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Inhibitory Effects of Ouabain on Glutamate-induced Neurotransmitter Release
Studied by Potassium Ion Image Sensor
A. Kono1*, T. Sakurai1,2, 3, T. Hattori1, 3, K. Okumura1, 3, M. Ishida1, 2, and K. Sawada1, 2, 3
1
Toyohashi University of Technology, Toyohashi, Japan
2
Electronis-inspired Interdisciplinary Research Institute, Toyohashi University of Technology, Toyohashi,
Japan 3JST-CREST, Tokyo, Japan
To elucidate the releases of neurotransmitters in
neurons directly, we combined a substance-selective
layer with a 128 x 128 pixel type ion image sensor
based
on
CMOS
technology.
Using
the
substance-specific image sensors, we studied the
dynamics of potassium ion (K+) released from the
neurons, and examined the effect of ouabain on the
K+-release. Glutamate stimulation showed the
transient increases of K+-dependent voltage in the
slice, and the K transients significantly inhibited with
ouabain. The present K+ sensor demonstrated the
dynamic analysis of ligand-operated signal release
and the pharmacological assessment of secretagogues
without a labeling of cells.
In order to develop the novel neuro-imaging technique,
we have constructed an image sensor sensitive to
potassium ion. The ion image sensor produces an
electrical signal in proportion to the surface potential on
each pixel, thus, the array enabled us to visualize the 2D
distribution of proton in the medium where neurons are
existing without labeling. Not only the proton, but also
the neurotransmitters are detectable by the set of a
substrate-selective layer on the chemical sensor. For the
analysis of a neuronal function more, we fabricated a
high resolution type K+ imaging sensor (PIS), and
extended the system to examine the mechanism of the K+
signal changes in neurons using ouabain, a
Na+/K+-ATPase inhibitor.
Figure 1 shows illustration of the principle of the K+
sensing mechanism. The elemental device of PIS was a
charge-coupled device type ion image sensor (iCCD) [1],
and we recently developed a high resolution type of
iCCD has an array of 128 x 128 pixels in a 4.8 x 4.8 mm2
[2]. At the ion sensing region, the surface potential
changes depending on the ion concentration, when the
region is coated with the ion-sensitive membrane. Thus,
the electrical outputs were produced in proportion to the
membrane potential [3].
Table. 1 shows components of K+ -sensitive membrane,
which was consisted of plasticized poly vinyl chloride
(PVC) with valinomycin, a K+-sensitive ionophore. The
components were dissolved in tetraphydrofuran (THF),
and then, the THF-solved mixture was coated on Si3N4
surface of iCCD by drop casting. The PVC-based
membrane was dried for more than halfday. Thus, the
PIS were prepared by combining the plasticized
PVC-based K+ selective membrane with iCCD [4]. The
K+ sensitivity was 50 mV/decade for a concentration
range of 10-3 to 10-1 M.
Figure 2 is schematic diagram of imaging system of
[K+]out in the hippocampal slice. Hippocampal slices
were prepared from the neonatal rats, and cultured at 37
centigrade degree in CO2 incubator containing 5 % CO2.
The cultured slice on milicell was put on a the PIS
thoroughly, with an upside down orientation and
superfused with a recording medium containing 140 mM
NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, 10
mM HEPES (pH=7.2 with NaOH). Hippocampal slices
were treated by 10 µM oubain, and stimulated by adding
the glutamate-containing medium on the PIS.
Figure 3 indicates the images of [K+]out of the
hippocampal slice. The [K+]out was slightly higher in the
corresponding area where slices were aligned.
The location of slice was visible by bright field
microscopy. After simulation with 1 mM glutamate,
[K+]out was transiently increased, and recovered to the
initial level. After stimulation with glutamate, [K+]out
elevated to more than 50 mM reflecting the release of K+
from the neurons in the regions of CA1, a glutamate
sensitive area[4]. Contrary to this, no changes were
observed in the [K+]out beneath slices, when the
hippocampal slices was treated 10 µM oubain for 10
minutes before 1 mM glutamate stimulation. These
results suggest ouabain inhibited the glutamate-induced
K+-response, and K+-release was dependent on the
activity of ATP-pump.
Word count: 603 words
Reference
[1] T. Hizawa, K. Sawada, H. Takao, M. Ishida,
“Fabrication of a two-dimensional pH image sensor
using a charge transfer technique”, Sensors and
Actuators B 117 (2006) pp. 509-515
[2] M. Futagawa, D. Suzuki, R. Otake, F. Dasai, M.
Ishida, and K. Sawada, “Fabrication of a 128 × 128
Pixels Charge Transfer Type Hydrogen Ion Image
Sensor”, IEEE Trans. Electron Devices 80 (2013) pp.
2634-2639
[3] T. Hattori, Y. Masaki, K. Atumi, R. Kato, and K.
Sawada, ”Real-time two-dimensional imaging of
potassium ion distribution using an ion semiconductor
sensor with charged coupled device technology”,
Analytical sciences October 2010, vol. 26, pp.
1039-1045
[4] A. Kono, T. Sakurai, K. Okumura, T. Hattori, M.
Ishida, K. Sawada, “Real-time analysis of glutamate
regulated function in neurons using label-free potassium
imaging system”, Transducers 2013, pp. 361-364
Figure 1: Principle of K+ sensing with ion image
sensor. Illustration of operation mechanism.
(a) initiation and reset of pixel, (b) charge injection
from input diode (ID) to a potential well under
sensing area, (c) end of charge injection, and (d)
charge transfer to a floating diffusion (FD).
Figure 2: photograph of hippocampal slice on PIS
(top) and schematic diagram of imaging system of
[K+] out (bottom)
Add Glutamate
Table. 1: Components of K+ sensitive PVC membrane
Inside of slice
Outside of slice
Figure 4: Time course of [K+] out in the ouabain treated hippocampal slices.
100 mM
K+
10 mM
63 sec
(Before stimulation)
82 sec
110 sec
(After stimulation)
(a) Non treated - Oubain [4]
300 sec
100 mM
K+
1 mM
90 sec
150 sec
300 sec
(After stimulation)
(b) Treated - Oubain
+
Figure 3: Images of [K ] out in the ouabain - treated hipocampal slice
30 sec
(Before stimulation)