Members
Hiromu Yawo (Neuron Network)
Dr. Hiromu Yawo graduated Kyoto University Graduate School of Medicine in 1981, being given PhD under the thesis ¡ÈHow a nerve fiber repairs its cut end: involvement of phospholipase A2.¡É He thereafter collaborated with Prof. Motoy Kuno in Kyoto University as an instructor of physiology. In 1985 he received an award from the Center for Cellular and Molecular Neurobiology in Washington University School of Medicine in St. Louis, USA (McDonnell Research Fellowship). Under collaboration with Prof. Dale Purves he started studies on the neuronal network plasticity. In 1995 he became a professor of Tohoku University School of Medicine. He has been a professor of Tohoku University Graduate School of Life Sciences since 2001. He organized a research team investigating ¡ÈPresynaptic mechanisms of learning and memory¡É as a Director of the Program of Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST) from 1999 to 2005. His major aim of research is to understand the molecular and cellular mechanisms underlying the neuronal network plasticity.
- Introduction of Research
-
The brain consists of many types of neurons which make a complex network. This idea was first proposed by Ramon y Cajal more than 100 years ago. However, it is still unresolved how the network activities are integrated into the brain¡Çs function, the mind. Using genetic engineering techniques we have developed new optical recording methods which visualizes the network activities (Araki et al. 2005). We plan to manipulate activities of individual neurons by light as well as to record them optically, and to reveal how they are integrated in the network. The neuronal network is also dynamically regulated by its environment as well as by its activity itself. We will investigate the cellular and molecular mechanisms regulating the network dynamics.
In the neuronal network of the central nervous system (CNS) the synaptic transmission is regulated by the vesicle exocytosis and endocytosis. This vesicular dynamics is associated with the changes of intra-vesicular pH and can be visualized by the fluorescence of synaptopHluorin (SpH), a pH-sensitive GFP fused to the lumenal aspect of VAMP-2. We have generated several SpH transgenic mouse lines in which the SpH expression is regulated by the neuron specific Thy-1.2 promoter or Cre/loxP recombination system. In one of them SpH was specifically expressed in the mossy fiber (MF) terminals of the hippocampus. Recently we found that there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool and that the fidelity of synaptic transmission is ensured by the rapid supply from the reserve subpopulation of releasable pool (Suyama et al. 2007).
During phototaxic and photophobic movements of unicellular green algae, light is perceived by archaeal-type rhodopsins which are localized in small regions of the plasma membrane, called eyespots. Two rhodopsins were isolated from Chlamydomonas reinhardii, channelrhodopsin (ChR) 1 and 2. ChR2 has a peak light absorbance at 460 nm and forms a non-selective cation channel, the gating of which is triggered by the photoisomerization of the all-trans retinal to 13-cis configuration. We expressed ChR2 exogenously in the hippocampal neurons of a living mouse (Ishizuka et al. 2006). A brief illumination by a blue LED light depolarized these neurons over threshold to evoke action potential which is phase-locked with the light pulses.
With its high resolution in space and time, its large dynamic range and its convenience this photostimulation method would fulfill all the requirements for artificial stimulation of neurons, namely generality, speed, localization and parallelism. Since ChR2 is relatively small and encoded in a single gene, it could be expressed in a specific subset of neurons under regulations of cell-type-specific promoters. It would, thus, open many potential applications, for both in vitro and in vivo studies of neuronal network, artificial manipulations of neuronal activity for the development of informational modules and possibly the development of non-invasive therapeutic instruments of bypassing interrupted neuronal connections.
Retinitis pigmentosa (RP) refers to a group of diseases in which a gene mutation results in death of rod photoreceptors followed by gradual death of cones. Approximately 1 in 4,000 people are affected by this disease. Symptoms include night blindness, loss of the peripheral visual field and of central vision. Although photoreceptor cells are almost degenerated in the eyes of RP patients, other retinal neurons including retinal ganglion cells (RGC) are preserved. These remaining neurons are possibly made photosensitive by genetically engineering to express ChR2. When ChR2 was expressed in the retina of the aged Royal College of Surgeons (RCS) rat, one of classic model animals of recessively inherited RP, some of visual responses were shown to be restored (Tomita et al. 2007). It is thus suggested that the ChR2 transduction method would provide a new strategy to treat some RP symptoms.
Optical analysis of synaptic acitivities using synaptopHluorin
We have generated a transgenic mouse which express synaptopHluorin (SpH, green), a optical probe reporting vesicular dynamics, selectively in the mossy fiber terminals of the hippocampus. Neurons are also stained their nuclei (DAPI, blue). The repetitive stimulation of mossy fiber axons increases the SpH fluorescence in the individual presynaptic terminals (insets left and middle). This change is shown in the difference image (inset right, pseudocolor ratings).
Photostimulation of neurons
Top, left: The hypothetical structure of channelrhodopsin 2 (ChR2, 1-315) which is tagged with Venus, one of GFP derivatives, at its C-terminus.
Bottom, left: ChR2 has a peak light absorbance at 460 nm and forms a non-selective cation channel, the gating of which is triggered by the photoisomerization of the all-trans retinal to 13-cis configuration.
Top, right: The dentate granule cells expressing ChR2-Venus in the hippocampus of a living mouse.
Bottom, right: Induction of patterned action potentials by blue LED light pulses (blue circles).
Spatial learning of a mouse
Morris water maze is one of current spatial learning tasks of rodents. Since the submerged platform is invisible for the mouse, its place has to be learned in relation to the around cues. This mouse had swimming tasks 3 times a day for 5 days, and learned the place of platform. The plasticity of neuronal networks in the hippocampus is involved in the establishment of spatial learning. - Articles
-
- "1. Suyama, S, Hikima, T., Sakagami, H., Ishizuka, T, Yawo, H. (2007). Synaptic vesicle dynamics in the mossy fiber-CA3 presynaptic terminals of mouse hippocampus. Neuroscience Research 59, 481-490. "
- 2. Tomita, H., Sugano, E., Yawo, H., Ishizuka, T., Isago, H., Narikawa, S., Ku¡¯gler, S., Tamai, M. (2007). Visual responses in aged dystrophic RCS rats were restored by AAV-mediated channelopsin-2 gene transfer. Invest Ophthalmol Vis Sci.¡¡ 48, 3821-3826.
- 3. Ishizuka, T., Kakuda, M., Araki, R., Yawo, H. (2006). Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels. Neuroscience Research 54, 85-94.
- 4. Araki, R., Sakagami, H., Yanagawa, Y., Hikima, T., Ishizuka, T., Yawo, H. (2005). Transgenic mouse lines expressing synaptopHluorin in hippocampus and cerebellar cortex. Genesis 42, 53-60.
- 5. Kamada, M., Li, R.-Y., Hashimoto, M., Kakuda, M., Okada, H., Koyanagi, Y., Ishizuka, T., Yawo, H. (2004). Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus. European Journal of Neuroscience 20, 2499-2508.