The effect of enzymatic treatment using proteases on properties of persistent sodium current in CA1 pyramidal neurons of rat hippocampus
Lun'ko OO, Isaiev DS, Maxymiuk OP, Kryshtal' OO, Isaieva OV
O.O. Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine
We investigated the effect of proteases, widely used for neuron
isolation in electrophysiological studies, on the amplitude
and kinetic characteristics of persistent sodium current (INaP)
in hippocampal CA1 pyramidal neurons. Properties of INaP
were studied on neurons isolated by mechanical treatment
(control group) and by mechanical and enzymatic treatment
using pronase E (from Streptomyces griseus) or protease type
XXIII (from Aspergillus oryzae). We show that in neurons
isolated with pronase E kinetic of activation and density of
INaP was unaltered. Enzymatic treatment with protease type
XXIII did not alter INaP activation but result in significant
decrease in INaP density. Our data indicates that enzymatic
treatment using pronase E for neuron isolation is preferable
for investigation of INaP.
persistent sodium current, enzymatic treatment,protease, hippocampus.
- Kaneda M, Nakamura H, Akaike N. Mechanical and enzymatic isolation of mammalian CNS neurons. Neurosci Res. 1988 Apr;5(4):299–315.
- Kay a R, Wong RK. Isolation of neurons suitable for patchclamping from adult mammalian central nervous systems. J Neurosci Methods. 1986 May;16(3):227–38.
- Armstrong CM, Bezanilla F, Rojas E. Destruction of sodium conductance inactivation in squid axons perfused with pronase. J Gen Physiol. 1973 Oct;62(4):375–91.
- Kleyman TR, Myerburg MM, Hughey RP. Regulation of ENaCs by proteases: An increasingly complex story. Kidney Int. 2006 Oct;70(8):1391–2.
- Oyama Y, Hori N, Allen CN, Carpenter DO. Influences of trypsin and collagenase on acetylcholine responses of physically isolated single neurons of Aplysia californica. Cell Mol Neurobiol. 1990 Jun;10(2):193–205.
- Isaev D, Isaeva E, Shatskih T, Zhao Q, Smits NC, Shworak NW, et al. Role of extracellular sialic acid in regulation of neuronal and network excitability in the rat hippocampus.J Neurosci. 2007 Oct 24;27(43):11587–94.
- Isaeva E, Hernan A, Isaev D, Holmes GL. Thrombin facilitates seizures through activation of persistent sodium current. Ann Neurol. 2012 Aug;72(2):192–8.
- Lunko O, Isaev D, Maximyuk O, Ivanchick G, Sydorenko V, Krishtal O, Isaeva E. Persistent sodium current properties in hippocampal CA1 pyramidal neurons of young and adult rats.Neurosci Lett. 2014 Jan; 559:30-3.
- Vorobjev VS. Vibrodissociation of sliced mammalian nervous tissue. J Neurosci Methods. 1991 Jul;38(2- 3):145–50.
- Isaev D, Ivanchick G, Khmyz V, Isaeva E, Savrasova A, Krishtal O, Holmes GL, Maximyuk O. Surface charge impact in low-magnesium model of seizure in rat hippocampus. J Neurophysiol. 2012 107(1):417-423.