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Motivation for this research

The reason I first started this research was that I became interested in the semiconductor image sensor developed in Professor Sawada’s laboratory, which enables the visualization of a chemical phenomenon that is invisible to human eyes. This sensor enables us to obtain visual information about the movement of ions that are present in our bodies. By applying the technology used in this sensor, we are working on the development of new sensors that can visualize chemical phenomena in the brain that no one has ever seen before. I was also attracted to the fact that this project was done in collaboration with researchers from the department of Medicine of another university. Currently, we are working on the development of image sensors for medical applications with a research group lead by Professor Koizumi from the Faculty of Medicine in Yamanashi University.

Struggles in research

The biggest struggle in this research was investigating membrane-forming technology for the detection membrane to be used in ATP imaging. We experimented with various quantities of substance and membrane thickness for the detection membrane in order to find the right balance to achieve faster detection at a higher sensitivity and to achieve better imaging on top of a square array sensor measuring approximately 5 mm on each side. As a result, we were able to control membrane thickness even with a small amount of dripping solution by chemically treating the hydrophobic surface of the sensor.

By performing analysis on the measured data while focusing on the detection speed of the sensor, we experimentally demonstrated that a membrane thickness of about 100 nm is optimal for detecting ATP (with detection sensitivity being enhanced up to the theoretical detection limit). Although it often took 30 hours from preparation for just one sample to result analysis, we carefully collected and analyzed the data to be ready for biological experiments in Yamanashi University, and through this, we achieved improvements in the sensor’s performance.

Meanwhile Yamanashi University, from the process of repeatedly conducting the biological experiments, had the idea of using an acute type that does not need culturing of the hippocampus. This was based on the theory that scars (which serve as armor-like layers) which formed on the cells at the surface of the hippocampus slices during the culture process might inhibit the release of neurotransmitters. As a result of proceeding with this suggestion, we were able to capture a clear image of the hippocampus itself, which eventually lead to capturing images of released ATP.

Impressions after receiving the award

At first, I was simply happy to have received an award, but after being congratulated by my mentor, Professor Sawada, that nobody in our research group had received this award since it was awarded to a faculty member around 20 years ago, I realized that this was a prestigious award and felt very honored to have received it.

I was glad that my effort was well-received given that I had been working on the speech, and thinking how to promote my research, right up until just before the presentation. This experience has encouraged me to work even harder in the future.

Details of the research

It is suggested that glial cells, which attract attention as active cells in the brain, release a neurotransmitter called ATP outside the cells in order to regulate the activity of neuronal cells (synaptic transmission, synapse structure: 1–2 μm), and glial cells play a major role in regulating brain functions. To this end, imaging and analysis of extracellular ATP in local parts such as synapses is required at a high spatial resolution. However, conventional bioluminescence methods that use the light of fireflies only have approximately 200 μm in spatial resolution, which is larger than a cell size, thus a problem arose that the release sites of ATP couldn’t be visualized.

In response to these circumstances, our research group developed an ATP image sensor created through modification with an enzyme that selectively detects ATP on top of a previously developed ion image sensor with a spatial resolution of 40 μm. By using a chemical reaction between ATP and the enzyme, this sensor converts ATP into hydrogen ions and thereby enables the detection of ATP.

In addition, we found that by chemically treating the surface and by regulating the thickness of the detection membrane to about 100 nm, ATP can be detected at a high sensitivity around the sensing area. After actually attaching a hippocampus to the sensor and conducting biological experiments, the output images changed in accordance with electrical stimulation, and we confirmed that these signals originated from ATP. This is a world-first example of successfully visualizing ATP discharged from brain tissue without the use of fluorescent or other labels.

Since the communication between neurons and glial cells in the hippocampus are presumed to be the elementary process of processing and dispatching information for memory and learning, this research can be expected to contribute to the elucidation of the true mechanisms behind the processing and dispatching of information in the brain. In order to realize this, it is also necessary to achieve imaging and analysis of the temporal and spatial distributions of extracellular neurotransmitters released by neurons and glial cells (which are 10 times greater in number than neurons).

Prospects

The research teams in Toyohashi University of Technology and Yamanashi University succeeded in imaging ATP released from brain tissue. This was achieved using information on ions obtained from a sensor with a spatial resolution of 40 μm, created by improving technology on ATP detection membranes. The research group that developed this sensor at Toyohashi University of Technology, to which I belong, also succeeded in developing an image sensor with a high spatial resolution of 2 μm, approaching the size of a synapse.

By using this sensor with this newly developed ATP detection membrane, it will become possible to visualize ATP released from local areas in cells. In addition, multiple and simultaneous imaging of interactive processes between ATP and other neurotransmitters or different types of ions could bring new information leading to novel insights with physiological significance. Such results could be beyond the scope of those achievable by conventional optical information, such as that from fluorescent microscopes.

This research was supported by Japan Science and Technology Agency (JST) CREST Grant Number JPMJCR14G2.