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2O1-1 Inter-laminar comparison of inter-areal circuit development in the mouse cerebral cortex Makoto Sato^1,2,3，Yuka Lin¹，Misaki Hattori¹，Sheena YX Tiong¹，Miyuki Doi¹，Tokuichi Iguchi¹，Yuichiro Oka^1,2 ¹Dept Anat & Neurosci, Grad Sch Med, Osaka Univ，²Div Dev Neurosci, United Grad Sch Child Dev, Osaka Univ，³Res Center for Child Mental Dev Direct ipsilateral connections between functional areas of the cerebral cortex, including long-range connections between different cortical lobes, should play an important role in integrating different sensory inputs with internal status to elicit an appropriate behavior. In mice, long association neurons (LANs) that connect two distant areas can be found in three layers: layers 2/3, 5a, and 6b. However, the developmental processes of, and the functional differences among, the neuronal pathways from these three layers are yet to be understood. To study the developmental dynamics and molecular mechanisms of axonal projection of the LANs in the three layers, it is critical to have a molecular tool to visualize and manipulate the LANs in each layer. By combining retrograde tracing from the primary motor cortex (M1) to the primary somatosensory cortex (S1) and in situ hybridization of the known layer-specific genes, we found that in S1 plxnd1 was expressed in LANs of layers 2/3 and 5a as well as in callosal neurons of layers 2/3, 4, and 5a. We labeled plxnd1-expressing neurons in layer 2/3 with EGFP expression by using the plxnd1 promoter together with our newly developed sparse labeling method based on the Cre-mediated recombination. A single plxnd1-neuron projected ipsilaterally to M1 and contralaterally to S1 on the other hemisphere. In contrast, plxnd1-expressing neurons in layer 5a, if not all, have a subcortical target in addition to those mentioned above. We report initial comparative analysis on the axonal paths from different layers and describe their developmental changes.		2O1-2 The development of screening methods to identify drugs, which can relieve ER stress, using wild-type and mutant serotonin transporter Norio Sakai，Kazusa Katarao，Seiya Murakawa，Masaya Asano，Toshihiko Shirafuji，Izumi Hide，Shigeru Tanaka Dept Mol and Pharmacol Neurosci, Grad Sch Biomed & Health Sci, Hiroshima Univ The function of serotonin transporter (SERT) is regulated by its membrane trafficking. Previously, we clarified the C-terminus-deleted mutant of SERT (SERTΔCT) showed the disturbance of membrane trafficking and subsequent retention at endoplasmic reticulum (ER). In addition, we found that proteasome inhibitor-induced ER stress resulted in the impairment of SERT membrane trafficking and retention of SERT at ER, the feature of which was very similar to that of SERTΔCT. Based on the result that chemical chaperone 4-phnylbutulic acid (4-PBA), which relieves ER stress, accelerated the membrane trafficking and up-regulated the SERT activity, we hypnotized that drugs that facilitates the membrane trafficking of SERT or SERTΔCT would have potential therapeutic effects on ER stress-related disease. In this study, we aimed to develop the simple screening methods for searching such drugs using SERT. We first validated the serotonin uptake assay using fluorescent substrates. This simple and reliable assay method was supposed to be useful for drug screening that affects the wild SERT but not SERTΔCT. In addition, we verified the assay focusing on the aggregate formation of SERTΔCT. The drugs 4-PBA and SKF-10047 facilitated the SERT membrane trafficking and reduced the SERTΔCT aggregates, indicating that these drugs would be potential candidates for ER stress relief. For both assays, we clarified the usefulness of a high content screening microscope. These results could pave the way for high-throughput screening of such drugs.

2O1-3 Mechanosensor function of TRPV2 is sensitized by high temperature region in growth cones, and promotes axonal outgrowth during development Koji Shibasaki¹，Shouta Sugio¹，Kohki Okabe²，Yuko Iwata³，Katsuhiko Ono⁴，Yasuki Ishizaki¹ ¹Dept Mol Cell Neurobiol. Gunma Univ Grad Sch Medicine，²Grad Sch Pharm Sci, The Univ Tokyo，³Dep Clin Res Dev, National Cerebral Cardiovascular Center Res Inst，⁴Dep Biol, Kyoto Pref Univ Medicine We previously reported that TRPV2 is a mechanosensor channel which contributes to axonal outgrowth in membrane stretch dependent manner (J. Neurosci. 2010, JPS 2016, FASEB J. 2017), although TRPV2 was originally cloned as a noxious heat sensor (>52°C). These results indicate that TRPV2 is an important component for the responses against the stretch. In this study, we examined the intracellular temperature distribution during axonal outgrowth by a temperature-imaging method. We found that specific heat spots (2~4°C higher) were occasionally localized in the growth cones. We hypothesized that the higher temperature might accelerate the sensitivity of TRPV2 for mechanical stimuli. Hence, we evaluated the mechanical stimuli-evoked TRPV2 currents at various temperatures by a whole cell patch-clamp recording. Surprisingly, physiological temperature (37°C) was insufficient to sensitize the TRPV2 activation. Unexpectedly, over 39°C condition dramatically accelerates the TRPV2 sensitivity for mechanical stimuli. These results suggest that the hot spots in growth cones contribute to accelerate axonal outgrowth through the TRPV2 sensitization. We compared the axonal outgrowth at 37°C or 39°C culture conditions, and found that the 39°C condition had significantly longer axons compared with 37°C  condition through the TRPV2 activation. To examine specific in vivo roles of TRPV2, we generated motor/sensory neuron-specific TRPV2CKO mice, and analyzed their specific defects. We found that TRPV2CKO mice had abnormal peripheral axons in embryonic primordial fingers. Taken together, the mechanosensor function of TRPV2 is sensitized by high temperature region (2~4°C higher) generated by growth cone movements, and is necessary to form long peripheral axons in embryonic stages.		2O1-4 A septin-mediated synaptic regulation required for spatial discrimination Makoto Kinoshita¹，Yugo Fukazawa²，Haruhiko Bito³，Natsumi Ageta-Ishihara¹ ¹Dept Mol. Biol., Nagoya Univ. Grad. Sch. Sci.，²Dept Neuroanat, Univ of Fukui Sch. Med.，³Dept Neurochemistry, Univ of Tokyo Grad Sch. Med A novel spatial context is recognized by differentiating firing patterns encoding the current unfamiliar surroundings and those of familiar ones. In theory, separation among similar but not identical patterns relies on decorrelation via high-threshold synapses. A representative case is those formed between the perforant path (pp) from the entorhinal cortex and granule cell (GC) dendrites in the hippocampal dentate gyrus (DG). Despite the unique low-firing property and physiological role, the pp-GC synapse is poorly characterized at molecular level. Here we show that mice that lack a pan-neuronal subunit of the septin cytoskeleton pass hippocampus-dependent tasks (e.g., spatial orientation and working memory, associative learning and memory). However, they consistently underperform in specific tasks that require discrimination among distinct spatial contexts (e.g., contextual fear conditioning, non-associative place recognition (NAPR)). AAV vector-mediated, DG neuron-selective supplementation of the subunit restores their performance in NAPR, while the local depletion recapitulates the defects in wild-type mice. Fine morphometry of asymmetric synapses in three major hippocampal regions (pp-GC, mf-CA3, sc-CA1) with ssTEM/3D reconstruction shows normal synapse density, PSD area, and spine volume, but reveals a significant scarcity of spine apparatus (endoplasmic reticulum (ER) in dendritic spines) that is most severe in the pp-GC synapse. Live imaging of the DG neurons reveals significantly infrequent ER entry from dendritic shafts into spines. These and other findings indicate a septin-mediated postsynaptic mechanism required in pp-GC synapses for spatial pattern separation. Molecular/subcellular mechanisms underlying the aberrant ER dynamics are under investigation.