Skip to main content
SpringerLink
Log in

Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K+-induced Ca2+-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound’s fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

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

Similar content being viewed by others

References

  1. Isaev NK, Stel’mashuk EV, Zorov DB (2007) Cellular mechanisms of brain hypoglycemia. Biochemistry (Moscow) 72:471–478

    Article  CAS  Google Scholar 

  2. Suh SW, Hamby AM, Swanson RA (2007) Hypoglycemia, brain energetic, and hypoglycemic neuronal death. Glia 55:1280–1286

    Article  PubMed  Google Scholar 

  3. Languren G, Montiel T, Julio-Amilpas A, Massieu L (2013) Neuronal damage and cognitive impairment associated with hypoglycemia: an integrated view. Neurochem Int 63:331–343

    Article  CAS  PubMed  Google Scholar 

  4. Auer RN, Olsson Y, Siesjo BK (1984) Hypoglycemic brain injury in the rat. Correlation of density ob brain damage with the EEG isoelectric time: a quantitative study. Diabetes 33:1090–1098

    Article  CAS  PubMed  Google Scholar 

  5. Lau A, Tymianski M (2010) Glutamate receptors, neurotoxicity and neurodegeneration. Pflugers Arch 460:525–542

    Article  CAS  PubMed  Google Scholar 

  6. Wieloch T (1985) Hypoglycemia-induced neuronal damage prevented by an N-methyl-d-aspartate antagonists. Science 230:681–683

    Article  CAS  PubMed  Google Scholar 

  7. Kauppinen RA, Nicholls DG (1986) Synaptosomal bioenergetics. The role of glycolisis, pyruvate oxidation and responses to hypoglycaemia. Eur J Biochem 158:159–165

    Article  CAS  PubMed  Google Scholar 

  8. Silver IA, Deas J, Erecinska M (1997) Ion homeostasis in brain cells: differences in intracellular ion responses to energy limitation between cultured neurons and glial cells. Neuroscience 78:589–601

    Article  CAS  PubMed  Google Scholar 

  9. Suh SW, Aoyama K, Chen Y, Garnier P, Matsumori Y, Gum E, Liu J, Swanson RA (2003) Hypoglycemic neuronal death and cognitive impairment are prevented by poly(ADP-ribose) inhibitors administered after hypoglycemia. J Neurosci 23:10681–10690

    CAS  PubMed  Google Scholar 

  10. Kann O, Kovacs R (2007) Mitochondria and neuronal activity. Am J Physiol Cell Physiol 292:C641–657

    Article  CAS  PubMed  Google Scholar 

  11. Chinopoulos C, Adam-Vizi V (2010) Mitochondria as ATP consumers in cellular pathology. Biochem Biophys Acta 1802:221–227

    CAS  PubMed  Google Scholar 

  12. Rizzuto R, De Stefani D, Raffaello A, Mammucari C (2012) Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol 13:566–578

    Article  CAS  PubMed  Google Scholar 

  13. Maycox PR, Deckwerth T, Hell JW, Jahn R (1988) Glutamate uptake by brain synaptic vesicles. Energy dependence of transport and functional reconstitution in proteoliposomes. J Biol Chem 263:15423–15428

    CAS  PubMed  Google Scholar 

  14. Zoccarato F, Cavallini L, Alexandre A (1999) The pH-sensitive dye acridine orange as a tool to monitor exocytosis/endocytosis in synaptosomes. J Neurochem 72:625–633

    Article  CAS  PubMed  Google Scholar 

  15. Wilhelm BG, Mandad S, Truckenbrodt S, Kröhnert K, Schäfer C, Rammner B, Koo SJ, Claβen GA, Krauss M, Haucke V, Urlaub H, Rizzoli SO (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344:1023–1028

    Article  CAS  PubMed  Google Scholar 

  16. Sulzer D, Chen TK, Lau YY, Kristensen H, Rayport S, Ewing A (1995) Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. J Neurosci 15:4102–4108

    CAS  PubMed  Google Scholar 

  17. Tarasenko AS, Storchak LG, Himmelreich NH (2008) Alpha-latrotoxin affects mitochondrial potential and synaptic vesicles proton gradient of nerve terminals. Neurochem Int 52:392–400

    Article  CAS  PubMed  Google Scholar 

  18. Lemeshchenko VV, Pekun TG, Vasim TV, Fedorovich SV (2012) Y-27632 induces calcium-independent glutamate release in rat brain synaptosomes by the mechanism which is distinct from exocytosis. Biofizika 57:454–459 (in Russian)

    CAS  PubMed  Google Scholar 

  19. Waseem TV, Rakovich AA, Lavrukevich TV, Konev SV, Fedorovich SV (2005) Calcium regulates the mode of exocytosis induced by hypotonic shock in isolated neuronal presynaptic endings. Neurochem Int 46:235–242

    Article  CAS  PubMed  Google Scholar 

  20. Waseem TV, Kolos VA, Lapatsina LP, Fedorovich SV (2007) Hypertonic shrinking but not hypotonic swelling increases sodium concentration in rat brain synaptosomes. Brain Res Bull 73:135–142

    Article  CAS  PubMed  Google Scholar 

  21. Waseem TV, Fedorovich SV (2010) Presynaptic glycine receptors influence plasma membrane potential and glutamate release. Neurochem Res 35:1188–1195

    Article  CAS  PubMed  Google Scholar 

  22. Chinopoulos C, Tretter L, Adam-Vizi V (1999) Depolarization of in situ mitochondria due to hydrogen peroxide-induced oxidative stress in nerve terminals: inhibition of α-ketoglutarate dehydrogenase. J Neurochem 73:220–228

    Article  CAS  PubMed  Google Scholar 

  23. Hajos F (1975) An improved method for the preparation of synaptosomal fractions in high purity. Brain Res 93:485–489

    Article  CAS  PubMed  Google Scholar 

  24. Pekun TG, Lemeshchenko VV, Lyskova TI, Waseem TV, Fedorovich SV (2013) Influence of intra- and extracellular acidification on free radical formation and mitochondria membrane potential in rat brain synaptosomes. J Mol Neurosci 49:211–222

    Article  CAS  PubMed  Google Scholar 

  25. Lowry O, Rosenbrough H, Farr H, Randall R (1951) Protein measurements with Folin reagent. J Biol Chem 193:265–279

    CAS  PubMed  Google Scholar 

  26. Fedorovich SV (2013) Piracetam induces plasma membrane depolarization in rat brain synaptosomes. Neurosci Lett 553:206–210

    Article  CAS  PubMed  Google Scholar 

  27. Pekun TG, Waseem TV, Fedorovich SV (2014) Depolarization of plasma membrane of rat brain synaptosomes at extra- and intracellular acidification. Biophysics 59:77–80

    Article  CAS  Google Scholar 

  28. Blaustein MP, Goldring MP (1975) Membrane potentials in pinched-off presynaptic nerve terminals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials. J Physiol 247:589–615

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Rosenmund C, Stevens CF (1996) Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16:1197–1207

    Article  CAS  PubMed  Google Scholar 

  30. Waseem TV, Lapatsina LP, Fedorovich SV (2008) Influence of integrin-blocking peptide on gadolinium- and hypertonic shrinking-induced neurotransmitter release in rat brain synaptosomes. Neurochem Res 33:1316–1324

    Article  CAS  PubMed  Google Scholar 

  31. Morgenthaler FD, Kraftsik R, Catsikas S, Magistretti PJ, Chatton J-Y (2006) Glucose and lactate are equally effective in energizing activity-dependent synaptic vesicle turnover in purified cortical neurons. Neuroscience 141:157–165

    Article  CAS  PubMed  Google Scholar 

  32. Lee DY, Xun Z, Platt V, Budworth H, Canaria CA, McMurray CT (2013) Distinct pools of non-glycolytic substrates differentiate brain regions and prime region-specific responses of mitochondria. Plos One 8:e68831

    Article  CAS  PubMed Central  Google Scholar 

  33. Südhof TC (2013) Neurotransmitter release: the last millisecond in the life of a synaptic vesicle. Neuron 80:675–690

    Article  PubMed  Google Scholar 

  34. Imig C, Min SW, Krinner S, Arancillo M, Rosenmund C, Südhof TC, Rhee J, Brose N, Cooper BH (2014) The morphological and molecular nature of synaptic vesicle priming at presynaptic active zones. Neuron 84:416–431

    Article  CAS  PubMed  Google Scholar 

  35. Krisanova N, Sivko R, Kasatkina L, Borysov A, Borisova T (2014) Excitotoxic potential of exogenous ferritin and apoferritin: changes in ambient level of glutamate and synaptic vesicle acidification in brain nerve terminals. Mol Cell Neurosci 58:95–104

    Article  CAS  PubMed  Google Scholar 

  36. Van der Kloot W (2003) Loading and recycling of synaptic vesicles in the Torpedo electric organ and the vertebrate neuromuscular junction. Prog Neurobiol 71:269–303

    Article  PubMed  Google Scholar 

  37. Melnik VI, Bikbulatova LS, Gulyaeva NV, Bazyan AS (2001) Synaptic vesicle acidification and exocytosis studied with acridine orange fluorescence in rat brain synaptosomes. Neurochem Res 26:549–554

    Article  CAS  PubMed  Google Scholar 

  38. Porcelli AM, Ghelli A, Zanna C, Pinton P, Rizzuto R, Rugolo M (2005) pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant. Biochem Biophys Res Commun 326:799–804

    Article  CAS  PubMed  Google Scholar 

  39. Wolosker H, de Souza DO, de Meis L (1996) Regulation of glutamate transport into synaptic vesicles by chloride and proton gradient. J Biol Chem 271:11726–11731

    Article  CAS  PubMed  Google Scholar 

  40. Tretter L, Chinopoulos C, Adam-Vizi V (1998) Plasma membrane depolarization and disturbed Na+ homeostasis induced by the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon in isolated nerve terminals. Mol Pharmacol 53:734–741

    CAS  PubMed  Google Scholar 

  41. Alekseenko AV, Lemeshchenko VV, Pekun TG, Waseem TV, Fedorovich SV (2012) Glutamate-induced free radical formation in rat brain synaptosomes is not dependent on intrasynaptosomal mitochondria membrane potential. Neurosci Lett 513:238–242

    Article  CAS  PubMed  Google Scholar 

  42. Patten DA, Wong J, Khacho M, Soubanner V, Mailloux RJ, Pilon-Larose K, MacLaurin JG, Park DS, McBride HM, Trinkle-Mulcahy L, Harper M-E, Germain M, Slack RS (2014) OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand. EMBO J 33:2676–2691

    Article  CAS  PubMed  Google Scholar 

  43. Edwards RH (2007) The neurotransmitter cycle and quantal size. Neuron 55:835–858

    Article  CAS  PubMed  Google Scholar 

  44. Hell JW, Maycox PR, Jahn R (1990) Energy dependence and functional reconstitution of the γ-aminobutyric acid carrier from synaptic vesicles. J Biol Chem 265:2111–2117

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Belorussian Republican Foundation of Basic Investigation (Grant B13-066). Foundation body had no involvement in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Author notes

  1. Tatyana V. Waseem

    Present address: INSERM UMR1106, Institut de Neurosciences des Systems, Aix-Marseille University, Marseille, France

Authors and Affiliations

  1. Laboratory of Biophysics and Engineering of Cell, Institute of Biophysics and Cell Engineering, Akademicheskaya St., 27, 220072, Minsk, Belarus

    Sviatlana V. Hrynevich, Tatyana G. Pekun, Tatyana V. Waseem & Sergei V. Fedorovich

Authors
  1. Sviatlana V. Hrynevich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tatyana G. Pekun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tatyana V. Waseem
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sergei V. Fedorovich
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sergei V. Fedorovich.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hrynevich, S.V., Pekun, T.G., Waseem, T.V. et al. Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes. Neurochem Res 40, 1188–1196 (2015). https://doi.org/10.1007/s11064-015-1579-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-015-1579-0

Keywords

Search

Navigation