Analysis of a ubiquitin ligase SCRAPPER with imaging mass spectrometry

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Abstracts

It is currently unclear how odor inputs are represented in higher association centers of the brain. We have used the simple olfactory system of Drosophila larvae to map the pathways linking identified olfactory sensory neurons (OSNs) to the input region (calyx) of the mushroom bodies, brain centers essential in associative olfactory learning. We mapped OSN projections in individual antennal lobe (AL) glomeruli, and then the stereotypic projections of single projection neurons (PNs) in AL and calyx glomeruli, thus generating the first comprehensive map of odor representation in a higher associative center. Stimulation of single OSNs evoked strong neuronal activity in one to three calyx glomeruli, showing limited divergence of odor representations when passing from OSNs to calyx glomeruli. Stereotypic representation of OSN input in calyx glomeruli provides a mechanism for MB neurons to detect and discriminate olfactory cues. doi:10.1016/j.neures.2009.09.245

O3-J4-1 Spatiotemporal activity of respiratory neurons in and near the phrenic nucleus Yoshio Shinozaki 1,4 , Shigefumi Yokota 2 , Fumikazu Miwakeichi 3 , Ryoma Aoyama 4 , Yoshitaka Oku 5 , Yasumasa Okada 6 1

Department Orthopaed. Surg, Hino Municipal Hosp, Tokyo, Japan; Department Anat. & Morphol. Neurosci, Shimane University, Izumo, Japan; 3 Grad. Sch. Eng., Chiba University, Chiba; 4 Department Orthopaed. Surg. Keio University, Tokyo, Japan; 5 Department Physiol. Hyogo. Col. Med., Nishinomiya, Japan; 6 Department Med. Keio University Tsukigase Rehab. Ctr., Izu, Japan 2

We analyzed spatiotemporal profiles of respiratory neuron activity in and near the phrenic nucleus by voltage-imaging. Two populations with inspiratory activity were recorded on the transverse plane at C4–C5 level and on the ventral surface at C3–C5 level of the spinal cord in the en bloc brainstem-spinal cord. Activity of one group rapidly emerged in the mid-ventral (MV) portion of the ventral horn, and the other appeared in the ventromedial (VM) portion. We also analyzed anatomical distributions of respiratory-related motoneurons by retrograde DiI-tracing. Distributions of DiI-labeled phrenic and scalene motoneurons coincided with optically detected MV and VM regions, respectively. Phrenic and scalene motoneurons have closely-related but distinct spatiotemporal activities, and together generate inspiratory output pattern. doi:10.1016/j.neures.2009.09.246

O3-J4-2 A screening of genes related to neuronal polarity or vesicle distribution by novel gene trap system Kazuhiro Muramatsu 1 , Takashi Sato 2 , Yukiko Hashimoto 2 , Yasumasa Ishida 3 , Hirokazu Arakawa 1 , Akihiro Harada 2 1

Department Pediatrics, Gunma University Grad. School of Medicine, Japan; Inst. for Mol. and Cell Regulation, Gunma University, Japan; 3 NARA Inst. of Sci. and Tech., Japan

2

We identified unknown important genes related to the polarity of neuron and the distribution and transportation of Synaptic vesicle (SV) using new gene trap method. In this system, retrovirus vector is inserted into genome of PC12 cells by infection. It is designed to inactivate the gene at the inserted locus. We can easily identify trapped gene locus using inserted vector. In addition, we can obtain revertant clones easily by Cre-loxP system to confirm whether the phenotype is caused by the trapped gene. We chose clones with obvious morphological changes, and showing changes in biogenesis or distribution of SV or secretory granules. We identified an inserted locus by 5 RACE and inverse PCR. Molecules with unknown functions were identified. One of them may be related to the function of dopaminergic neuron.Having identified novel genes by this system in PC12, we expect that many unknown genes will be identified by this system in any cell lines. doi:10.1016/j.neures.2009.09.247

O3-J4-3 Analysis of a ubiquitin ligase SCRAPPER with imaging mass spectrometry Ikuko Yao 1,2 , Yuki Sugiura 2,3,4 , Mitsutoshi Setou 2,3 1

Kansai Medical University, Osaka, Japan; 2 Mitsubishi Kagaku Inst. of Life Sciences, Tokyo, Japan; 3 Hamamatsu Med. University, Hamamatsu, Japan; 4 Tokyo Inst. of Technology, Yokohama, Japan Focused on protein degradation system, we identified an E3 ligase that we named SCRAPPER. SCRAPPER is an ubiquitin E3 ligase that is involved in decomposition of RIM (Rab3-interacting molecule) 1, an important regulator of synaptic plasticity, and thus regulates synaptic transmissions (Yao et al., Cell 2007). We applied imag-

ing mass spectrometry (IMS) for Scrapper-gene knockout (SCR-KO) mice brain for the comparison between wild-type mice brain. Moreover, we employed principal component analysis (PCA) along with multivariate analysis to identify protein alterations between samples. As a result, we could find numerous alterations in the KO mouse brain. Furthermore, we could also know the information on the position of altered substances all together. By application of in situ proteomics with IMS and PCA, it will be easier to analyze the hylic alterations in mutant mice. In addition, this technique may be useful for not only analyses of mutant mice but also those of human diseases. doi:10.1016/j.neures.2009.09.248

O3-J4-4 Recombinant rabies virus vectors Takuma Mori, Edward M. Callaway Salk Institute, United States We have developed recombinant rabies viruses that can express various transgenes and are therefore useful for linking together cell types, connectivity, and function. By introducing the coding sequence for other genes (than GFP) into the genome of the glycoprotein-deleted rabies virus, we have created additional vectors with further utility. New recombinant rabies viruses include one encoding an epitope-tagged red fluorescent protein mCherry-myc (SAD-G-mCherry-myc), and another encoding the troponin C based calcium biosensor TN-XL (SAD-G-TN-XL). Both viruses can be used as described previously for GFP expressing virus, to label specific types of projection neurons. The SAD-G-mCherry-myc virus has the additional advantage that it can be used in combination with GFP expressing transgenic mice or viral vectors. The SAD-G-TN-XL virus can be used not only to visualize the morphology of neurons as finely as with SAD-G-GFP, but it would also allow optical monitoring of the activity of specific projection neurons or interconnected populations. These new rabies viral vectors will therefore enable us to understand morphological and physiological aspects of neural circuits at a finer scale. doi:10.1016/j.neures.2009.09.249

O3-J4-5 Light-driven control of receptor tyrosine kinase signaling Masayuki Isa, Yusaku Ohta, Shigeyuki Namiki, Kenzo Hirose Department Neurobiol, Tokyo University Grad. Sch. Med., Tokyo, Japan Receptor tyrosine kinase (RTK) plays important roles in a nervous system. However, physiological roles of each RTKs subtype largely remain unclear. Here we developed a system which can subtype-specifically activate RTK signaling. We designed chimeric receptor constructs by replacing the extracellular domain of PDGF receptor ␤ subtype (PDGFR-␤) with an anti-fluorescein single-chain antibody. In this system, we expected that multiply fluorescein-labeled BSA (BSA-FL) triggers PDGFR-␤ specific signal transduction. To test whether this system can work as expected, intracellular Ca2+ response, Cdc42 activation and cell migration were examined in NIH3T3 cells expressing the chimeric receptor; all the three tested responses were observed upon BSA-FL application. Furthermore, UV irradiation following application of caged-fluorescein-labeled BSA elicited increase in intracellular Ca2+ concentrations. The result indicates that our strategy enables spatially controlled activation of specific RTK signaling by light. doi:10.1016/j.neures.2009.09.250

O3-J4-6 Transgenic zebrafish expressing ChR2: a useful tool to analyze neural circuit for motor behaviors Tamami Yamamoto, Kohei Hatta Grad. Sch. of Life Science, University of Hyogo, Hyogo, Japan Identification of the role of each neuron and muscle in animal behaviors is one of the goals of neuroscience, still difficult to achieve. To solve this, we are using optogenetic approach in zebrafish, a simple vertebrate model to study neural development and function. Channel Rhodopsin 2 (ChR2), an opsin originally isolated from unicellular green algae Chlamydomonas reinhardii, functions as light-gated non-selective cation channels. Here we created transgenic zebrafish lines in which ChR2 is expressed in neurons and/or muscles by using Gal4/UAS system. When blue light is applied on the brain or spinal cord, different types of motor behaviors are induced such as escape and swimming, as well as abnormal behaviors including bilateral twitching and shivering. These results indicate that these transgenic fish are useful for analyzing neural circuits that control motor behaviors. We hope to identify neurons that are responsible in creating different patterns of behaviors at single cell resolution. doi:10.1016/j.neures.2009.09.251