光による神経細胞機能制御 Optical control of neuronal function
S1-1-2-1 オプトジェネティクスを用いた神経細胞内CaMKII活性の操作 Optogenetic manipulation of CaMKII activity in neuron ○村越秀治1,2 ○Hideji Murakoshi1,2 生理学研究所　脳機能計測・支援センター1, 科学技術振興機構さきがけ2 National Institute for Physiological Sciences1, PRESTO, JST, Saitama, Japan2 Dendritic spines, small bulbous postsynaptic compartments emanating from neuronal dendrites, have been thought to serve as basic units of memory storage. In spine, thousands of species of proteins exist, including receptors, channels, scaffolding proteins and signaling enzymes. One of the most important molecules is Ca2+/Calmodulin-dependent kinase II (CaMKII) which is required for long-term potentiation (LTP) and associated spine enlargement underlying learning and memory in the mammalian forebrain. Previously, it has been demonstrated that exogenous CaMKII activity lasts for ~1 min during induction of spine-specific LTP and spine enlargement. Here, to determine the precise duration of endogenous CaMKII activation required for CaMKII-dependent synaptic plasticity, we developed a genetically encoded light-inducible inhibitor of CaMKII activation by fusing autocamtide inhibitory peptide 2 (AIP2) to LOV2 derived from phototropin1. We applied it to the study of structural plasticity of single dendritic spines by using 2-photon glutamate uncaging, and found that ~10 s of CaMKII activation is sufficient for inducing transient spine enlargement, while ~60 s is required for sustained spine enlargement. The different temporal requirement between these two phases must be due to differences in the integration properties of downstream signaling.	S1-1-2-2 Photocontrol of protein function in living cells ○Arnaud Gautier1 École Normale Supérieure, Paris, France1 Our ability to modulate protein activity and observe the effects produced by its intentional activation or inhibition is essential to better understand the function, mechanism of action, and regulation of a specific protein. Light represents a unique external control element as its intensity can be easily controlled with high spatial and temporal resolution. Precise photochemical control of biomolecule function can be achieved through the site-specific introduction of a light-removable protecting group, a so called caging group, which can render a biologically active molecule temporarily inactive by preventing molecular interactions with its biological partners. During this talk, I will present technologies that enable to control protein function with light and I will show how these technologies can be used to precisely control with light cellular signaling pathways.
S1-1-2-3 光活性化蛋白質によるシナプス構造可塑性制御 Regulation of synapse structural plasticity using photoactivatable signaling proteins ○実吉岳郎1, 林康紀1 ○Takeo Saneyoshi1, Yasunori Hayashi1 理化学研究所　脳科学総合研究センター 1 RIKEN Brain Science Institute1 The structure of dendritic spines is dynamically regulated by plastic changes at the synapse. The size of a spine correlates with the strength of synaptic transmission. However, the mechanism by which induction of synaptic plasticity leads to changes in spine structure and subsequent maintenance of long-term structural alterations are not yet fully understood. Synaptic activation can initiate intracellular signaling cascades that can lead to modifications of spine structure. However, traditional pharmacological or genetic approaches do not have the required spatiotemporal resolution necessary to examine the exact role of each molecule within the signaling cascade. With photoactivatable (PA)-molecule, we could isolate the role of specific molecules within a network of multiple parallel signaling cascades present in a single spine. We choose CaMKII and Rac as model to investigate their roles in structural plasticity using PA-molecule. CaMKII plays pivotal roles in various type of neuronal plasticity. Inhibition of CaMKII activity impairs functional and structural synaptic plasticity. To investigate the roles of CaM-KII in complex signaling cascades, we developed PA-CaMKII. Using PA-CaMKII under 2-photon microscopy, local activation of CaMKII within a single spine induced spine enlargement. The PA-CaMKII could reconstitute the CaMKII functions in a space and time in a single spine. Rac is known as important player for actin dynamics in variety of cellular systems including neuron. We examined the role of Rac in synapse structural plasticity. Rac inhibitor blocked 2-photon glutamate uncaging-induced structural plasticity. Activation of Rac by PA-Rac induced spine enlargement, which was blocked by PAK inhibitor. Moreover, Rac activation induced actin polymerization in a spine by means of fluorescence lifetime imaging microscopy (FLIM)-FRET. Thus, these data indicate that Rac regulates structural plasticity through activating PAK and subsequent actin polymerization.	S1-1-2-4 新規シナプス光感受性プローブを用いた“Synaptic Optogenetics”の開発 AS-PA (Activated Synapse targeting PhotoActivatable) probe: a novel synaptic probe for "Synaptic Optogenetics" ○林（高木）朗子1,2 ○Akiko Hayashi-Takagi1,2 東京大学大学院医学系研究科・疾患生命工学センター・構造生理部門1, 科学技術振興機構、さきがけ「脳神経回路の形成・動作と制御」領域2 Lab of Structural Physiology, Univ of Tokyo, Tokyo1, PRESTO, JST, Kawaguchi, Japan2 Optogenetics is a powerful tool to control the action potential of neuron. However, a distinct subset of synapses are activated in a different context, indicating that not only an entire neuron level, but also a synaptic level of photo-manipulation would be a complementary tool to elucidate the nature of neural ensemble. For this aim, namely "Synaptic Optogenetics", we here developed a novel probe that is targeted only in the recently enlarged spine (AS-PaRac1: Activated Synapse targeting PhotoActivatable Rac1) to manipulate the structure/function of the dendritic spine in the active circuit. Activity-specificity was confirmed as follows: (1) AS-PaRac1 is strongly co-localized with SEP-GluA1, a marker for AMPA receptor exocytosis. (2) A single spine LTP protocol by glutamate uncaging induced the recruitment of AS-PaRac1 in the stimulated spine. (3) Motor learning induced AS-PaRac1 accumulation in the M1 cortex, and the extent of spine enlargement and the increase in the fluorescence intensity of AS-PaRac1 after learning was highly correlated. Taken together, we concluded that AS-PaRac1 is a reliable marker of recently activated synapse. Of note, in vivo photoactivation induced spine shrinkage only for the AS-PaRac1 containing spines, while spines without AS-PaRac1 were intact. Finally, mice were subjected to RotaRod motor learning, followed by photostimulation over M1 cortex to elicit the shrinkage of newly generated or enlarged spine during learning. Mice, which was injected with AAV containing AS-PaRac1, displayed disrupted learning after photostimulation, while general activity was not affected, suggesting the spines delineated by AS-PaRac1 spines were necessary structures of the learning engram. This novel approach, Synaptic Optogenetics, combined with various tasks could be a powerful tool to uncover the causal relationship between the synapse and behavioral manifestations.
S1-1-2-5 シナプス可塑性の制御技術の開発 Development of the technology for controlling synaptic plasticity ○松田信爾1,2, 柚崎通介1 ○Shinji Matsuda1,2, Michisuke Yuzaki1 慶應義塾大学　医学部生理学教室1, 科学技術振興機構2 Department of Physiology, Keio University School of Medicine1, JST, PRESTO2 Synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD), is believed to underlie learning and memory processes. However, whether and how synaptic plasticity at specific synapses is related to learning and learning process in vivo remains largely unclear. To address this fundamental question, development of technologies that could regulate synaptic plasticity in a temporally and spatially controlled manner is desired. While recently developed optogenetic tools can be used to induce or inhibit the generation of action potentials in specific neurons, they cannot directly control synaptic plasticity. It has become recently clear that synaptic plasticity is largely mediated by neuronal activity-dependent trafficking of postsynaptic AMPA-type glutamate receptors (AMPA receptors). For example, during LTD induction, AMPA receptors are removed from the postsynaptic membrane by clathrin mediated endocytosis and transported to the lysosomes. On the other hand, LTP is induced by AMPA receptor exocytosis. Therefore, we hypothesized that synaptic plasticity could be regulated by optogenetic modification of these processes. Recently, we have developed the technique to control the function of intracellular organelles that play critical roles for the intracellular trafficking of AMPA receptors. We would like to introduce our new technique in this symposium.	S1-1-2-6 オプトジェネティクスを用いた本能行動調節機構の解明 Optogenetical approach to reveal regulatory mechanism of instinctive behaviors ○山中章弘1, 常松友美1 ○Akihiro Yamanaka1, Tomomi Tsunematsu1 名古屋大学環境医学研究所神経系分野Ⅱ1 Research Institute of Environmental Medicine, Nagoya University1 Instinctive behaviors, such as feeding and drinking behaviors, sleep/wakefulness and sexual behavior, are important to survive or to keep species. These behaviors are regulated by the neurons in the hypothalamus. Some neurons in the hypothalamus contain neuropeptides and release them as neurotransmitter. Recent studies revealed that these neuropeptides have a crucial role in the regulation of instinctive behaviors. In spite of its physiological and biological importance, little is known about the regulatory mechanism of instinctive behaviors since these behaviors are only exhibited in the whole animal. Optogenetics is a powerful too to study regulatory mechanism of these behaviors in vivo. We generated new transgenic mice use tetracycline gene expression system which enable express channelrhodopsin2 (ChR2) or Archaerhodopsin-3 (ArchT) in the specific type of cells expressing tTA. We generated tTA mice lines which specifically express tTA in the peptide containing neurons in the hypothalamus using its promoter, such as prepro-orexin promoter or melanin concentrating hormone promoter. Immunohistochemical studies revealed that ChR2 or ArchT is exclusively expressed in these neurons. And slice patch clamp studies confirmed that ChR2 and ArchT correctly function in the transgenic mice. We controlled instinctive behaviors by manipulating the activity of peptide containing neurons by illuminating light into the hypothalamus. In this symposium, I will discuss physiological importance of the activity of these neurons in the regulation of instinctive behavior using optogenetics.

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