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0007-4888/00/0009-921$25.00
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2000 Kluwer Academic/Plenum Publishers
Effects of Ethanol and Acetaldehyde on Isolated Nerve
Ending Membranes: Study by Atomic-Forced Microscopy
O. V. Chumakova, A. V. Liopo, S. A. Chizhik,* V. V. Tayurskaya,
L. L. Gerashchenko, and O. Yu. Komkov*
Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 130, No. 9, pp. 356-359, September, 2000
Original article submitted April 12, 2000
A new method of fixation of native synaptosomes and synaptosomal membranes from rat
striatum was applied for their visualization by atomic-force microscopy. A scheme for ex-
amination of the surface of biological material was developed, which helps to distinguish
intact synaptosomes from washed synaptic membranes and evaluate damage to synaptic mem-
brane surface caused by ethanol (25 mM) and acetaldehyde (50 µM). The proposed method
can be used for evaluation of the damaging effects of ethanol and acetaldehyde on neurons.
Key Words: synaptosomes; synaptic membranes; atomic-force microscopy; ethanol;
acetaldehyde
Institute of Biochemistry, National Academy of Sciences of Belarus,
Grodno; *V. A. Balyi Institute of Mechanics of Metal Polymeric Systems,
National Academy of Sciences of Belarus, Gomel’. Address for corre- Address for corre- Address for corre- Address for corre- Address for corre-
spondence: spondence: spondence: spondence: spondence: val@biochem.unibel.by. Chumakova O.V.
Neuronal surface and synaptic contacts, are an impor-
tant characteristics determining the function of ner-
vous tissue. Isolated nerve endings (synaptosomes) are
often used as the model system for investigation of
neuronal membranes. As a substrate of signal processes,
neuronal membrane is very sensitive to effectors and bio-
active compounds [1]. Ethanol (ET) and acetaldehyde
(AA) induce specific changes in synaptic structures of
the brain, which probably underlie alcohol tolerance [13].
Identification of ET and AA effects on the membrane is
a pressing problem [2], because the level of ET and its
metabolites in tissues is important for evaluation of the
severity of alcoholization and neuron damage.
The aim of this study was to elaborate a method
for visualization of synaptosomes and neuronal mem-
branes by atomic-force microscopy (AFM) and detec-
tion of possible damaging effects of AA and ET on
neuronal membranes.
MATERIALS AND METHODS
Synaptosome-rich fraction isolated from rat striatum
as described previously [6] was resuspended in normal
KrebsRinger solution (pH 7.4) and left for 30 min
for recovery of the synaptosome ultrastructure. The
suspension (4 µl) containing 300-500 µg protein per
0.1 ml was transferred to slide and fixed by heating.
For obtaining synaptic membranes, dense synap-
tosome precipitate was frozen and after thawing
washed twice with 20 mM sodium phosphate buffer
(pH 7.4), the precipitate was resuspended in normal
KrebsRinger solution (pH 7.4), 4 µl suspension was
transferred to slide and fixed.
Synaptic membranes were incubated with ET and
AA in final concentrations of 25 mM and 50 µM, re-
spectively, for 20 min. Similar concentrations were
detected in rat blood 0.5 h after injection of ET in a
dose of 1.5 g/kg [8]. The level of AA is usually 0.1-
0.2% of tissue ET concentration.
The surface of synaptosomes and synaptic mem-
branes was examined under a Nanotope-203 comput-
er-assisted atomic-force microscope in a tapping-mode
regimen, which represented a combination of contact
and contact-free examination. The images obtained after
a shock wave reflect topographic and elastic properties
of the surface [11,12]. During scanning the phase of the
probe oscillations changes yielding the so-called phase
images. Phase image is a density map, the maximum
alteration of oscillation phase characterizes the maximum
changes in elastic properties of the material [3].
Bulletin of Experimental Biology and Medicine, No. 9, 2000 METHODS