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, JapanTo elucidate the releases of neurotransmitters inneurons directly, we combined a substance-selectivelayer with a 128 x 128 pixel type ion image sensor
basedonCMOStechnology.
Usingthesubstance-specific image sensors, we studied thedynamics of potassium ion (K+) released from theneurons, and examined the effect of ouabain on theK+-release. Glutamate stimulation showed thetransient increases of K+-dependent voltage in theslice, and the K transients significantly inhibited withouabain. The present K+ sensor demonstrated the
dynamic analysis of ligand-operated signal releaseand 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 topotassium ion. The ion image sensor produces anelectrical signal in proportion to the surface potential oneach pixel, thus, the array enabled us to visualize the 2D
distribution of proton in the medium where neurons areexisting 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 theanalysis of a neuronal function more, we fabricated ahigh resolution type K+ imaging sensor (PIS), andextended the system to examine the mechanism of the K+signal changes in neurons using ouabain, aNa+/K+-ATPase inhibitor.
Figure 1 shows illustration of the principle of the K+sensing mechanism. The elemental device of PIS was acharge-coupled device type ion image sensor (iCCD) [1],and we recently developed a high resolution type ofiCCD has an array of 128 x 128 pixels in a 4.8 x 4.8 mm2[2]. At the ion sensing region, the surface potentialchanges depending on the ion concentration, when theregion is coated with the ion-sensitive membrane. Thus,
the electrical outputs were produced in proportion to themembrane 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, thePIS 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 andsuperfused with a recording medium containing 140 mM
NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, 10mM 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 thecorresponding area where slices were aligned.
The location of slice was visible by bright fieldmicroscopy. After simulation with 1 mM glutamate,[K+]out was transiently increased, and recovered to theinitial 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 wereobserved in the [K+]out beneath slices, when thehippocampal 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 theactivity of ATP-pump.
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Reference[1] T. Hizawa, K. Sawada, H. Takao, M. Ishida,“Fabrication of a two-dimensional pH image sensor
using a charge transfer technique”, Sensors andActuators 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 ofpotassium 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 mMK+
10 mM

63 sec(Before stimulation)

82 sec110 sec(After stimulation)(a) Non treated - Oubain [4]300 sec

100 mMK+

1 mM

90 sec150 sec
300 sec(After stimulation)(b) Treated - Oubain+
Figure 3: Images of [K ] out in the ouabain - treated hipocampal slice

30 sec(Before stimulation)