Research On Bitter Taste Cell-based Sensing Technology And Its Application In Bitter Taste Transduction Mechanism

Taste receptor cells are basic units for taste sensation. There are various specific receptors expressed on cell membrane, which are the targets for different tastants. Studies have shown that bitter, sweet and umami could elicit action potentials from taste receptor cells. In particular, bitter, sweet and umami tastants could activate the G-protein-coupled taste receptors and then elicit the intracellular signaling cascades, among which calcium ions are released from endoplasmic reticulum calcium store and action potentials are generated. In this way, the chemical information from tastants is converted to electrophysiological signals. There are a lot of similarities between the the responses from taste receptor cells and those from taste nerves, indicating that the action potentials from taste receptor cells may be the main component of taste information on taste nerves. Thus the multiple parameters, such as calcium response in signaling pathway and the membrane potentials, reflecting the characteristics of taste cellular responses to taste stimuli, can be employed in taste coding research. At present, little is known about firing patterns and mechanism underlying interaction between taste receptor cells and bitter taste stimuli. Therefore multiple approaches including molecular biological technologies and cell-based biosensors should be employed to monitor the intracellular and extracellular responses of taste receptor cells in order to investigate the mechanism under the bitter taste sensation.The main innovative content of this paper is listed as follows.1. The extracellular electrophysiological characteristics of taste bud cell were carefully investigated. A taste bud cell-based biosensor was established.Based on the electrophysiological characteristics of taste receptor cells and the development of micro-electromechanical systems (MEMS), we designed a micro-electrode array chip which can simultaneously record the extracellular potential of taste receptor cells with taste stimuli in a non-invasive way. Taste bud cells isolated from mouse taste epithelium were immobilized on the MEA surface. The extracellular potential changes of taste bud cells in response to three bitter stimuli (quinine, cycloheximide and denatonium) were recorded and were discriminated through principle component analysis.2. A germ cell-based bitter biosensor was proposed for the first time. The characteristics of bitter receptor cells in a non-taste tissue were carefully examined.The signaling molecules changes in the taste receptor signaling pathway also reflects the receptor activation. Thus calcium imaging technology was used to monitor the calcium level induced by bitter taste receptor activation. Cell-based impedance sensor was used to monitor the changes in the microenvironment of cell-electrode interface, which also affected by the cellular responses of bitter receptor activation.In this way, taste information from multi-scales was acquired.Bitter receptors in mouse germ cells were functionally characterized by calcium imaging, examining the functional expression of bitter receptor in mouse germ cells. The cell-impedance responses of germ cells to four bitter stimuli (denatonium, PTC, PROP and quinine) were monitored. On this basis, a germ-cell-based taste biosensor was established and was proved effective in bitter detection among five basic tastants.3. A salicin-specific T2R16 bitter receptor cells was built by bioengineering technology. A salicin-specific bitter receptor cell-based biosensor was established combined with cell-impedance sensors.The structural and functional expression of T2R16 in heterologous HEK-293 cells was characterized by immunostainning and calcium imaging. Then the specific T2R16 bitter receptor cell-based biosensor was first designed combined with cell-impedance sensor. The specific detection of T2R16 ligand, salicin, was carried out by this biosensor.4. A hippocampal neuronal network-based taste sensor was designed and established by employing multi-site recording for hippocampal neuronal networks.To further study the signal processing and transmission of bitter information in taste center, we used hippocampal neuronal network as model. The electrophysiological characteristics of neuronal network were preliminarily investigated. The signal transmission mechanism of hippocampal neurons and networks was also analyzed with neurotransmitter treatment. Simulated electrical stimuli originated from bitter responses of taste bud cells were applied to neuronal network in order to monitor and investigate the network response characteristics.Primary hippocampal neurons were cultured on the surface of microelectrode array. With the multi-site recording, the firing characteristics of neurons and networks were investigated intensively. The signal transmission of bitter signal in central taste system was explored preliminarily.