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386 Abstracts / Neuroscience R

3-i13 A novel membrane protein #123 specificallyxpressed in olfactory sensory neuronsomomi Kaneko-Goto 1 , Yuki Sato 1, Sayako Katada 1,2, Sei-ichioshihara 1, Atsushi Nishiyori 1, Mitsuhiro Kimura 1, Kazushigeouhara 2, Randall R. Reed 3, Yoshihiro Yoshihara 1

Lab. Neurobiology of Synapse, RIKEN BSI, Wako, Japan 2 Grad. School ofgri Life Sci, University of Tokyo, Tokyo 3 Center for Sensory Biology, Johnsopkins Sch. of Med, MD, USA

embrane proteins play important roles in various aspects of cellularunctions such as intercellular communication, signal transduction, andntracellular trafficking. To make a comprehensive survey of secreted and

embrane proteins expressed in the olfactory sensory neurons, we per-ormed a signal sequence trap screening in yeasts. From a cDNA library ofhe mouse olfactory epithelium at postnatal day 3, we discovered twelveovel molecules with a canonical signal sequence at the N-terminus. Amonghem, we focused on the clone #123 because of its specific and abundantxpression in the olfactory epithelium as assessed by Northern blot analysis.123 cDNA encodes a type I integral membrane protein with no signifi-ant homology to any other proteins. In situ hybridization analysis revealedhat #123 mRNA is specifically expressed in the olfactory and vomeronasalensory neurons. This tightly regulated expression was confirmed by the123 gene enhancer/promoter analysis in transgenic mice. #123 mRNA

s first detectable at embryonic day 11.5 in the olfactory epithelium andt embryonic day 13.5 in the vomeronasal organ, much earlier than thensets of olfactory marker protein in these epithelia. The expression of #123RNA increases toward birth and a high level of expression persist into

dulthood. Immunohistochemical analysis revealed that #123 protein is pre-ominantly localized to the Golgi apparatus in both immature and matureensory neurons. To clarify the physiological function of this unique protein,e generated gene-targeted null mutant mice of #123. The #123-deficientice are viable and fertile, but exhibit abnormalities in protein localization,

ellular morphology, and odor responses of the olfactory sensory neurons.hese results suggest that #123 protein is required for the proper signalransduction and physiological function of the olfactory sensory neurons.

oi:10.1016/j.neures.2010.07.1707

3-i14 Alkaline pH sensation mediated by a Caenorhabditislegans transmembrane guanylyl cyclaseakashi Murayama 1 , Mayuki Fujiwara 1, Jun Takayama 2, Ichiroaruyama 1

Information Processing Biology Unit, Okinawa Institute of Science andechnology, Okinawa, Japan 2 Department of Biophysics and Biochemistry,raduate School of Science, The University of Tokyo, Tokyo, Japan

nimals can survive in a narrow pH range by monitoring the pH in theirnvironment and body fluids. However, little is known about how animalsncluding humans detect extracellular alkaline pH although channels detectntracellular alkaline pH have recently been found. The nematode Caenorhab-itis elegans (C. elegans) is attracted to alkaline pH lower than pH 10.4. Tonvestigate cellular and molecular modes underlying this chemosensation,

e have used an agar plate assay with a linear pH gradient. Along the pHradient from pH 6.8 to pH 8.5, wild-type worms were attracted to higher pHegions, whereas mutants defective in ASE chemosensory neurons failed too so. By imaging of membrane voltage changes using the voltage-sensitiveuorescent protein Mermaid, furthermore, we have found that a pair of ASEeurons sense alkaline pH up to pH 10.0. ASE-left (ASEL) is activated by pHp-shift, and ASE-right (ASER) is activated and inactivated by pH down- andp-shift, respectively. Through phenotypic analysis of mutants, we also foundhat a transmembrane guanylyl cyclase, GCY-14, and cGMP-gated cationhannel proteins, TAX-2 and TAX-4, are required for the alkaline pH sensationn ASEL. As expected, GFP-tagged GCY-14 was localized to ASEL sensory cilia,nd in the gcy-14 mutant, ASEL did not respond to pH up-shift. While ASI andSK sensory neurons did not respond to pH up-shift, furthermore, ASI andSK neurons ectopically expressing GCY-14 were activated by pH up-shift

rom pH 7 to pH10. This indicates that GCY-14 is sufficient for the alkalineH sensation. Indeed, point mutations of conserved cysteines on the extra-ellular domain of GCY-14 could not rescue the chemotaxis defect of gcy-14.

hese results indicate that the transmembrane guanylyl cyclase GCY-14 actss an alkaline pH sensor, and an increased concentration of cGMP opens theGMP-gated cation channel TAX-2/TAX-4 for the activation of ASEL.

oi:10.1016/j.neures.2010.07.1708

ch 68S (2010) e335–e446

P3-i15 Tbr2 is required for proper differentiation ofmitral/tufted cells in the mouse olfactory bulbRumiko Mizuguchi , Yoshihiro YoshiharaLab Neurobiology of Synapse, RIKEN BSI, Wako, Japan

Tbr2 (Eomes) is a member of the T-box transcription factor family that isknown to regulate various aspects of differentiation of both neurons andhematopoietic cells. In the mouse olfactory bulb, Tbr2 and its two relatedmembers, Tbr1 and Tbx21, are expressed in the projection neurons, mitraland tufted cells, in an overlapping and differential manner. Their expres-sions dynamically change during development, implying their functionsin those neurons. Because Tbr2-null mice die at early embryonic stagesdue to defects in trophoectoderm differentiation, we generated conditionalTbr2-knockout mice using Cre/loxP system in which Tbr2 gene is deletedspecifically in mitral/tufted cells. These mice are viable and fertile, and showno obvious abnormality in the gross morphology of the brain includingthe olfactory bulb. Immunohistochemistry and in situ hybridization analysisrevealed that the loss of Tbr2 in mitral/tufted cells leads to the upregula-tion of Tbr1 and VGLUT2 in a cell-autonomous manner. Concomitantly, weobserved anatomical disorganization of the external plexiform layer, wheresecondary dendrites of mitral/tufted cells make connections with varioustypes of interneurons, indicating that dendritic morphology and/or neuronalconnectivity patterns of the mitral/tufted cells are altered in conditionalTbr2-knockout mice. Severer phenotypes were observed in the tufted cellsthan in the mitral cells, implicating functional redundancy among T-box fam-ily members in the mitral cells. These results suggest that there are at leasttwo types of tufted cells, whose molecular and morphological properties aredifferent, and that the loss of Tbr2 results in the unbalanced differentiation ofthe tufted cell subtypes. Detailed molecular and anatomical characterizationand elucidation of functional roles of those tufted cells are currently underinvestigation.

doi:10.1016/j.neures.2010.07.1709

P3-i16 Axonal projection of mitral and tufted cells in theventral zone of olfactory bulbMyungho An , Kei Igarashi, Nao Ieki, Kensaku MoriDepartment Physiol, Univ of Tokyo, Tokyo

The glomerular sheet of the olfactory bulb (OB) forms odorant receptor maps.Recent studies demonstrate that glomeruli in the dorsal (D) zone of the OBare required for innate aversive response to predator odors. On the otherhand, glomeruli in the ventral (V) zone of the OB are thought to be associatedwith food odor information processing. Here we report the axonal projec-tion pattern to the olfactory cortex (OC) of individual mitral/tufted (M/T)cells associated with V-zone glomeruli and responding to Ester molecule (acomponent of food odor). Ester-responsive M/T cells were juxtacellularlylabeled and then labeled axons were histochemically visualized and 3D-reconstructed. The results showed that Mitral cell extended axon collateralsto multiple areas of the OC, including the anterior olfactory nucleus (AON),piriform cortex (PC), olfactory tubercle (OT), tenia tecta (TT), amygdaloidcortex, and lateral entorhinal cortex, while axon collaterals of Tufted cellprojected to anterior region of OC, including AON, anterior piriform cortex(APC), OT, TT. Especially, axon collaterals of tufted cell formed high-densityclusters in the ventral part of TT. These results provide initial steps towardunderstanding how the food odor signals are sent to specific regions of theOC to induce feeding behavioral responses.

doi:10.1016/j.neures.2010.07.1710

P3-i18 Effects of menthol on food intake and intestine ingoldfishTsuneo Hirochi 1 , Kosuke Kamitahira 2, Masanori Kasai 1

1 Graduate School of Science and Engneering, Kagoshima University2 Department of Chemistry and Bioscience, Faculty of Science, KagoshimaUniversity, Kagoshima, Japan

Menthol is widely used in food, cigarettes and pharmaceutical industry. Itinduces cold and pain sensations in subjects. Application of relatively low

doses of menthol or icilin is associated with a cooling sensation or pain relief,while at higher doses, menthol causes burning, irritation, and pain. Men-thol activates TRPM8 or TRPA1 receptors which is a member of the transientreceptor potential (TRP) channel family that causes cold sensation or coldpain. We showed that exposure to menthol decreased food intake followed

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Abstracts / Neuroscience R

y vomiting in goldfish. To know the action site of the menthol effect, gold-sh was applied by food pellets which contained menthol and the effect ofenthol on contractile responses was observed in isolated goldfish intestine

reparations. The intestine was surgically isolated from goldfish. Intesti-al movement was recorded as changes in isotonic tension.Food intake was

nhibited when goldfish ate menthol containing pellet and the pellet inducedvomiting 10 min later after eating. Menthol (0.1 mM) induced an inhibitionf spontaneous contraction of intestine but not affect to intestinal movement.igher concentration of menthol (1.0 mM) induced the inhibition of sponta-eous contraction and a contractile response on intestine. The result suggestshat vomiting by menthol may be due to direct action to the intestine.

oi:10.1016/j.neures.2010.07.1712

3-i19 Territory of gustatory area in the rat thalamusaruki Iwai , Takahiro Sonomura, Atsushi Yamanaka, Masanoriemura

Department of Anatomy for Oral Sciences, Kagoshima University, Kagoshima,apan

rojection fibers from the parvicellular part of the posteromedial ventralhalamic nucleus (VPMpc), a gustatory relay nucleus, have been shown toerminate in the insular cortex (IC), a ventral part of the caudate putamenvCPu), and the central nucleus of the amygdala (CeA) in the previous studyNakashima et al., 2000). Since this study has indicated that projection neu-ons to the vCPu and CeA were widely distributed in the thalamic area medialo the VPMpc as well as the VPMpc, the medial thalamic area raised the pos-ibility that relayed to gustation. In the present study, an anterograde tracerbiotinylated dextran amine) was injected into the gustatory parabrachialrea (GPB) of the rat’s gustatory relay area, to ascertain whether the thala-ic area medial to the VPMpc concerns with a gustatory relay. This terminal

abeling from the PBG was observed in the VPMpc and thalamic area medial tohe VPMpc bilaterally. Hense, the thalamic area medial to the VPMpc wouldoncern with a gustatory relay, receive gustatory information from the PBG,nd send it to the IC, vCPu, and CeA, similarly to the VPMpc.

oi:10.1016/j.neures.2010.07.1713

3-i20 Functional difference between global presynapticnd postsynaptic inhibition in the Drosophila olfactory cir-uitasafumi Oizumi 1 , Ryota Satoh 1, Hokto Kazama 2, Masatokada 1,3

The University of Tokyo, Chiba 2 Department of Neurobiology, Harvard Med-cal School, Boston, USA 3 RIKEN, Brain Science Institute, Saitama

ecent investigations have indicated that lateral presynaptic inhibitory inputediates gain control in the Drosophila antennal lobe. They have shown

hat the lateral presynaptic inhibitory input to projection neurons (PNs)oughly scales with total feedforward input from olfactory receptor neu-ons (ORNs). This gain control mechanism is considered to be necessary forhe olfactory system to deal with a wide variety of odors. We investigatedow this gain control mechanism contributes to the discriminability of odoresponses of PNs by simulating a network model of the Drosophila antennalobe with actual physiological data. To clarify the functional significance ofpre”synaptic inhibitory gain control mechanism, which indirectly inhibitsNs by regulating the release probability of the synapse between ORNs andNs, we also considered the case that inhibition acts “post”synaptically,hich directly decreases the membrane potential of PNs. Postsynaptic inhi-

ition can similarly control the gain in the sense that it can also properlyegulate the overall firing rates of PNs. We addressed whether there is quali-ative difference between presynaptic inhibition and postsynaptic inhibitionn terms of odor discrimination. We first showed that the presynaptic globalnhibition sharpened the odor tuning of PNs whereas the postsynaptic globalnhibition did not. This means that presynaptic inhibition can reduce theimilarity of mean PN responses to various odors. By applying a classificationechnique developed in machine learning, we next demonstrated that presy-aptic gain control enhanced the accuracy of odor discrimination whereasostsynaptic gain control only diminished it. Our results would provide a rea-on why presynaptic but not postsynaptic gain control exists in the actual

rosophila antennal lobe from the point of view of realizing accurate odoriscrimination.

oi:10.1016/j.neures.2010.07.1714

h 68S (2010) e335–e446 e387

P3-i21 Spatiotemporal characteristics of taste-sensitiveneurons in the rostral nucleus of the solitary tract in theratTatsuko Yokota , Katsunari HirabaDepartment Physiol, Sch. Dent., Aichi-Gakuin University, Nagoya

The rostral nucleus of the solitary tract (rNST), the first-order taste relay,receives spatially organized projections from plural taste nerves. The spatiallocation and temporal firing pattern of taste-sensitive neurons have beenstudied separately in the rat rNST. Here we examined relationships betweenthe temporal characteristics (onset latency and response duration) and local-ization of rNST taste-sensitive neurons. Multi-barrel glass micropipetteswere used to record extracellularly single unit activity under urethane anes-thesia. The recording sites marked by dye spots (ejected from the recordingelectrodes) were reconstructed on the rostrocaudal (RC), mediolateral (ML)and dorsoventral (DV) axes. Taste solutions were applied to the anteriortongue and oral cavity. Forty-five taste-sensitive neurons were classified into17 NaCl (N)-best, 12 NH-best, 10 HCl (H)-best, 5 sucrose (S)-best, 1 SH-bestand 0 quinine (Q)-best. In N-best neurons, the average duration of responsesto NaCl (4.1 s) was significantly longer than that to HCl (1.4 s) (paired t-test,p < 0.01) with no significant difference in onset latency. Most N-best neuronswere observed in the rostral half of rNST. In contrast, in H-best neurons theaverage onset latency (1.2 s) of response was significantly longer to HCl thanto NaCl (0.7 s) (paired t-test, p < 0.05) with no difference in response duration.H-best neurons tended to localize more caudally than N-best neurons, butthe two distributions overlapped extensively. In S-best neurons, the averageonset latency of response was longer to sucrose (1.6 s) than in neurons ofother best taste categories to their best tastes. S-best neurons were mainlyfound in the caudal half of rNST apart from N-best neurons. Distributionsof neurons of the different best tastes did not differ markedly on the ML orDV axis. These results suggest that taste qualities may be represented byspatiotemporal patterns of neuronal activities along the RC axis in rNST.

doi:10.1016/j.neures.2010.07.1715

P3-i22 Fos expression in the brain following microinjec-tion of GABAA receptor agonist muscimol into the centralnucleus of amygdala in sodium depleted ratsQian Wang , Jianqun Yan, Jinrong Li, Xuejuan Yang, Ke Chen,Shiru Zhao, Huiling Sun, Bo SunPhysiology and Pathophysiology, Xi’an Jiaotong University School ofMedicine

Our previous study showed that activating GABAA receptor within the centralnucleus of amygdala (CeA) could inhibit sodium intake in sodium depletedrats. In the present study we examined changes in neuronal activaties ofthe nucleus tractus solitarius (NTS), the parabrachial nuclei (PBN), the par-aventricular hypothalamic nucleus (PVN) and the supraoptic nucleus (SON),which have extensive communications with the CeA and are associated withthe regulation of sodium intake, following bilateral microinjection of musci-mol (GABAA receptor agonist) into the CeA using Fos immunohistochemistry,and identified the possible role of such involved brain regions in regula-tion of sodium intake. All rats were subjected to furosemide (s.c.) combinedwith 24 h sodium deficient diet before microinjection. 90 min after musci-mol(0.20 nmol/0.2 �l) microinjected into the CeA, the number of Fos-likeimmunoreactive (FLI) neurons increased in the caudal and intermediate partof the NTS (cNTS and iNTS) and the lateral PBN (LPBN)(t = 10.42, p < 0.001;t = 5.99, p < 0.001 and t = 12.04, p < 0.001, respectively). A few FLI-positiveneurons were scattered in the PVN and the SON, but had no evident differ-ence between groups. These results suggest that activating GABAA receptorwithin the CeA probably modulate sodium intake, which is partly mediatedby activating the neurons in the cNTS, iNTS and LPBN.

doi:10.1016/j.neures.2010.07.1716

P3-i23 Stereological estimation of the olfactory receptorneurons for the olfactory discrimination of cycloheximideKyutaro Kawagishi 1 , Kumiko Yokouchi 1, Yasuyuki Sekiguchi 2,Akira Kakegawa 1, Nanae Fukushima 1, Tetsuji Moriizumi 1

1 Dept Anatomy, Shinshu Univ, Nagano, Japan 2 Dept Neurosurgery, ShinshuUniv, Nagano, Japan

We have recently determined functionally essential neuronal population(approximately 50% of the normal value) of the unilateral olfactory epithe-