e esear

PtM

SwsmtisssawsiotifTpatopasv

d

PmK

SgtrptppriefitsCgvpbtemn

d

PcT1

2

Rcfl

336 Abstracts / Neuroscience R

3-a04 Formation and elimination of excitatory synapses inhe developing neocortex in vivoasaaki Isshiki , Shigeo Okabe

Department Cellular Neurobiology, University of Tokyo, Tokyo, Japan

pines are specialized dendritic protrusions for excitatory synaptic contactsith nearby target axons. In the mature neocortex, most of the excitatory

ynapses are formed onto spines and few shaft synapses exists. Spines in theature neocortex are thought to be mainly derived from selective stabiliza-

ion of early motile filopodia. This model has multiple supportive evidencesn reduced preparations of dissociated neurons and slice preparations, buthould also be critically evaluated in vivo. Indeed, previous electron micro-copic analyses in the hippocampus showed relative abundance of shaftynapses in the postnatal development and may indicate the presence of anlternative pathway from shaft synapses to spines in vivo. Another aspecthich requires information in vivo is the temporal relationship between

pine formation and the formation of functional synapses. In previous imag-ng studies, there is no consensus in the extent of delay between the initialutgrowth of nascent spines and completion of synapse formation, indicatinghat the time course of synaptogenesis may be sensitive to culture and imag-ng conditions. In this sense, information in time-course of spine/filopodiaormation and maturation of synapses in vivo should be highly valuable.o address these questions, we performed time-lapse two-photon imaging ofyramidal neurons expressing both cytosolic red fluorescent protein (RFP)nd GFP-tagged postsynaptic scaffolding proteins (PSD-95 or Homer1c) inhe layer II/III of developing mouse barrel cortex. There was rapid increasef spine density in the cortical pyramidal neurons during the early postnataleriod and we could successfully visualize development of spine structurend clustering of PSDs in vivo. Analysis of time-lapse sequences indicatedeveral unique features of synapse formation and dendrite remodeling inivo.

oi:10.1016/j.neures.2010.07.1485

3-a05 Preferable binding of a novel synaptic adhesionolecule, LRRTM2 with SAP102

eiichiro Minatohara , Yoshinori Fujiyoshi, Tomoko DoiDept Biophys, Univ of Kyoto, Kyoto

ynaptically localized adhesion molecules, such as neurexins and neuroli-ins, play important roles in the formation, maturation and plasticity ofhe synaptic connection. Our proteomic analyses have found out leucine-ich repeat transmembrane proteins (LRRTMs), highly enriched in theostsynaptic density (PSD) fraction of rat forebrain. LRRTMs have 4 sub-ypes, LRRTM1–4, each composed of an N-terminal extracellular domainossessing 10 leucine-rich repeats, one transmembrane region and a cyto-lasmic domain with the C-terminal PDZ binding motif. Recent studieseveal that LRRTMs bind to presynaptically localized neurexin-1B andnduce synapse formation as neuroligin. To investigate functional differ-nces between neuroligins and LRRTMs in the synapse connection, werst generated antibodies against LRRTMs, and then assessed the localiza-ion of LRRTMs in the brain sections by immunohistochemistry. LRRTM2howed an extensive expression in the stratum lucidum of hippocampusA3, distinct from neuroligin-1. Moreover, LRRTM2 bound to the PSD-95roup proteins of membrane-associated guanylate kinase (MAGUK) familyia the PDZ-2 domain. Particularly, LRRTM2 bound to synapse-associatedrotein102 (SAP102) with a 4-fold higher affinity than that of PSD-95, proveny immunoprecipitation of proteins expressed in HEK-293 cells. Amonghe PSD-95 group proteins, SAP102 is an only postsynaptic scaffold proteinxpressed from a postnatal early period. These results suggest that LRRTM2ay induce synapse formation in the different regions independently from

euroligin-1 and at the early developmental stage of the brain.

oi:10.1016/j.neures.2010.07.1486

3-a06 Cell diversity and connection specificity betweenallosal projection neurons in the frontal cortexakeshi Otsuka 1,2 , Yasuo Kawaguchi 1,2

Division of Cerebral Circuitry, National Institute for Physiological Sciences

JST, CREST, Japan

ecent advances have established that multiple subnetworks of synapti-ally coupled excitatory neurons provide distinct pathways for informationow through the cortical circuit. These excitatory subnetworks are now

ch 68S (2010) e335–e446

assumed to consist of functionally segregated channels corresponding toprojection systems to subcortical areas. The cortex is, however, composedof two hemispheres connected through the corpus callosum. Callosal dis-connection causes dramatic sensation and perceptual deficits, indicatingthe importance of information transfer and integration between corticalhemispheres. To understand how the commissural system organizes theintracortical excitatory subnetworks, we investigated physiological and mor-phological properties of callosal projection neurons and the local synapticconnections among them. Callosal projection neurons were identified by theinjection of retrograde fluorescent tracer to the contralateral frontal cor-tex, and found in both supra- and infragranular layers. We then obtainedwhole-cell recordings from labeled cells in the slice preparations. Our resultssuggest that callosal projection neurons are heterogeneous in morphologicaland physiological properties, and form subnetworks between them.

doi:10.1016/j.neures.2010.07.1487

P3-a07 Subcellular localization of intracortical excitatorysynaptic inputs onto layer 2/3 pyramidal neurons in neo-cortexNao Nakagawa 1,2 , Yumiko Yoshimura 1,2,3

1 National Institutes of Natural Sciences, Okazaki Institute for IntegrativeBioscience, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan 2 Department of Physiological Sciences, Graduate Universityfor Advanced Studies (SOKENDAI), Hayama, Kanagawa 240-0193, Japan3 PRESTO, JST, Saitama 332-0012, Japan

Neocortical pyramidal neurons receive numerous synaptic inputs on thedendrites. Their subcellular localization may be crucial for understand-ing integration mechanisms. It is, however, difficult to identify functionalsynaptic sites from various sources onto single neurons.We developed anovel method to determine whether synaptic inputs occur in apical orbasal dendrites, using whole-cell voltage-clamp recordings of excitatorypostsynaptic currents (EPSCs) simultaneously from the soma and proximal(∼20 �m distant) apical dendrite of single layer 2/3 pyramidal neurons inrat visual cortical slices. Spontaneous EPSCs were concurrently recorded andthe amplitude ratios of the dendritic to the somatic EPSCs were clearly sep-arated into two groups. EPSCs with a large ratio may be of apical dendriticorigin, while EPSCs with a small ratio may be of basal dendritic origin. Weconfirmed this supposition by recording inward currents directly evoked byfocal uncaging of glutamate in either apical or basal dendrites in the pres-ence of tetrodotoxin. As expected, the amplitude ratio of the dendritic to thesomatic response was larger for the stimulation of apical dendrites than thatof basal dendrites. Using this simultaneous recording method, we analyzedEPSCs evoked by laser scanning photostimulation of presynaptic neurons inlayers 2/3, 4 and 5. The results indicated that the localization pattern of inputsfrom layer 2/3 cells varied among the cells; excitatory inputs were locatedpreferentially on the apical dendrites in about half of the recorded cells, whilethey were located on both apical and basal dendrites in the remaining cells.On the other hand, excitatory inputs from layer 4 and 5 cells were mostlylocated on the basal dendrite.This subcellular localization of synaptic inputsmay provide a basis for signal integration of different sources of synapticinputs in layer 2/3 pyramidal neurons.

doi:10.1016/j.neures.2010.07.1488

P3-a09 Triadic synaptic interactions of large corticothala-mic terminals in non-lemniscal thalamic nuclei of the catauditory systemHisayuki Ojima 1 , Kunio Murakami 2

1 Cognitive Neurobiology, Graduate School of Medical and Dental Sciences,Tokyo Medical and Dental University, Tokyo, Japan 2 Department of Anatomy,Toho University School of Medicine, Tokyo, Japan

Large corticothalamic (CT) terminals, presumed to originate from corticallayer-5 pyramidal cells, are distributed predominantly in non-specific thala-mic nuclei in mammals. In the auditory system, little is known about whetherthese CT projections participate in the synaptic aggregation referred to asthe triad. We studied synaptic interactions of these terminals with neuronal

elements in one of the auditory non-lemniscal thalamic nuclei, the dorsalnucleus of the medial geniculate complex (MGC), in cats. After injections of ananterograde tracer in the primary auditory cortex, areas containing labeledlarge terminals were examined using an electron microscope. It was revealedthat a fraction of large CT terminals participated in complicated synaptic