Advertisement

Recording Activity-Dependent Release of BDNF from Hippocampal Neurons

  • Tanja BrigadskiEmail author
  • Petra Lichtenecker
  • Volkmar LessmannEmail author
Protocol
Part of the Neuromethods book series (NM, volume 143)

Abstract

Release of brain-derived neurotrophic factor (BDNF) shapes development and synaptic plasticity of neuronal circuits, thereby controlling brain functions. Analysis of activity-dependent BDNF release can be performed by enzyme-linked immunosorbent assay (ELISA) detection methods or western blot analysis. But these methods require sensitive and specific antibodies directed against the protein of interest and do not allow time-resolved subcellular analysis of protein dynamics. However, knowing the kinetics of activity-dependent secretion as well as subcellular localization of BDNF secretion is important to elucidate the pivotal effects of BDNF on synaptic functions in response to changes in synaptic input activity and activity states of the releasing cells.

Here, we describe an assay which allows for analysis of neuropeptide and protein release on the timescale of several hundred milliseconds and at a resolution of 0.5 μm, thus enabling observation of release from single exocytotic vesicles. To this aim, we constructed plasmids coding for BDNF, tagged at the C-terminus with green fluorescence protein (GFP) or mCherry. Following transfection of dissociated rat hippocampal neurons with one of these plasmids, BDNF secretion can be monitored using wide-field fluorescence live cell imaging. Patterns of electrical activity triggering BDNF secretion were identified by parallel whole-cell patch clamp recordings of releasing neurons. Repetitive depolarizing stimulation as well as the generation of repetitive action potentials (APs) led to a robust BDNF release from neuronal processes. Altogether, these results indicate that BDNF release can be elicited with physiological levels of electrical stimulation.

Keywords

Activity-dependent release BDNF Fusion protein GFP Live cell imaging Neuron Neurotrophins Secretion Time-lapse fluorescence microscopy 

Notes

Acknowledgments

The authors wish to thank Regina Ziegler and Sabine Eichler for excellent technical assistance and Dr. Amelie Baschwitz for comments on the manuscript.

References

  1. 1.
    Klein R (1994) Role of neurotrophins in mouse neuronal development. FASEB J 8:738–744CrossRefGoogle Scholar
  2. 2.
    Huang EJ, Reichardt LF (2001) Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 24:677–736CrossRefGoogle Scholar
  3. 3.
    Lessmann V, Brigadski T (2009) Mechanisms, locations, and kinetics of synaptic BDNF secretion: an update. Neurosci Res 65:11–22CrossRefGoogle Scholar
  4. 4.
    Yang F, Je HS, Ji Y, Nagappan G, Hempstead B, Lu B (2009) Pro-BDNF-induced synaptic depression and retraction at developing neuromuscular synapses. J Cell Biol 185:727–741CrossRefGoogle Scholar
  5. 5.
    Park H, Poo M-M (2013) Neurotrophin regulation of neural circuit development and function. Nat Rev Neurosci 14:7–23CrossRefGoogle Scholar
  6. 6.
    Edelmann E, Lessmann V, Brigadski T (2014) Pre- and postsynaptic twists in BDNF secretion and action in synaptic plasticity. Neuropharmacology 76:610–627CrossRefGoogle Scholar
  7. 7.
    Haubensak W, Narz F, Heumann R, Lessmann V (1998) BDNF-GFP containing secretory granules are localized in the vicinity of synaptic junctions of cultured cortical neurons. J Cell Sci 111:1483–1493PubMedGoogle Scholar
  8. 8.
    Hartmann M, Heumann R, Lessmann V (2001) Synaptic secretion of BDNF after high-frequency stimulation of glutamatergic synapses. EMBO J 20:5887–5897CrossRefGoogle Scholar
  9. 9.
    Kohara K, Kitamura A, Morishima M, Tsumoto T (2001) Activity-dependent transfer of brain-derived neurotrophic factor to postsynaptic neurons. Science 291:2419–2423CrossRefGoogle Scholar
  10. 10.
    Brigadski T, Hartmann M, Lessmann V (2005) Differential vesicular targeting and time course of synaptic secretion of the mammalian neurotrophins. J Neurosci 25:7601–7614CrossRefGoogle Scholar
  11. 11.
    Matsuda N, Lu H, Fukata Y, Noritake J, Gao H, Mukherjee S, Nemoto T, Fukata M, Poo M-M (2009) Differential activity-dependent secretion of brain-derived neurotrophic factor from axon and dendrite. J Neurosci 29:14185–14198CrossRefGoogle Scholar
  12. 12.
    Edelmann E, Cepeda-Prado E, Franck M, Lichtenecker P, Brigadski T, Lessmann V (2015) Theta burst firing recruits BDNF release and signaling in postsynaptic CA1 neurons in spike-timing-dependent LTP. Neuron 86:1041–1054CrossRefGoogle Scholar
  13. 13.
    Brigadski T, Lessmann V (2014) BDNF: a regulator of learning and memory processes with clinical potential. e-Neuroforum 5:1–11CrossRefGoogle Scholar
  14. 14.
    Kolarow R, Brigadski T, Lessmann V (2007) Postsynaptic secretion of BDNF and NT-3 from hippocampal neurons depends on calcium calmodulin kinase II signaling and proceeds via delayed fusion pore opening. J Neurosci 27:10350–10364CrossRefGoogle Scholar
  15. 15.
    Eckenstaler R, Lessmann V, Brigadski T (2016) CAPS1 effects on intragranular pH and regulation of BDNF release from secretory granules in hippocampal neurons. J Cell Sci 129(7):1378–1390CrossRefGoogle Scholar
  16. 16.
    Seifert B, Eckenstaler R, Rönicke R, Leschik J, Lutz B, Reymann K, Lessmann V, Brigadski T (2016) Amyloid-beta induced changes in vesicular transport of BDNF in hippocampal neurons. Neural Plast 2016:4145708CrossRefGoogle Scholar
  17. 17.
    Lessmann V, Dietzel ID (1995) Two kinetically distinct 5-hydroxytryptamine-activated Cl- conductances at Retzius P-cell synapses of the medicinal leech. J Neurosci 15:1496–1505CrossRefGoogle Scholar
  18. 18.
    Lessmann V, Heumann R (1997) Cyclic AMP endogenously enhances synaptic strength of developing glutamatergic synapses in serum-free microcultures of rat hippocampal neurons. Brain Res 763:111–122CrossRefGoogle Scholar
  19. 19.
    Lessmann V, Heumann R (1998) Modulation of unitary glutamatergic synapses by neurotrophin-4/5 or brain-derived neurotrophic factor in hippocampal microcultures: presynaptic enhancement depends on pre-established paired-pulse facilitation. Neuroscience 86:399–413CrossRefGoogle Scholar
  20. 20.
    Kohrmann M, Haubensak W, Hemraj I, Kaether C, Lessmann VJ, Kiebler MA (1999) Fast, convenient, and effective method to transiently transfect primary hippocampal neurons. J Neurosci Res 58:831–835CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  1. 1.Department of Informatics and Microsystems TechnologyUniversity of Applied Science KaiserslauternKaiserslauternGermany
  2. 2.Medical FacultyInstitute of Physiology, Otto-von-Guericke-UniversityMagdeburgGermany
  3. 3.Center for Behavioral Brain Sciences (CBBS)MagdeburgGermany

Personalised recommendations