Synaptic plasticity has been suggested to be a cellular counterpart for learning and memory. However, it is practically impossible to visually monitor synapses actually undergoing synaptic plasticity at a given memory paradigm in a given neuronal network. This project proposal, written in response to the program announce- ment """"""""Developing Novel Genetic Methods For Mapping Functional Neuronal Circuits and Synaptic Change"""""""", describes the development of a technology for visualizing synapses that are undergoing synaptic plasticity in neurons in living animal. This goal will be accomplished by a combination of two technologies: fluorescence resonance energy transfer (FRET) and two-photon laser scanning microscopy. We will first develop a FRET-based construct for optically measuring the CaMKII activity and actin polymerization/depolymerization equilibrium, which will allow non-destructive optical detection of CaMKII activation in intact neurons. Since the enzymatic activity of CaMKII is constitutively enhanced after the induction of NMDA receptor-mediated syn- aptic plasticity and this activity is required for maintaining synaptic potentiation, we expect that the activation of CaMKII will be a good indicator of synaptic plasticity. In contrast, in a work which we published, we found actin polymerization/depolymerization equilibrium can be detected with FRET and it follow LTP and LTD respec- tively. To detect FRET at the synaptic structure, we will take advantage of a two-photon microscope. We will then test the feasibility of our strategy by expressing the construct in neurons and induce synaptic plasticity by either high-speed ionophoretic stimulation of individual synapses or local electrical stimulation or combined with detection of structural plasticity. Finally, we will generate a transgenic animal expressing this construct. The barrel cortex of the resultant animal will be observed with the two-photon microscope. Paradigms known to induce synaptic plasticity in this structure such as sensory deprivation will be tested to see whether synaptic plasticity is reflected by FRET. In summary, our technology will provide a unique system for detecting NMDAR-mediated synaptic plas- ticity with spatial resolution at the single synapse level on a sub-second time scale. This technique, in com- bination with technologies currently under development in other laboratories, such as in vivo two-photon im- aging in freely moving animals and deep-structure two-photon microscope imaging with a relay lens, will pro- vide a versatile system for monitoring synaptic plasticity that cannot be achieved with existing experimental systems. In the future, this technique could be applied to higher mammals, such as macaque monkeys, after viral or transgenic introduction of our reporter construct. Itmay be possible to have monkeys perform a task and observe synaptic plasticity in the brain during learning of the task.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MDCN-K (94))
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Wu, Da-Yu
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Institute of Physical and Chemical Research
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Kim, Karam; Saneyoshi, Takeo; Hosokawa, Tomohisa et al. (2016) Interplay of enzymatic and structural functions of CaMKII in long-term potentiation. J Neurochem 139:959-972
Hosokawa, Tomohisa; Mitsushima, Dai; Kaneko, Rina et al. (2015) Stoichiometry and phosphoisotypes of hippocampal AMPA-type glutamate receptor phosphorylation. Neuron 85:60-7
Sakaguchi, Masanori; Kim, Karam; Yu, Lily Mae Yee et al. (2015) Inhibiting the Activity of CA1 Hippocampal Neurons Prevents the Recall of Contextual Fear Memory in Inducible ArchT Transgenic Mice. PLoS One 10:e0130163
Kim, Karam; Lakhanpal, Gurpreet; Lu, Hsiangmin E et al. (2015) A Temporary Gating of Actin Remodeling during Synaptic Plasticity Consists of the Interplay between the Kinase and Structural Functions of CaMKII. Neuron 87:813-26
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Martinez-Lozada, Zila; Waggener, Christopher T; Kim, Karam et al. (2014) Activation of sodium-dependent glutamate transporters regulates the morphological aspects of oligodendrocyte maturation via signaling through calcium/calmodulin-dependent kinase II?'s actin-binding/-stabilizing domain. Glia 62:1543-1558
Ueda, Yoshibumi; Kwok, Showming; Hayashi, Yasunori (2013) Application of FRET probes in the analysis of neuronal plasticity. Front Neural Circuits 7:163
Ueda, Yoshibumi; Hayashi, Yasunori (2013) PIP? regulates spinule formation in dendritic spines during structural long-term potentiation. J Neurosci 33:11040-7
Wang, Dan Ohtan; Matsuno, Hitomi; Ikeda, Shuji et al. (2012) A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes. RNA 18:166-75
Bosch, Miquel; Hayashi, Yasunori (2012) Structural plasticity of dendritic spines. Curr Opin Neurobiol 22:383-8

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