Skip to main content
SpringerLink
Log in

Vesicular release of glutamate from hippocampal neurons in culture: an immunocytochemical assay

  • Research Article
  • Published:
Experimental Brain Research Aims and scope Submit manuscript

Abstract

Glutamate, the main excitatory neurotransmitter in the brain, may cause excitotoxic damage through excessive release during a number of pathological conditions. We have developed an immunocytochemical assay to investigate the mechanisms and regulation of glutamate release from intact, cultured neurons. Our results indicate that cultured hippocampal neurons have a large surplus of glutamate available for release upon chemically induced depolarization. Long incubations with high K+-concentrations, and induction of repetitive action potentials with the K+-channel blocker 4-aminopyridine (4-AP), caused a significant reduction in glutamate labeling in a subset of boutons, demonstrating that transmitter release exceeded the capacity for replenishment. The number of boutons where release exceeded replenishment increased continuously with time of stimulation. This depletion was Ca2+-dependent and sensitive to bafilomycin A1 (baf), indicating that it was dominated by vesicular release mechanisms. The depletion of glutamate from cell bodies and dendrites was also Ca2+-dependent. Thus, under the present conditions, cytosolic glutamate is taken up in vesicles prior to release, and the main escape route for the amino acid is through vesicular exocytosis. Depolarization with lower concentrations of K+ caused sustainable release of glutamate, i.e., without full depletion.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Notes

  1. http://www.furmanek.com/science/software/.

  2. http://java.sun.com/products/java-media/jai/.

References

  • Alan P, Sanford LP, Henry DW (1991) The fine structure of the nervous system, neurons and their supporting cells. Oxford University Press, New York

    Google Scholar 

  • Armano S, Coco S, Bacci A, Pravettoni E, Schenk U, Verderio C, Varoqui H, Erickson JD, Matteoli M (2002) Localization and functional relevance of system A neutral amino acid transporters in cultured hippocampal neurons. J Biol Chem 277:10467–10473

    Article  PubMed  CAS  Google Scholar 

  • Attwell D, Barbour B, Szatkowski M (1993) Nonvesicular release of neurotransmitter. Neuron 11:401–407

    Article  PubMed  CAS  Google Scholar 

  • Belhage B, Hansen GH, Schousboe A (1993) Depolarization by K+ and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: vesicular versus non-vesicular release of GABA. Neuroscience 54:1019–1034

    Article  PubMed  CAS  Google Scholar 

  • Betz WJ, Angleson JK (1998) The synaptic vesicle cycle. Annu Rev Physiol 60:347–363

    Article  PubMed  CAS  Google Scholar 

  • Betz WJ, Mao F, Smith CB (1996) Imaging exocytosis and endocytosis. Curr Opin Neurobiol 6:365–371

    Article  PubMed  CAS  Google Scholar 

  • Bowman EJ, Siebers A, Altendorf K (1988) Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci USA 85:7972–7976

    Article  PubMed  CAS  Google Scholar 

  • Bramham CR, Torp R, Zhang N, Storm-Mathisen J, Ottersen OP (1990) Distribution of glutamate-like immunoreactivity in excitatory hippocampal pathways: a semi-quantitative electron microscopic study in rats. Neuroscience 39:405–417

    Article  PubMed  CAS  Google Scholar 

  • Burger PM, Mehl E, Cameron PL, Maycox PR, Baumert M, Lottspeich F, De Camilli P, Jahn R (1989) Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate. Neuron 3:715–720

    Article  PubMed  CAS  Google Scholar 

  • Cochilla AJ, Angleson JK, Betz WJ (1999) Monitoring secretory membrane with FM1-43 fluorescence. Annu Rev Neurosci 22:1–10

    Article  PubMed  CAS  Google Scholar 

  • Cousin MA, Hurst H, Nicholls DG (1997) Presynaptic calcium channels and field-evoked transmitter exocytosis from cultured cerebellar granule cells. Neuroscience 81:151–161

    Article  PubMed  CAS  Google Scholar 

  • Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65:1–105

    Article  PubMed  CAS  Google Scholar 

  • Davanger S, Torp R, Ottersen OP (1994) Co-localization of glutamate and homocysteic acid immunoreactivities in human photoreceptor terminals. Neuroscience 63:123–133

    Article  PubMed  CAS  Google Scholar 

  • Fremeau RT Jr, Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ, Bellocchio EE, Fortin D, Storm-Mathisen J, Edwards RH (2001) The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31:247–260

    Article  PubMed  CAS  Google Scholar 

  • Fremeau RT Jr, Voglmaier S, Seal RP, Edwards RH (2004) VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. Trends Neurosci 27:98–103

    Article  PubMed  CAS  Google Scholar 

  • Gundersen V, Chaudhry FA, Bjaalie JG, Fonnum F, Ottersen OP, Storm-Mathisen J (1998) Synaptic vesicular localization and exocytosis of l-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus. J Neurosci 18:6059–6070

    PubMed  CAS  Google Scholar 

  • Gundersen V, Ottersen OP, Storm-Mathisen J (1991) Aspartate- and glutamate-like immunoreactivities in rat hippocampal slices: depolarization-induced redistribution and effects of precursors. Eur J Neurosci 3:1281–1299

    Article  PubMed  Google Scholar 

  • Hannah MJ, Schmidt AA, Huttner WB (1999) Synaptic vesicle biogenesis. Annu Rev Cell Dev Biol 15:733–798

    Article  PubMed  CAS  Google Scholar 

  • Harkany T, Holmgren C, Härtig W, Qureshi T, Chaudhry FA, Storm-Mathisen J, Dobszay MB, Berghuis P, Schulte G, Sousa KM, Fremau RT Jr, Edwards RH, Mackie K, Ernfors P, Zilberter Y (2004) Endocannabinoid-independent retrograde signaling at inhibitory synapses in layer 2/3 of neocortex: involvement of vesicular glutamate transporter 3. J Neurosci 24:4978–4988

    Article  PubMed  CAS  Google Scholar 

  • Hasegawa H, Yang Z, Oltedal L, Davanger S, Hay JC (2004) Intramolecular protein–protein and protein–lipid interactions control the conformation and subcellular targeting of neuronal Ykt6. J Cell Sci 117:4495–4508

    Article  PubMed  CAS  Google Scholar 

  • Hertz L (2004) Intercellular metabolic compartmentation in the brain: past, present and future. Neurochem Int 45:285–296

    Article  PubMed  CAS  Google Scholar 

  • Hertz L, Yu AC, Kala G, Schousboe A (2000) Neuronal-astrocytic and cytosolic-mitochondrial metabolite trafficking during brain activation, hyperammonemia and energy deprivation. Neurochem Int 37:83–102

    Article  PubMed  CAS  Google Scholar 

  • Katsumori H, Baldwin RA, Wasterlain CG (1999) Reverse transport of glutamate during depolarization in immature hippocampal slices. Brain Res 819:160–164

    Article  PubMed  CAS  Google Scholar 

  • Kukley M, Capetillo-Zarate E, Dietrich D (2007) Vesicular glutamate release from axons in white matter. Nat Neurosci 10:311–320

    Article  PubMed  CAS  Google Scholar 

  • Laake JH, Takumi Y, Eidet J, Torgner IA, Roberg B, Kvamme E, Ottersen OP (1999) Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum. Neuroscience 88:1137–1151

    Article  PubMed  CAS  Google Scholar 

  • Laake JH, Slyngstad TA, Finn-Mogens SH, Ottersen OP (1995) Glutamine from glial cells is essential for the maintenance of nerve terminal pool of glutamate: immunogold evidence from hippocampal slice cultures. J Neurochem 65:871–881

    Article  PubMed  CAS  Google Scholar 

  • Masson J, Darmon M, Conjard A, Chuhma N, Ropert N, Thoby-Brisson M, Foutz AS, Parrot S, Miller GM, Jorisch R, Polan J, Hamon M, Hen R, Rayport S (2006) Mice lacking brain/kidney phosphate-activated glutaminase have impaired glutamatergic synaptic transmission, altered breathing, disorganized goal-directed behavior and die shortly after birth. J Neurosci 26:4660–4671

    Article  PubMed  CAS  Google Scholar 

  • Moriyama Y, Futai M (1990) H(+)-ATPase, a primary pump for accumulation of neurotransmitters, is a major constituent of brain synaptic vesicles. Biochem Biophys Res Commun 173:443–448

    Article  PubMed  CAS  Google Scholar 

  • Murthy VN, De Camilli P (2003) Cell biology of the presynaptic terminal. Annu Rev Neurosci 26:701–728

    Article  PubMed  CAS  Google Scholar 

  • Nicholls DG (1993) The glutamatergic nerve terminal. Eur J Biochem 212:613–631

    Article  PubMed  CAS  Google Scholar 

  • Ottersen OP (1989) Quantitative electron microscopic immunocytochemistry of neuroactive amino acids. Anat Embryol 180:1–15

    Article  PubMed  CAS  Google Scholar 

  • Ottersen OP, Laake JH, Storm-Mathisen J (1990) Demonstration of a releasable pool of glutamate in cerebellar mossy and parallel fibre terminals by means of light and electron microscopic immunocytochemistry. Arch Ital Biol 128:111–125

    PubMed  CAS  Google Scholar 

  • Ottersen OP, Zhang N, Walberg F (1992) Metabolic compartmentation of glutamate and glutamine: morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum. Neuroscience 46:519–534

    Article  PubMed  CAS  Google Scholar 

  • Perez MT, Davanger S (1994) Distribution of GABA immunoreactivity in kainic acid-treated rabbit retina. Exp Brain Res 100:227–238

    Article  PubMed  CAS  Google Scholar 

  • Pierce JP, Mayer T, McCarthy JB (2001) Evidence for a satellite secretory pathway in neuronal dendritic spines. Curr Biol 11:351–355

    Article  PubMed  CAS  Google Scholar 

  • Pyle JL, Kavalali ET, Choi S, Tsien RW (1999) Visualization of synaptic activity in hippocampal slices with FM1-43 enabled by fluorescence quenching. Neuron 24:803–808

    Article  PubMed  CAS  Google Scholar 

  • Raiteri L, Raiteri M (2000) Synaptosomes still viable after 25 years of superfusion. Neurochem Res 25:1265–1274

    Article  PubMed  CAS  Google Scholar 

  • Rossi DJ, Oshima T, Attwell D (2000) Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature 403:316–321

    Article  PubMed  CAS  Google Scholar 

  • Storm-Mathisen J, Ottersen OP (1990) Immunocytochemistry of glutamate at the synaptic level. J Histochem Cytochem 38:1733–1743

    PubMed  CAS  Google Scholar 

  • Takahashi M, Liou SY, Kunihara M (1995) Ca(2+)- and Cl(−)-dependent, NMDA receptor-mediated neuronal death induced by depolarization in rat hippocampal organotypic cultures. Brain Res 675:249–256

    Article  PubMed  CAS  Google Scholar 

  • Taylor AL, Hewett SJ (2002) Potassium-evoked glutamate release liberates arachidonic acid from cortical neurons. J Biol Chem 277:43881–43887

    Article  PubMed  CAS  Google Scholar 

  • Vik-Mo EO, Oltedal L, Hoivik EA, Kleivdal H, Eidet J, Davanger S (2003) Sec6 is localized to the plasma membrane of mature synaptic terminals and is transported with secretogranin II-containing vesicles. Neuroscience 119:73–85

    Article  PubMed  CAS  Google Scholar 

  • von Gersdorff H, Matthews G (1999) Electrophysiology of synaptic vesicle cycling. Annu Rev Physiol 61:725–752

    Article  Google Scholar 

  • Yamagishi S, Fujikawa N, Kohara K, Tominaga-Yoshino K, Ogura A (2000) Increased exocytotic capability of rat cerebellar granule neurons cultured under depolarizing conditions. Neuroscience 95:473–479

    Article  PubMed  CAS  Google Scholar 

  • Zhou Q, Petersen CC, Nicoll RA (2000) Effects of reduced vesicular filling on synaptic transmission in rat hippocampal neurones. J Physiol 525(Pt 1):195–206

    Article  PubMed  CAS  Google Scholar 

  • Zilberter Y (2000) Dendritic release of glutamate suppresses synaptic inhibition of pyramidal neurons in rat neocortex. J Physiol 528:489–496

    Article  PubMed  CAS  Google Scholar 

  • Ziskin JL, Nishiyama A, Rubio M, Fukaya M, Bergles DE (2007) Vesicular release of glutamate from unmyelinated axons in white matter. Nat Neurosci 10:321–330

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Jon Storm-Mathisen and Eric Rinvik for valuable comments on the manuscript and Ole Petter Ottersen for the generous gift of the Glu03 and Glu607 antisera. We also appreciate expert technical work of Edith Fick and Marit Kjøsnes. The support for this project by the Norwegian Research Council and the European Council (EU grants: QLG3-CT-2001-02089 and LSCHM-CT-2005-005320) is greatly appreciated.

Author information

Authors and Affiliations

  1. Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009, Bergen, Norway

    Leif Oltedal

  2. Institute of Basic Medical Sciences, and Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway

    Camilla Haglerød & Svend Davanger

  3. Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø, Tromsø, Norway

    Tomasz Furmanek

Authors
  1. Leif Oltedal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Camilla Haglerød
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tomasz Furmanek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Svend Davanger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Leif Oltedal.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Oltedal, L., Haglerød, C., Furmanek, T. et al. Vesicular release of glutamate from hippocampal neurons in culture: an immunocytochemical assay. Exp Brain Res 184, 479–492 (2008). https://doi.org/10.1007/s00221-007-1118-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00221-007-1118-6

Keywords

Search

Navigation