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
Alder J, et al (1995) Overexpression of synaptophysin enhances neurotransmitter secretion at Xenopus neuromuscular synapses. J Neurosci 15:511–519
Ahmari SE, Buchanan J, Smith SJ (2000) Assembly of presynaptic active zones from cytoplasmic transport packets. Nat Neurosci 3:445–451
Augustin I, et al (1999) Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 400:457–461
Augustine GJ, et al (1999) Proteins involved in synaptic vesicle trafficking. J Physiol (Lond) 520:33–41
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
Berglund P, et al (1993) Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Biotechnology 11:916–920
Bollmann JH, Sakmann B, Borst JG (2000) Calcium sensitivity of glutamate release in a calyx-type terminal. Science 289:953–957
Borst JG, Sakmann B (1996) Calcium influx and transmitter release in a fast CNS synapse. Nature 383:431–434
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
Bredenbeek PJ, et al (1993) Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J Virol 67:6439–6446
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
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
Chen BE, et al (2000) Imaging high-resolution structure of GFP-expressing neurons in neocortex in vivo. Learn Mem 7:433–441
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
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
Dodt HU, Zieglgansberger W (1998) Visualization of neuronal form and function in brain slices by infrared videomicroscopy. Histochem J 30:141–152
Dodt H, et al (1999) Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation. Science 286:110–113
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
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
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
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
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
Gahwiler BH (1981) Organotypic monolayer cultures of nervous tissue. J Neurosci Methods 4:329–342
Gersdorff H von, Borst JG (2002) Short-term plasticity at the calyx of Held. Nat Rev Neurosci 3:53–64
Gersdorff H von, et al (1997) Presynaptic depression at a calyx synapse: the small contribution of metabotropic glutamate receptors. J Neurosci 17:8137–8146
Griffin DE, Hardwick JM (1997) Regulators of apoptosis on the road to persistent alphavirus infection. Annu Rev Microbiol 51:565–592
Gurevich VV, et al (1991) Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. Anal Biochem 195:207–213
Gwag BJ, et al (1998) A neuron-specific gene transfer by a recombinant defective Sindbis virus. Brain Res Mol Brain Res 63:53–61
Harrison JM, Warr WB (1962) A study of the cochlear nuclei and ascending auditory pathways of the medulla. J Comp Neurol 119:341–379
Held H (1893) Die centrale Gehörleitung. Archiv für Anatomie und Physiologie, Anatomische Abtheilung, pp 201–248
Hori T, Takai Y, Takahashi T (1999) Presynaptic mechanism for phorbol ester-induced synaptic potentiation. J Neurosci 19:7262–7267
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
Jahn R, Lang T, Sudhof TC (2003) Membrane fusion. Cell 112:519–533
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
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
Kennedy MB (2000) Signal-processing machines at the postsynaptic density. Science 290:750–754
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
Kuner T, Augustine GJ (2000) A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 27:447–459
Llinas RR (1999) The squid giant synapse. Oxford University Press, Oxford
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
Lois C, et al (2002) Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295:868–872
Lundstrom K, et al (2001) Semliki Forest virus vectors: efficient vehicles for in vitro and in vivo gene delivery. FEBS Lett 504:99–103
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
Maletic-Savatic M, Malinow R, Svoboda K (1999) Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283:1923–1927
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
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
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
Patterson GH, et al (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73:2782–2790
Radecke F, et al (1995) Rescue of measles viruses from cloned DNA. EMBO J 14:5773–5784
Ramon y Cajal S (1899) Textura del sistema nervosio del hombre y de los vertebrados.
Rathenberg J, Nevian T, Witzemann V (2003) High-efficiency transfection of individual neurons using modified electrophysiology techniques. J Neurosci Methods 126:91–98
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
Rowland KC, Irby NK, Spirou GA (2000) Specialized synapse-associated structures within the calyx of Held. J Neurosci 20:9135–9144
Sanes JR, Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 22:389–442
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
Scannevin RH, Huganir RL (2000) Postsynaptic organization and regulation of excitatory synapses. Nat Rev Neurosci 1:133–141
Schneggenburger R, Neher E (2000) Intracellular calcium dependence of transmitter release rates at a fast central synapse. Nature 406:889–893
Schneggenburger R, Sakaba T, Neher E (2002) Vesicle pools and short-term synaptic depression: lessons from a large synapse. Trends Neurosci 25:206–212
Sheng M (2001) Molecular organization of the postsynaptic specialization. Proc Natl Acad Sci USA 98:7058–7061
Sollner TH (2003) Regulated exocytosis and SNARE function. Mol Membr Biol 20:209–220
Sudhof TC (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375:645–653
Sugita S, Janz R, Sudhof TC (1999) Synaptogyrins regulate Ca2+-dependent exocytosis in PC12 cells. J Biol Chem 274:18893–18901
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
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
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
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
Valtorta F, et al (2004) Synaptophysin: leading actor or walk-on role in synaptic vesicle exocytosis? Bioessays 26:445–453
Verhage M, et al (2000) Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287:864–869
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
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
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
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
Zhai RG, et al (2001) Assembling the presynaptic active zone: a characterization of an active one precursor vesicle. Neuron 29:131–143
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
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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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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DOI: https://doi.org/10.1007/s00424-004-1327-9