, Li Z, Baj G, Braiuca P, Wu Y, Lee FS, Gardossi L, Baraban JM, Tongiorgi E (2009) Dendritic trafficking of BDNF mRNA is mediated by translin and blocked by the G196A (Val66Met) mutation. Proc Natl Acad Sci U S A 106(38):16481–16486Google Scholar
  22. 22.
    Aliaga EE, Mendoza I, Tapia-Arancibia L (2009) Distinct subcellular localization of BDNF transcripts in cultured hypothalamic neurons and modification by neuronal activation. J Neural Transm (Vienna) 116(1):23–32Google Scholar
  23. 23.
    Tongiorgi E (2008) Activity-dependent expression of brain-derived neurotrophic factor in dendrites: facts and open questions. Neurosci Res 61(4):335–346Google Scholar
  24. 24.
    Tongiorgi E, Baj G (2008) Functions and mechanisms of BDNF mRNA trafficking. Novartis Found Symp 289:136–147 discussion 147–151, 193–195. ReviewGoogle Scholar
  25. 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
  26. 26.
    Wang F, Flanagan J, Su N, Wang L-C, Bui S, Nielson A, Wu X, Vo H-T, Ma X-J, Luo Y (2012) RNAscope®: a novel in situ RNA analysis platform for formalin-fixed paraffin-embedded tissues J Mol Diagn 14:22–29Google Scholar
  27. 27.
    Baj G, Patrizio A, Montalbano A, Sciancalepore M, Tongiorgi E (2014) Developmental and maintenance defects in Rett syndrome neurons identified by a new mouse staging system in vitro. Front Cell Neurosci 8:18Google Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  • Andrea Colliva
    • 1
  • Kristen R. Maynard
    • 2
    • 3
  • Keri Martinowich
    • 2
    • 3
  • Enrico Tongiorgi
    • 1
    Email author
  1. 1.Department of Life Sciences, BRAIN Center for NeuroscienceUniversity of TriesteTriesteItaly
  2. 2.Lieber Institute for Brain DevelopmentBaltimoreUSA
  3. 3.Department of Psychiatry and Behavioral SciencesThe Johns Hopkins University School of MedicineBaltimoreUSA

Personalised recommendations