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Yasunori Hayashi, M.D., Ph.D.
Assistant Professor of Neurobiology

Department of Brain and Cognitive Sciences
Building: 46-4243
Lab: Hayashi Lab
*Please note that the Hayashi Lab will be moving to Riken-Japan in Spring 2009.

Synaptic Plasticity and Glutamate Receptor

Topic 1: Molecular Mechanism of Synaptic Plasticity
In the mammalian hippocampus, a brief tetanic stimulation of input fibers leads to a long-term potentiation (LTP) of excitatory synaptic transmission. LTP has been widely studied as a cellular and synaptic model for learning and memory. The LTP induction in hippocampal CA1 region requires post-synaptic NMDA receptor activation and a resultant influx of Ca2+ ions. One consequence of this rise in postsynaptic Ca2+ concentration is to trigger an increase of the transmission mediated by AMPA receptors via an activation of various Ca2+-dependent protein kinases. However, it has not been clear how this increase in transmission is attained. We suggested that an activity-induced translocation of the AMPA receptor from the extrasynaptic location to the postsynaptic site explains an increase in AMPA receptor transmission after LTP induction. We demonstrated this by using GFP and electrophysiologically tagged AMPA receptor molecules. On the other hand, recent application of the yeast two-hybrid screening method disclosed a complex network of protein-protein interaction underlying excitatory synapse. This structural meshwork is not rigid but is dynamically regulated by various stimuli. It is therefore highly likely that the dynamics of AMPA receptors which themselves are embedded in this meshwork are regulated as a part of this dynamism of the meshwork. The principal aim of our research project is to understand the protein-protein interactions regulating AMPA receptor dynamics during LTP. For this purpose, we employ multidisciplinary approaches including biochemical isolation of glutamate receptor binding protein, construction of various mutants of glutamate receptor itself and binding proteins, gene introduction into neurons, and electrophysiological recording of those neurons.

Topic 2: Molecular mechanism spine formation
Spines are major site of excitatory synaptic transmission in hippocampal pyramidal cells. Their peculiar shape was first described as early as 19th century but the knowledge how it is formed and how its number and shapes are regulated are still limited. This is mainly due to a technical difficulty in accessing this structure. However, recent advance in imaging technique, mainly two-photon microscope, enabled direct visualization of spines in living neurons. The number and shape of the spines is regulated during development as well as by synaptic activity. Hippocampal CA1 pyramidal cells are virtually aspiny during the first two postnatal weeks except for filopodial structures, which are much longer and thinner than typical spine structure. After this period, typical spines with head and neck structure start emerging. In addition, tetanic stimulation induces new spines. In order to understand the mechanism involved in spine formation, we express various proteins found in spines and to see if they can form spine by themselves or change the morphology of spines. The spines are visualized with green fluorescent protein (GFP) under two-photon microscope. When GFP is expressed in neuron, it fills cytosol and thus depict the tiny protrusions of the cells including spines as if Golgi staining. Electrophysiological recording and Ca2+ imaging will be combined with GFP imaging to assess functional aspect of spines.

Topic 3: Structural approach to understand postsynapse
To understand the biophysical properties of synaptic protein from different aspect, we started a project to reveal 3D structures of those proteins. We will express various postsynaptic proteins in bacteria in large quantity, purify them, and make crystals. The resultant protein crystal will be analyzed with X-ray defraction. This approach is auxiliary the functional approach described above. For example, once functional unit of a protein is identified the physiological experiment, the relevant fragment will be used for crystallization trials. On the other hand, once we solved the structure and find a motif which seems to have functional significance, we can make mutants for further physiological analyses.

Topic 4: Role of adult neurogenesis in learning and memory
One interesting feature of hippocampus is a continual regeneration of neurons that takes place in adulthood. Placing an animal in a rich environment (for example, provided with a place to hide etc.) increases neurogenesis. The neurogenesis also takes place in olfactory bulb. However, there are no direct proof that the newly generated neurons are actually involved in formation of neuronal circuit and hence in a process of learning and memory. In order to test this, we are currently making a transgenic animal model where neuronal stem cell can be removed by infusion of cytotoxic substance. The resulting animal will be tested for memory paradigms such as Morris water maze task or pregnancy block.

Topic 5: Further application of our knowledge to clinical medicine
Amyotrophic lateral sclerosis (ALS) and motor neuron disease are neurological diseases that affect over 350,000 of the world’s population, and kill over 100,000 every year. In the patients, spinal motor neurons degenerate resulting in a loss of control of muscles and, eventually, in a tragic death due to inability of respiration. Of the total ALS cases, 90% is sporadic and 10% has family history. Some populations of familial ALS cases have mutation in cytosolic Cu/Zn superoxide dismutase, an enzyme that converts free radical oO2 to H2O2 and O2. However, it accounts for only 15-20% of the familial cases and the cause the rest of familial and also most of sporadic cases is not known. We are interested in looking for gene involved in such familial ALS cases which cannot be attributed to SOD mutation. The discovery of a gene responsible for ALS will help to understand the pathogenesis of ALS and motor neuron diseases, or even neurodegenerative disease in general.

Recent original papers

Nishi, M., Hinds, H., Lu, H. P., Kawata, M., and Hayashi, Y. (2001). Motoneuron-specific expression of NR3B, a novel NMDA-type glutamate receptor subunit that works in a dominant-negative manner, J Neurosci 21 , RC185.

Sala, C., Futai, K., Yamamoto, K., Worley, P. F., Hayashi, Y. , Sheng, M. (2003) Inhibition of dendritic spine morphogenesis and synaptic transmission by activity-inducible protein Homer1a. J Neurosci 23 , 6327-6337.

Okamoto, K., Nagai, T., Miyawaki, A. & Hayashi, Y. (2004) Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nat Neurosci 7:1104-1112.

Nakagawa, T., Futai, K., Lashuel, H.A., Lo, I., Okamoto, K., Walz, T., Hayashi, Y. , Sheng, M. Quaternary structure, protein dynamics and synaptic function of SAP97 controlled by L27 domain interactions. Neuron 44:453-467.

Li, Z., Okamoto, K., Hayashi, Y ., and Sheng, M. (2004). The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119, 873-887.

Takao, K., Okamoto, K., Nakagawa, T., Neve, R. L., Nagai, T., Miyawaki, A., Hashikawa, T., Kobayashi, S., and Hayashi, Y. (2005). Visualization of synaptic Ca 2+ /calmodulin-dependent protein kinase II activity in living neurons. J Neurosci 25, 3107-3112.

Fukaya, M., Hayashi, Y. , and Watanabe, M. (2005). NR2 to NR3B subunit switchover of NMDA receptors in early postnatal motoneurons. Eur J Neurosci 21, 1432-1436.

Howarth, M., Takao, K., Hayashi, Y., and Ting, A. Y. (2005) Targeting quantum dots to surface proteins in living cells by using biotin ligase. Proc Natl Acad Sci 102, 7583-7588

Hayashi, MK, Ames , H., and Hayashi, Y. (2006) Tetrameric hub structure of postsynaptic scaffolding protein Homer. J Neurosci 23, 8492-8501

Okamoto, K. and Hayashi, Y. (2006) Visualization of F-actin and G-actin equilibrium using fluorescence resonance energy transfer (FRET) in cultured cells and neurons in slices. Nature Protocols 1, 912-919 .

Futai, K., Kim, KJ., Hashikawa, T., Sheng, M., Scheiffele, M., Hayashi, Y. (2007) Retrograde modulation of presynaptic release probability through PSD-95-neuroligin mediated signaling. Nat. Neurosci. 10, 186-195.

Okamoto, K.-I., Narayanan, R., Lee, S.-H., Murata, K., Hayashi, Y. The role of CaMKII as an F-actin bundling protein crucial for maintenance of dendritic spine structure. Proceedings of the National Academy of Science USA 104: 6418-6423 (2007).

Niemann S, Landers JE, Churchill MJ, Hosler B, Sapp P, Speed WC, Lahn BT, Kidd KK, Brown RH Jr, Hayashi Y.* Motoneuron-specific NR3B gene: no association with ALS and evidence for a common null allele. Neurology. 2008 Feb 26;70 (9):666-76. Advanced E Publication . 2007 Aug. 8*


Hayashi, Y. Majewska, A. (2005) Dendritic spine geometry: Functional implication and regulation. Neuron 44, 529-532