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Targeted in vivo expression of proteins in the calyx of Held

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Abstract

The calyx of Held serves as a model for synaptic transmission in the mammalian central nervous system. While offering unique access to the biophysics of presynaptic function, studies addressing the molecular mechanisms of neurotransmitter exocytosis in this model have been mainly limited to pharmacological interventions. To overcome this experimental limitation we used stereotaxic delivery of viral gene shuttles to rapidly and selectively manipulate protein composition in the calyx terminal in vivo. Sindbis or Semliki Forest viruses encoding enhanced green fluorescent protein (EGFP) were injected into the ventral cochlear nucleus (VCN) of rats (postnatal days 7–21) and yielded bright fluorescence in cells of the VCN, including globular bushy cells with their axon and calyx terminal. Fluorescence imaging and three dimensional reconstructions visualized developmental changes in calyx morphology. Small cytoplasmic and synaptic vesicle proteins were successfully overexpressed in the calyx. We extended two-photon microscopy to obtain simultaneous fluorescence and infrared scanning gradient contrast images, allowing for efficient patch-clamp recordings from EGFP-labelled calyces in acute brain slices (postnatal days 9–14). Recordings of spontaneous miniature excitatory postsynaptic currents and short-term depression in synapses overexpressing EGFP or synaptophysin-EGFP revealed normal synaptic function. Thus, Sindbis and Semliki Forest virus-directed overexpression of proteins in the calyx of Held provides a new avenue for molecular structure-function studies of mammalian central synapses.

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References

  1. Alder J, et al (1995) Overexpression of synaptophysin enhances neurotransmitter secretion at Xenopus neuromuscular synapses. J Neurosci 15:511–519

    CAS  PubMed  Google Scholar 

  2. Ahmari SE, Buchanan J, Smith SJ (2000) Assembly of presynaptic active zones from cytoplasmic transport packets. Nat Neurosci 3:445–451

    Article  CAS  PubMed  Google Scholar 

  3. Augustin I, et al (1999) Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 400:457–461

    Article  CAS  PubMed  Google Scholar 

  4. Augustine GJ, et al (1999) Proteins involved in synaptic vesicle trafficking. J Physiol (Lond) 520:33–41

    Google Scholar 

  5. Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 97:11984–11989

    Article  CAS  PubMed  Google Scholar 

  6. Berglund P, et al (1993) Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Biotechnology 11:916–920

    Article  CAS  PubMed  Google Scholar 

  7. Bollmann JH, Sakmann B, Borst JG (2000) Calcium sensitivity of glutamate release in a calyx-type terminal. Science 289:953–957

    Article  CAS  PubMed  Google Scholar 

  8. Borst JG, Sakmann B (1996) Calcium influx and transmitter release in a fast CNS synapse. Nature 383:431–434

    Article  CAS  PubMed  Google Scholar 

  9. Borst JG, Helmchen F, Sakmann B (1995) Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat. J Physiol (Lond) 489:825–840

    Google Scholar 

  10. Bredenbeek PJ, et al (1993) Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J Virol 67:6439–6446

    CAS  PubMed  Google Scholar 

  11. Burns ME, Augustine GJ (1999) Functional studies of presynaptic proteins at the squid giant synapse. In: Bellen H (ed) Neurotransmitter release. Oxford University Press, Oxford, pp 237–264

  12. Cant NB, Benson CG (2003) Parallel auditory pathways: projection patterns of the different neuronal populations in the dorsal and ventral cochlear nuclei. Brain Res Bull 60:457–474

    Article  PubMed  Google Scholar 

  13. Chen BE, et al (2000) Imaging high-resolution structure of GFP-expressing neurons in neocortex in vivo. Learn Mem 7:433–441

    Article  CAS  PubMed  Google Scholar 

  14. Cao G, et al (2002) In vivo delivery of a Bcl-xL fusion protein containing the TAT protein transduction domain protects against ischemic brain injury and neuronal apoptosis. J Neurosci 22:5423–5431

    CAS  PubMed  Google Scholar 

  15. D’Apuzzo M, et al (2001) Abundant GFP expression and LTP in hippocampal acute slices by in vivo injection of sindbis virus. J Neurophysiol 86:1037–1042

    PubMed  Google Scholar 

  16. Dodt HU, Zieglgansberger W (1998) Visualization of neuronal form and function in brain slices by infrared videomicroscopy. Histochem J 30:141–152

    Article  CAS  PubMed  Google Scholar 

  17. Dodt H, et al (1999) Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation. Science 286:110–113

    Article  CAS  PubMed  Google Scholar 

  18. Ehrengruber MU, et al (1999) Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proc Natl Acad Sci USA 96:7041–7046

    Article  CAS  PubMed  Google Scholar 

  19. Ehrengruber MU, et al (2001) Gene transfer into neurons from hippocampal slices: comparison of recombinant Semliki Forest virus, adenovirus, adeno-associated virus, lentivirus, and measles virus. Mol Cell Neurosci 17:855–871

    Article  CAS  PubMed  Google Scholar 

  20. Forsythe ID (1994) Direct patch recording from identified presynaptic terminals mediating glutamatergic EPSCs in the rat CNS, in vitro. J Physiol (Lond) 479:381–387

    Google Scholar 

  21. Friauf E, Ostwald J (1988) Divergent projections of physiologically characterized rat ventral cochlear nucleus neurons as shown by intra-axonal injection of horseradish peroxidase. Exp Brain Res 7:263–284

    Google Scholar 

  22. Frolov I, Schlesinger S (1994) Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells. J Virol 68:1721–1727

    CAS  PubMed  Google Scholar 

  23. Gahwiler BH (1981) Organotypic monolayer cultures of nervous tissue. J Neurosci Methods 4:329–342

    Article  PubMed  Google Scholar 

  24. Gersdorff H von, Borst JG (2002) Short-term plasticity at the calyx of Held. Nat Rev Neurosci 3:53–64

    Article  PubMed  Google Scholar 

  25. Gersdorff H von, et al (1997) Presynaptic depression at a calyx synapse: the small contribution of metabotropic glutamate receptors. J Neurosci 17:8137–8146

    PubMed  Google Scholar 

  26. Griffin DE, Hardwick JM (1997) Regulators of apoptosis on the road to persistent alphavirus infection. Annu Rev Microbiol 51:565–592

    Article  CAS  PubMed  Google Scholar 

  27. Gurevich VV, et al (1991) Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. Anal Biochem 195:207–213

    CAS  PubMed  Google Scholar 

  28. Gwag BJ, et al (1998) A neuron-specific gene transfer by a recombinant defective Sindbis virus. Brain Res Mol Brain Res 63:53–61

    Article  CAS  PubMed  Google Scholar 

  29. Harrison JM, Warr WB (1962) A study of the cochlear nuclei and ascending auditory pathways of the medulla. J Comp Neurol 119:341–379

    CAS  PubMed  Google Scholar 

  30. Held H (1893) Die centrale Gehörleitung. Archiv für Anatomie und Physiologie, Anatomische Abtheilung, pp 201–248

  31. Hori T, Takai Y, Takahashi T (1999) Presynaptic mechanism for phorbol ester-induced synaptic potentiation. J Neurosci 19:7262–7267

    CAS  PubMed  Google Scholar 

  32. Iwasaki S, Takahashi T (2001) Developmental regulation of transmitter release at the calyx of Held in rat auditory brainstem. J Physiol (Lond) 534:861–871

    Google Scholar 

  33. Jahn R, Lang T, Sudhof TC (2003) Membrane fusion. Cell 112:519–533

    Article  CAS  PubMed  Google Scholar 

  34. Kaether C, Skehel P, Dotti CG (2000) Axonal membrane proteins are transported in distinct carriers: a two-color video microscopy study in cultured hippocampal neurons. Mol Biol Cell 11:1213–1224

    CAS  PubMed  Google Scholar 

  35. Kandler K, Friauf E (1993) Pre- and postnatal development of efferent connections of the cochlear nucleus in the rat. J Comp Neurol 328:161–184

    CAS  PubMed  Google Scholar 

  36. Kennedy MB (2000) Signal-processing machines at the postsynaptic density. Science 290:750–754

    Article  CAS  PubMed  Google Scholar 

  37. Kim J, et al (2004) Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons. J Neurosci Methods 133:81–90

    Article  CAS  PubMed  Google Scholar 

  38. Kuner T, Augustine GJ (2000) A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 27:447–459

    Google Scholar 

  39. Llinas RR (1999) The squid giant synapse. Oxford University Press, Oxford

  40. Lohmann C, Ilic V, Friauf E (1998) Development of a topographically organized auditory network in slice culture is calcium dependent. J Neurobiol 34:97–112

    Article  CAS  PubMed  Google Scholar 

  41. Lois C, et al (2002) Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295:868–872

    Article  CAS  PubMed  Google Scholar 

  42. Lundstrom K, et al (2001) Semliki Forest virus vectors: efficient vehicles for in vitro and in vivo gene delivery. FEBS Lett 504:99–103

    Article  CAS  PubMed  Google Scholar 

  43. Lundstrom K, et al (2003) Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature sensitivity for long-term enhancement of transgene expression. Mol Ther 7:202–209

    Article  CAS  PubMed  Google Scholar 

  44. Maletic-Savatic M, Malinow R, Svoboda K (1999) Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283:1923–1927

    Article  CAS  PubMed  Google Scholar 

  45. Meinrenken CJ, Borst JG, Sakmann B (2003) Local routes revisited: the space and time dependence of the Ca2+ signal for phasic transmitter release at the rat calyx of Held. J Physiol (Lond) 547:665–689

    Google Scholar 

  46. Morest DK (1968) The growth of synaptic endings in the mammalian brain: a study of the calyces of the trapezoid body. Z Anat Entwicklungsgesch (3):201–220

    Google Scholar 

  47. Niswender KD, et al (1995) Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits. J Microsc 180:109–116

    CAS  PubMed  Google Scholar 

  48. Patterson GH, et al (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73:2782–2790

    CAS  PubMed  Google Scholar 

  49. Radecke F, et al (1995) Rescue of measles viruses from cloned DNA. EMBO J 14:5773–5784

    CAS  PubMed  Google Scholar 

  50. Ramon y Cajal S (1899) Textura del sistema nervosio del hombre y de los vertebrados.

  51. Rathenberg J, Nevian T, Witzemann V (2003) High-efficiency transfection of individual neurons using modified electrophysiology techniques. J Neurosci Methods 126:91–98

    Article  PubMed  Google Scholar 

  52. Romand R, Avan P (1997) Anatomical and functional aspects of the cochlear nucleus. In: Ehret G, Romand R (eds) The central auditory system. Oxford University Press, Oxford, pp 97–192

  53. Rowland KC, Irby NK, Spirou GA (2000) Specialized synapse-associated structures within the calyx of Held. J Neurosci 20:9135–9144

    CAS  PubMed  Google Scholar 

  54. Sanes JR, Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 22:389–442

    Article  CAS  PubMed  Google Scholar 

  55. Satzler K, et al (2002) Three-dimensional reconstruction of a calyx of Held and its postsynaptic principal neuron in the medial nucleus of the trapezoid body. J Neurosci 22:10567–10579

    CAS  PubMed  Google Scholar 

  56. Scannevin RH, Huganir RL (2000) Postsynaptic organization and regulation of excitatory synapses. Nat Rev Neurosci 1:133–141

    Article  CAS  PubMed  Google Scholar 

  57. Schneggenburger R, Neher E (2000) Intracellular calcium dependence of transmitter release rates at a fast central synapse. Nature 406:889–893

    Article  CAS  PubMed  Google Scholar 

  58. Schneggenburger R, Sakaba T, Neher E (2002) Vesicle pools and short-term synaptic depression: lessons from a large synapse. Trends Neurosci 25:206–212

    Article  CAS  PubMed  Google Scholar 

  59. Sheng M (2001) Molecular organization of the postsynaptic specialization. Proc Natl Acad Sci USA 98:7058–7061

    Article  CAS  PubMed  Google Scholar 

  60. Sollner TH (2003) Regulated exocytosis and SNARE function. Mol Membr Biol 20:209–220

    Article  PubMed  Google Scholar 

  61. Sudhof TC (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375:645–653

    Article  CAS  PubMed  Google Scholar 

  62. Sugita S, Janz R, Sudhof TC (1999) Synaptogyrins regulate Ca2+-dependent exocytosis in PC12 cells. J Biol Chem 274:18893–18901

    Article  CAS  PubMed  Google Scholar 

  63. Takahashi T, Kajikawa Y, Tsujimoto T (1998) G-protein-coupled modulation of presynaptic calcium currents and transmitter release by a GABAB receptor. J Neurosci 18:3138–3146

    CAS  PubMed  Google Scholar 

  64. Taschenberger H, von Gersdorff H (2000) Fine-tuning an auditory synapse for speed and fidelity: developmental changes in presynaptic waveform, EPSC kinetics, and synaptic plasticity. J Neurosci 20:9162–9173

    CAS  PubMed  Google Scholar 

  65. Terry BR, Matthews EK, Haseloff J (1995) Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy. Biochem Biophys Res Commun 217:21–27

    Article  CAS  PubMed  Google Scholar 

  66. Tsujimoto T, et al (2002) Neuronal calcium sensor 1 and activity-dependent facilitation of P/Q-type calcium currents at presynaptic nerve terminals. Science 295:2276–2279

    Article  CAS  PubMed  Google Scholar 

  67. Valtorta F, et al (2004) Synaptophysin: leading actor or walk-on role in synaptic vesicle exocytosis? Bioessays 26:445–453

    Article  CAS  PubMed  Google Scholar 

  68. Verhage M, et al (2000) Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287:864–869

    Article  CAS  PubMed  Google Scholar 

  69. Wimmer VC, Sakmann B, Kuner T (2002a) The calyx of Held as a model system of neurotransmitter release: virus-mediated perturbation of protein function in a giant presynaptic terminal. Pflugers Arch 443:S300

    Google Scholar 

  70. Wimmer VC, Sakmann B, Kuner T (2002b) Expression of engineered proteins in the calyx of Held by stereotaxic injection and virus-mediated gene transfer. Society for Neuroscience Abstracts 28

  71. Wu SH, Kelly JB (1993) Response of neurons in the lateral superior olive and medial nucleus of the trapezoid body to repetitive stimulation: intracellular and extracellular recordings from mouse brain slice. Hear Res 68:189–201

    Article  CAS  PubMed  Google Scholar 

  72. YamashitaT, Ishikawa T, Takahashi T (2003) Developmental increase in vesicular glutamate content does not cause saturation of AMPA receptors at the calyx of Held synapse. J Neurosci 23:3633–3638

    PubMed  Google Scholar 

  73. Zhai RG, et al (2001) Assembling the presynaptic active zone: a characterization of an active one precursor vesicle. Neuron 29:131–143

    Article  CAS  PubMed  Google Scholar 

  74. Zrachia A, et al (2002) Infection of glioma cells with Sindbis virus induces selective activation and tyrosine phosphorylation of protein kinase C delta. Implications for Sindbis virus-induced apoptosis. J Biol Chem 277:23693–23701

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to Bert Sakmann for generous support and critical discussions. We thank Pavel Osten for advice on the Sindbis virus system and making available several viral constructs and virus preparations, Peter H. Seeburg for providing access to the S2 cell culture laboratory, Harald Hutter for sharing the Volocity software, Günter Giese for support with confocal microscopy, Heinz Horstmann for electron microscopy and Johann Bollmann for help with calyx preparation and discussions. We thank Markus Ehrengruber (University of Zürich) for sharing the SFV vectors and for expert advice. We would like to thank Rudolf Leube (University of Mainz), Jun Chen (University of Pittsburg), Martin Billeter (University of Zürich), Hildegard Büning (University of Munich), Carlos Lois (Caltech), Andrew Matus (Friedrich-Miescher-Institute Basel), Christian Rosenmund, Erwin Neher, Nils Brose (Göttingen), Hiroshi Tokumaru and George Augustine (Duke University Medical Center) for supplying materials.

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  1. Abteilung Zellphysiologie, Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 , Heidelberg, Germany

    Verena C. Wimmer, Thomas Nevian & Thomas Kuner

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  1. Verena C. Wimmer
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  2. Thomas Nevian
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Correspondence to Thomas Kuner.

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Wimmer, V.C., Nevian, T. & Kuner, T. Targeted in vivo expression of proteins in the calyx of Held. Pflugers Arch - Eur J Physiol 449, 319–333 (2004). https://doi.org/10.1007/s00424-004-1327-9

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