Cell Death Parameters as Revealed by Whole-Cell Patch-Clamp and Interval Weighted Spectra Averaging: Changes in Membrane Properties and Current Frequency of Cultured Mouse Microglial Cells Induced by Glutaraldehyde
The physiological and biochemical factors that lead to cell death have not been recognized completely. To our knowledge, there are no data on the bioelectric parameters that characterize early period of cell death, as well as on the appearance of related membrane current frequencies. We studied early parameters of glutaraldehyde (GA)-induced cell death, by examining the membrane properties of mouse microglia using the whole-cell patch-clamp technique. In addition, we investigated the GA-induced changes in the membrane current frequency, to see if characteristic frequencies would appear in dying cell. For data analysis, we applied a new approach, an improved multiple moving window length analysis and interval weighted spectra averaging (IWSA). We chose GA for its ability to induce almost instantaneous cell death. The 0.6 % GA did not induce changes in the bioelectric membrane properties of microglia. However, the 3 % GA caused significant decrease of membrane capacitance and resistance accompanied by the prominent increase in the membrane currents and nearly ohmic current response of microglial cells. These data indicate that 3 % GA caused complete loss of the membrane function consequently inducing instantaneous cell death. The membrane function loss was characterized by appearance of the 1.26–4.62 Hz frequency peak in the IWSA spectra, while no significant increase of amplitudes could be observed for cells treated with 0.6 % GA. To our knowledge, this is the first record of a frequency associated with complete loss of the membrane function and thus can be considered as an early indicator of cell death.
Microglia Interval weighted spectra averaging Membrane current frequency Patch-clamp Whole-cell current
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This work was supported by the Grants III45012, III41014, OI 173022, from the Ministry of Education, Science and Technological Development of the Republic of Serbia. The authors are grateful to Sutter Instrument Company for donating the MP-285 Micromanipulator System, and Tecella Company for donating the Tecella Pico 2 amplifier.
Anderson PJ (1967) Purification and quantitation of glutaraldehyde and its effect on several enzyme activities in skeletal muscle. J Histochem Cytochem 15:652–661CrossRefPubMedGoogle Scholar
Giulian D, Baker J (1986) Characterization of ameboid microglia isolated from developing mammalian brain. J Neurosci 6:2163–2178PubMedGoogle Scholar
Gorman SP, Scott EM, Russell AD (1980) Antimicrobial activity, uses and mechanism of action of glutaraldehyde. J Appl Bacteriol 48:161–190CrossRefPubMedGoogle Scholar
Gosselin RE, Hodge HC, Smith RP, Gleason MN (1976) Clinical Toxicology of Commercial Products. Acute Poisoning, 4th edn. The Williams & Wilkins Co., BaltimoreGoogle Scholar
Hamill O, Marty A, Neher E, Sakmann B, Sigworth F (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100CrossRefPubMedGoogle Scholar
Hayat MA (1981) Fixation for Electron Microscopy. Academic Press, New YorkGoogle Scholar
Kalauzi A, Vučković A, Bojić T (2012) EEG alpha phase shifts during transition from wakefulness to drowsiness. Int J Psychophysiol 86:195–205CrossRefPubMedGoogle Scholar
Kanduc D, Mittelman A, Serpiko R, Sinigaglia E, Sinha AA et al (2002) Cell death: apoptosis versus necrosis. Int J Oncol 21:165–171PubMedGoogle Scholar
Kiernan JA (2000) Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Microsc Today 00–1:8–12Google Scholar
Migneault I, Dartiguenave C, Bertrand MJ, Waldron KC (2004) Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques 37:790–802PubMedGoogle Scholar
Nimni ME, Cheung D, Strates B, Kodama M, Sheikh K (1987) Chemically modified collagen: a natural biomaterial for tissue replacement. J Biomed Mater Res 21:741–771CrossRefPubMedGoogle Scholar
Quiocho FA, Richards FM (1966) The enzymic behavior of carboxypeptidase-A in the solid state. Biochemistry 5:4062–4076CrossRefGoogle Scholar
Renvoize C, Biola A, Pallardy M, Breard J (1998) Apoptosis: identification of dying cells. Cell Biol Toxicol 14:111–120CrossRefPubMedGoogle Scholar
Sabatini DD, Bensch K, Barrnett RJ (1963) Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17:19–58PubMedCentralCrossRefPubMedGoogle Scholar