- Email: [email protected]
Quantitative imaging of synaptic receptor dynamics using a fluorescence activation-coupled protein labeling (FAPL) method
Abstracts / Neuroscience Research 71S (2011) e108–e415
characterizations of this non-neuronal tissue may pave the way for novel perspectives on the central nervous system physiology and pathology. We have demonstrated previously that choroid plexus epithelial cells (CPECs) express multiple primary cilia, and that the cilia modulate the cellular transport activity across the epithelial layer. Since the mode of ciliary expression is highly unusual, we performed a proteomic analysis of CPEC cilia to elucidate the molecular basis of extraordinariness. CPEC cilia were isolated and purified from swine choroid plexus tissue. The subsequent proteomic analysis identified ∼1,100 proteins. Among them, we found several molecules implicated in ciliary motility. Real-time PCR also validated the expression of these molecules in CPECs. Immunostaining for one of them, radial spoke head 9 homolog (Rsph9), demonstrated that the molecule localized to some, but not all, swine CPEC cilia. Surprisingly, live imaging of mouse choroid plexus demonstrated that CPEC cilia could beat at the neonatal stage. The beating pattern appeared to be different from that of nodal cilia. By contrast, motile cilia were not found in adult choroid plexus. In conclusion, our CPEC ciliome revealed a novel heterogeneous nature of CPECs in terms of ciliary motility and the subsequent analyses demonstrated striking postnatal transition of this property. These results also suggested that CPEC cilia could be classified as either hybrid or mosaic type. We propose to rearrange the concept of ciliary subtype classifications. Research fund: KAKENHI 22770190. doi:10.1016/j.neures.2011.07.1391
P4-b06 Putative function of TRPV4 on the choroid plexus primary cilia Shohei Sasamoto , Keishi Narita, Sen Takeda Department of Anatomy and Cell Biology, Faculty of Medicine, University of Yamanashi Primary cilia are eukaryotic cell organelle projecting from the cell surface. It has been shown that primary cilia, in general, function as a biosensor to sense diverse mechanical and chemical stimuli from extracellular environment. However, the detailed molecular mechanisms of cilia-mediated sensing as well as its diversity among different cell types have not been fully characterized. Recently, we performed a proteomic analysis of primary cilia isolated from choroid plexus epithelial cells (CPECs), which produce the cerebrospinal fluid (CSF) of the ventricular system in vertebrate brain, and identified the transient receptor potential channel vanilloid superfamily type 4 (TRPV4), a divalent cation channel activated by various mechanical and chemical stresses. In the present study, we hypothesized that TRPV4 in CPEC primary cilia may play a role in regulation of CSF production. We demonstrated that TRPV4 was localized in primary cilia of CPECs by immunocytochemistry. TRPV4 was present in 75% of total CPEC primary cilia. The intracellular Ca2+ levels in CPECs were elevated by GSK1016790A, a TRPV4 agonist, with the EC50 value of 34 nM, validating the function of TRPV4 in CPECs. A 24-hr treatment with 4 mM chloral hydrate, a potent deciliation reagent, reduced the number of cilia-positive cells as well as the TRPV4 protein levels in cultured CPECs. In addition, we found that GSK1016790A increased the basal-to-apical fluid transcytosis, which reflects CSF production activities in vitro. In conclusion, our study revealed that functional TRPV4 is expressed in CPECs, and that its activity is abolished by deciliation. Our study also suggested that TRPV4 in CPEC primary cilia may play an important role in the regulation of CSF homeostasis. doi:10.1016/j.neures.2011.07.1392
P4-b07 Identification and characterization of a novel splicing variant, short form sigma-1 receptor Norifumi Shioda , Kiyoshi Ishikawa, Kohji Fukunaga Dept. Pharmacol., Tohoku University Grad. Sch. Pharm. Sci., Sendai, Japan Sigma-1 receptor (Sigma-1R) is characterized a endoplasmic reticulum (ER)associated chaperone protein, regulating ER-mitochondrial interorganellar Ca2+ mobilization through type-3 inositol 1,4,5-trisphosphate receptors (IP3 R3) and promoting cell survival. We identified a novel splice variant of Sigma-1R, termed short form Sigma-1R (Sigma-1SR) from mouse brain cDNA libraries. Sigma-1SR lacks 50 ribonucleotides that encode the second transmembrane domain, resulting in a frame shift and giving rise to a novel 4 amino acid sequence in the C-terminus of the protein that not seen in Sigma-1R reported previously. The Sigma-1SR protein expression level is 30–40% of Sigma-1R in mouse cortex, hippocampus and striatum. When Sigma-1SR was expressed in neuroblastoma Neuro-2a cells, it localized on ER and golgi apparatus, and interacted with Sigma-1R, but not with IP3 R3. The stable expression of Sigma-1SR reduced the efficiency of
e319
mitochondrial Ca2+ uptake in response to IP3 R-driven stimuli. In addition, coexpression of Sigma-1R with Sigma-1SR blocked its action on mitochondrial Ca2+ mobilization. Most importantly, Sigma-1R overexpression suppressed hydrogen peroxide-induced ATP depletion and cell apoptosis, whereas Sigma-1SR overexpression had opposite effects in Neuro-2a cells. Taken together, Sigma-1SR dimerizes with Sigma-1R and inhibits its function, thereby promoting apoptosis in neurons. Research fund: KAKENHI23110501. doi:10.1016/j.neures.2011.07.1393
P4-b08 Dimension of GluAs reconstituted in lipid bilayer Nahoko Kasai 1 , Chandra S. Ramanujan 2 , Jelena Barnovic 2 , John F. Ryan 2 , Koji Sumitomo 1 , Keiichi Torimitsu 1 1
NTT Basic Research Labs 2 University of Oxford
As one of ligand-gated ion channel glutamate receptors, ␣-amino-3hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) are present throughout the vertebrate central nervous system, located mainly at postsynaptic membranes where they act as the main mediators of fast excitatory neurotransmission. AMPAR is a tetrameric which consists of four subunits (GluA1–GluA4). AMPAR density is reported ∼1000/m2 in a synapse of Purkinje cells in rat cerebellum, and in dentate gyrus in hippocampus the density becomes 5–10 times less in extra-synaptic membrane than in synaptic membrane. This density is also revealed to relate to short-term memory formation. We have focused on imaging single, full-length AMPARs, which were purified and reconstituted into artificial lipid membrane, using atomic force microscopy (AFM) in near physiological condition. In the previous study we reported that the single molecule measurements of the reconstituted homomeric GluA3 revealed the AMPAR extracellular domain observed in AFM was clearly tetrameric, but it appeared to be somewhat smaller in height and more extended laterally in the membrane than expected on the basis of cryo-EM and X-ray measurements. In this study, we examined the dimension of the reconstituted AMPARs in different receptor density in lipid bilayer. Full-length GluA3 homomer were overexpressed in High Five cells, purified, and reconstituted into lipid bilayer consisting phosphatidylcholine from egg yolk and phosphatidylserine from porcine brain (3:1). AFM (Dimension 3100TM , Digital Instruments Veeco Metrology, Woodbury, USA) measurements were made in tapping mode in buffer solution (30 mM HEPES (pH7.4), 260 mM KCl and 40 mM NaCl). We observed the smaller height under our low receptor density conditions, and larger height under the higher receptor density. This result implied the different receptor interaction in different density, suggesting that different receptor function at different density. Research fund: This research was supported in part by KAKENHI (B20360014). doi:10.1016/j.neures.2011.07.1394
P4-b09 Quantitative imaging of synaptic receptor dynamics using a fluorescence activation-coupled protein labeling (FAPL) method Yuichiro Ishii 1 , Hiroyuki Okuno 1 , Toru Nagano 2 , Yasuteru Urano 3 , Haruhiko Bito 1,4
Komatsu 2 , Tetsuo
1
Dep. of Neurochemistry, Grad. Sch. of Med, Univ. of Tokyo, Tokyo, Japan Lab. of Chemistry and Biology, Grad. Sch. of Pharmaceutical Sci., Univ. of Tokyo, Tokyo, Japan 3 Lab. of Chemical Biology and Molecular Imaging, Grad. Sch. of Med, Univ. of Tokyo, Tokyo, Japan 4 CREST-JST 2
Long-term potentiation (LTP) of glutamatergic synaptic transmission represents an elementary adaptive response that is essential for learning and memory. One class of glutamate-gated ion channels, the AMPA-type glutamate receptors (AMPARs), plays a central role in this process, and mechanisms that allow AMPARs to enter synapses, such as vesicular trafficking, exocytosis and lateral diffusion, are critical in controlling and altering the strength of synaptic transmission. Multiple methods have been developed to measure AMPARs trafficking in real-time, but it still remains a challenge to dissect the contribution of distinct mechanisms of AMPAR synaptic delivery. In particular, it has been difficult to segregate new AMPAR insertion by exocytosis from lateral diffusion of preexisting AMPARs into the synaptic membranes. To begin to address this issue, we here designed a novel strategy to measure AMPARs dynamics with single synapse resolution. We tagged GluA1, an AMPAR subunit, by inserting into its extracellular N-terminus an enzymatic tag SNAP, which covalently binds benzylguanine-substituted organic-dyes. We then used a novel membrane-impermeable SNAP ligand with improved fluorescence properties (Komatsu et al. JACS, 2011), and
e320
Abstracts / Neuroscience Research 71S (2011) e108–e415
imaged SNAP-GluA1 transfected in cultured hippocampal neurons. A sizable portion of surface GluA1 subunits were labeled within 10–30 min with a low background. GluA1 puncta were distributed throughout the dendrites, especially at spines, and largely overlapped with an excitatory presynaptic marker, VGluT1. Live imaging revealed active GluA1 puncta movement along the dendritic shafts and across spines during basal activity. This new FAPL labeling method paves the way to fast pulse-chase labeling of newly membrane-inserted receptors and will help distinguish AMPARs exocytosis from lateral diffusion events in and out of the synapses, both at rest and during plasticity. doi:10.1016/j.neures.2011.07.1395
P4-b10 GABA conditioning regulated by the transporters in cultured cerebellar glial cells Yui Kurimoto 1 , Yu Yonezawa 1 , Kazuto Kobayashi 2 , Naohiro Hozumi 1 , Chitoshi Takayama 3 , Atsuo Fukuda 4 , Seiji Yamamoto 4 , Sachiko Yoshida 1 1
Dep. Env. & Life Sci., Toyohashi University Tech., Toyohashi, Japan 2 Honda Electronics Co., Ltd., Toyohashi 3 Dep. of Anat. 2, Univ. of the Ryukyu Fac. of Med. Okinawa 4 Hamamatsu Univ. Sch. of Med. Hamamatsu ␥-Amino butyric acid (GABA) is known to play important roles as a modulator in the survival and development of cerebellar cortex. To assist an observation of GABAergic neurons, we produced the vesicular GABA transporter (VGAT)-Venus (VGAT-Venus) transgenic rats. Recently, we have observed distribution and elimination of VGAT in culture of dissociated glial cells and organotypic culture of neonatal cerebellar cortex. This ectopic VGAT expression was also observed even in the undifferentiated dissociated glial culture, which was remained for several weeks. Differentiated fibrous glial cells in vitro, however, exhibited weak Venus fluorescence. To induce differentiation for short time, we applied dibutyryl cyclic AMP (dBcAMP) to cultured glia. Cultures are promoted phosporilation by cAMP. To investigate the source of GABA, we observed GABA release from dissociated glial culture without GABAergic neurons using the enzyme-linked photo assay technique, and confirmed small amount of GABA was released from glial cells. Undifferentiated and VGAT expressed glial cells released more GABA than differentiated fibrous glial cells. Glial differentiation could be also observed using the highresolution impedance microscopy. We would report an interaction between GABA release and transporter expression in glial cells, and discuss physiological mechanism of glial GABA regulation. doi:10.1016/j.neures.2011.07.1396
P4-b11 Molecular sequence and distribution of vesicular glutamate transporter 2 mRNA in the brain of the zebra finch (Taeniopygia guttata) Mohammad Rabiul Karim , Shouichiro Saito, Yasuro Atoji Laboratory of Veterinary Anatomy, Gifu University, Gifu, Japan Among songbirds, the zebra finch is an excellent model system for investigating the neural mechanisms underlying complex behaviors such as vocal communication and learning. The glutamatergic transmission is mediated mainly by the vesicular glutamate transporters (VGLUTs) and VGLUT1 and 2 have been considered the most specific marker for glutamatergic neurons. Here we determined the complementary DNA (cDNA) sequences of zebra finch VGLUT2 (zVGLUT2) gene and mapped the expression pattern of the corresponding mRNA within the zebra finch brain. We identified a full-length cDNA sequence of zVGLUT2 gene and the predicted zVGLUT2 protein showed 94% identity with human VGLUT2 and 98% identity with pigeon and chicken VGLUT2 protein. In situ hybridization autoradiograms showed differential expression patterns for VGLUT2 mRNA in the zebra finch brain. In the telencephalon, zVGLUT2 mRNA was expressed exclusively in the pallium but the subpallium was totally devoid of zVGLUT2 mRNA including area X. In the diencephalon, zVGLUT2 mRNA was more densely distributed in the thalamus than in the hypothalamus. In the mesencephalon and rhombencephalon, rich zVGLUT2 mRNA expression was observed in the optic tectum, and granular layer of the cerebellum. These results suggested that cDNA sequence of zVGLUT2 is comparable to those of VGLUT2 in birds and mammals and the distribution of zVGLUT2 mRNA resembles that of VGLUT2 in pigeon. doi:10.1016/j.neures.2011.07.1397
P4-b12 Modulating effect of Bacopa monniera against monocrotophos induced cholinergic and dopaminergic dysfunctions in rats Rajesh S. Yadav , Rajendra K. Shukla, Madhu L. Sankhwar, Aditya B. Pant, Vinay K. Khanna Development Toxicology Division, Indian Institute of Toxicology Research, Lucknow, India Human exposure to monocrotophos (MCP) is quite imminent and associated with neurological disorders involving cognitive deficits. In view of increasing human health risk following exposure to MCP, studies have been carried out to investigate the neuroprotective effect of Bacopa monniera (Brahmi) in MCP induced neurobehavioral toxicity in rats. A significant impairment in motor activity and rota-rod performance (64%) associated with decreased binding of dopamine-(D2) receptors in corpus striatum (20%) was observed in rats treated with monocrotophos (1.0 mg/kg body weight, p.o., 28 days) as compared to controls. These rats also exhibited a significant decrease in grip strength (19%) and impaired learning activity (49%) associated with decreased binding of muscarinic-cholinergic receptors (49%) and activity of acetylcholinesterase (48%) in hippocampus as compared to controls. A significant protection in motor activity, rota-rod performance, grip strength (21%) and learning activity (53%) was observed in rats simultaneously treated with monocrotophos and brahmi (50 mg/kg, body weight, p.o., 28 days) as compared to rat treated with MCP alone. Binding of dopamine-(D2) receptor in corpus striatum (22%) and muscuranic-cholinergic receptor in hippocampus (28%) was also found to be increased in rats simultaneously treated with MCP and brahmi as compared to those treated with MCP alone. An increase in the activity of acetylcholinesterase in hippocampus was observed in rats as compared to controls. The result of the present study indicates that treatment with Bacopa monniera significantly modulates the cholinergic and dopaminergic functionsand imparts its protective efficacy against MCP induced neurotoxicity in rats. Research fund: Supra Institution Project (SIP - 008). doi:10.1016/j.neures.2011.07.1398
P4-b13 Abnormality in the SNARE-related complex in the syntaxin-1A (R151G) knock-in mice Yumi Watanabe 1,2,3 , Michitoshi Watanabe 1 , Kenji Sakimura 4 , Michihiro Igarashi 1,3 1
Div. of Mol. Cell. Biol., Grad. Sch. of Med. and Dent. Sci., Niigata Univ., Japan JSPS Research Fellow 3 Transdisc. Res. Prog., Niigata Univ., Japan 4 Dept. of Cell. Neurobiol., Brain Res. Inst., Niigata Univ., Japan
2
We previously found that the autophosphorylated CaMKII interacts with syntaxin-1A (Stx1A), a t-SNARE operating the vesicle docking and fusion, in the presence of Ca2+ , and demonstrated at the cellular level that this binding modulates the frequency of exocytosis. This interaction is strongly dependent upon R151 of Stx1A, a highly conserved residue localized in the linker domain of Stx1A, and Stx1A (R151G) completely lost the CaMKIIbinding activity in in vitro experiments. To understand the significance of this interaction more in detail, we produced the knock-in (KI) mice having Stx1A (R151G) of the C57/BL6-strain. As we showed in the former JNS meeting, the immunoprecipitation study using the synaptosomal fraction revealed the significantly reduced interaction between Stx1A and CaMKII in the homozygous KI mice. We also examined the effect of this mutation upon exocytosis and synaptic vesicle recycling using the hippocampal neurons of the KI mice: FM 4-64 uptake during high K+ exposure was significantly decreased in the KI-mice derived neurons, although the KI-mice-derived neurons developed without apparent abnormality in the presynaptic terminal. Furthermore, immunofluorescent experiments showed the weaker fluorescent intensity against anti-Stx1A specific antibody repeatedly in the synaptic area of KImouse derived neurons stimulated with high K+ . These results indicate that the R151G mutation is physiologically involved in the dynamics of the Stx1A-containing protein complex formation. In the current presentation, we examined the Stx1A associated protein between R151G mutant and the wild-type both in synaptosome and in in vitro reconstitution assay. Both experiments revealed the reduced association of complexin to Stx1A (R151G). We also studied the difference of native syntaxin-1A-associated complex in the mutant. The observed abnormality in the SNARE-associated complex partly, if not all, account the defects in the exocytosis observed in the KI-mice. Research fund: KAKENHI (17023019). doi:10.1016/j.neures.2011.07.1399
characterizations of this non-neuronal tissue may pave the way for novel perspectives on the central nervous system physiology and pathology. We have demonstrated previously that choroid plexus epithelial cells (CPECs) express multiple primary cilia, and that the cilia modulate the cellular transport activity across the epithelial layer. Since the mode of ciliary expression is highly unusual, we performed a proteomic analysis of CPEC cilia to elucidate the molecular basis of extraordinariness. CPEC cilia were isolated and purified from swine choroid plexus tissue. The subsequent proteomic analysis identified ∼1,100 proteins. Among them, we found several molecules implicated in ciliary motility. Real-time PCR also validated the expression of these molecules in CPECs. Immunostaining for one of them, radial spoke head 9 homolog (Rsph9), demonstrated that the molecule localized to some, but not all, swine CPEC cilia. Surprisingly, live imaging of mouse choroid plexus demonstrated that CPEC cilia could beat at the neonatal stage. The beating pattern appeared to be different from that of nodal cilia. By contrast, motile cilia were not found in adult choroid plexus. In conclusion, our CPEC ciliome revealed a novel heterogeneous nature of CPECs in terms of ciliary motility and the subsequent analyses demonstrated striking postnatal transition of this property. These results also suggested that CPEC cilia could be classified as either hybrid or mosaic type. We propose to rearrange the concept of ciliary subtype classifications. Research fund: KAKENHI 22770190. doi:10.1016/j.neures.2011.07.1391
P4-b06 Putative function of TRPV4 on the choroid plexus primary cilia Shohei Sasamoto , Keishi Narita, Sen Takeda Department of Anatomy and Cell Biology, Faculty of Medicine, University of Yamanashi Primary cilia are eukaryotic cell organelle projecting from the cell surface. It has been shown that primary cilia, in general, function as a biosensor to sense diverse mechanical and chemical stimuli from extracellular environment. However, the detailed molecular mechanisms of cilia-mediated sensing as well as its diversity among different cell types have not been fully characterized. Recently, we performed a proteomic analysis of primary cilia isolated from choroid plexus epithelial cells (CPECs), which produce the cerebrospinal fluid (CSF) of the ventricular system in vertebrate brain, and identified the transient receptor potential channel vanilloid superfamily type 4 (TRPV4), a divalent cation channel activated by various mechanical and chemical stresses. In the present study, we hypothesized that TRPV4 in CPEC primary cilia may play a role in regulation of CSF production. We demonstrated that TRPV4 was localized in primary cilia of CPECs by immunocytochemistry. TRPV4 was present in 75% of total CPEC primary cilia. The intracellular Ca2+ levels in CPECs were elevated by GSK1016790A, a TRPV4 agonist, with the EC50 value of 34 nM, validating the function of TRPV4 in CPECs. A 24-hr treatment with 4 mM chloral hydrate, a potent deciliation reagent, reduced the number of cilia-positive cells as well as the TRPV4 protein levels in cultured CPECs. In addition, we found that GSK1016790A increased the basal-to-apical fluid transcytosis, which reflects CSF production activities in vitro. In conclusion, our study revealed that functional TRPV4 is expressed in CPECs, and that its activity is abolished by deciliation. Our study also suggested that TRPV4 in CPEC primary cilia may play an important role in the regulation of CSF homeostasis. doi:10.1016/j.neures.2011.07.1392
P4-b07 Identification and characterization of a novel splicing variant, short form sigma-1 receptor Norifumi Shioda , Kiyoshi Ishikawa, Kohji Fukunaga Dept. Pharmacol., Tohoku University Grad. Sch. Pharm. Sci., Sendai, Japan Sigma-1 receptor (Sigma-1R) is characterized a endoplasmic reticulum (ER)associated chaperone protein, regulating ER-mitochondrial interorganellar Ca2+ mobilization through type-3 inositol 1,4,5-trisphosphate receptors (IP3 R3) and promoting cell survival. We identified a novel splice variant of Sigma-1R, termed short form Sigma-1R (Sigma-1SR) from mouse brain cDNA libraries. Sigma-1SR lacks 50 ribonucleotides that encode the second transmembrane domain, resulting in a frame shift and giving rise to a novel 4 amino acid sequence in the C-terminus of the protein that not seen in Sigma-1R reported previously. The Sigma-1SR protein expression level is 30–40% of Sigma-1R in mouse cortex, hippocampus and striatum. When Sigma-1SR was expressed in neuroblastoma Neuro-2a cells, it localized on ER and golgi apparatus, and interacted with Sigma-1R, but not with IP3 R3. The stable expression of Sigma-1SR reduced the efficiency of
e319
mitochondrial Ca2+ uptake in response to IP3 R-driven stimuli. In addition, coexpression of Sigma-1R with Sigma-1SR blocked its action on mitochondrial Ca2+ mobilization. Most importantly, Sigma-1R overexpression suppressed hydrogen peroxide-induced ATP depletion and cell apoptosis, whereas Sigma-1SR overexpression had opposite effects in Neuro-2a cells. Taken together, Sigma-1SR dimerizes with Sigma-1R and inhibits its function, thereby promoting apoptosis in neurons. Research fund: KAKENHI23110501. doi:10.1016/j.neures.2011.07.1393
P4-b08 Dimension of GluAs reconstituted in lipid bilayer Nahoko Kasai 1 , Chandra S. Ramanujan 2 , Jelena Barnovic 2 , John F. Ryan 2 , Koji Sumitomo 1 , Keiichi Torimitsu 1 1
NTT Basic Research Labs 2 University of Oxford
As one of ligand-gated ion channel glutamate receptors, ␣-amino-3hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) are present throughout the vertebrate central nervous system, located mainly at postsynaptic membranes where they act as the main mediators of fast excitatory neurotransmission. AMPAR is a tetrameric which consists of four subunits (GluA1–GluA4). AMPAR density is reported ∼1000/m2 in a synapse of Purkinje cells in rat cerebellum, and in dentate gyrus in hippocampus the density becomes 5–10 times less in extra-synaptic membrane than in synaptic membrane. This density is also revealed to relate to short-term memory formation. We have focused on imaging single, full-length AMPARs, which were purified and reconstituted into artificial lipid membrane, using atomic force microscopy (AFM) in near physiological condition. In the previous study we reported that the single molecule measurements of the reconstituted homomeric GluA3 revealed the AMPAR extracellular domain observed in AFM was clearly tetrameric, but it appeared to be somewhat smaller in height and more extended laterally in the membrane than expected on the basis of cryo-EM and X-ray measurements. In this study, we examined the dimension of the reconstituted AMPARs in different receptor density in lipid bilayer. Full-length GluA3 homomer were overexpressed in High Five cells, purified, and reconstituted into lipid bilayer consisting phosphatidylcholine from egg yolk and phosphatidylserine from porcine brain (3:1). AFM (Dimension 3100TM , Digital Instruments Veeco Metrology, Woodbury, USA) measurements were made in tapping mode in buffer solution (30 mM HEPES (pH7.4), 260 mM KCl and 40 mM NaCl). We observed the smaller height under our low receptor density conditions, and larger height under the higher receptor density. This result implied the different receptor interaction in different density, suggesting that different receptor function at different density. Research fund: This research was supported in part by KAKENHI (B20360014). doi:10.1016/j.neures.2011.07.1394
P4-b09 Quantitative imaging of synaptic receptor dynamics using a fluorescence activation-coupled protein labeling (FAPL) method Yuichiro Ishii 1 , Hiroyuki Okuno 1 , Toru Nagano 2 , Yasuteru Urano 3 , Haruhiko Bito 1,4
Komatsu 2 , Tetsuo
1
Dep. of Neurochemistry, Grad. Sch. of Med, Univ. of Tokyo, Tokyo, Japan Lab. of Chemistry and Biology, Grad. Sch. of Pharmaceutical Sci., Univ. of Tokyo, Tokyo, Japan 3 Lab. of Chemical Biology and Molecular Imaging, Grad. Sch. of Med, Univ. of Tokyo, Tokyo, Japan 4 CREST-JST 2
Long-term potentiation (LTP) of glutamatergic synaptic transmission represents an elementary adaptive response that is essential for learning and memory. One class of glutamate-gated ion channels, the AMPA-type glutamate receptors (AMPARs), plays a central role in this process, and mechanisms that allow AMPARs to enter synapses, such as vesicular trafficking, exocytosis and lateral diffusion, are critical in controlling and altering the strength of synaptic transmission. Multiple methods have been developed to measure AMPARs trafficking in real-time, but it still remains a challenge to dissect the contribution of distinct mechanisms of AMPAR synaptic delivery. In particular, it has been difficult to segregate new AMPAR insertion by exocytosis from lateral diffusion of preexisting AMPARs into the synaptic membranes. To begin to address this issue, we here designed a novel strategy to measure AMPARs dynamics with single synapse resolution. We tagged GluA1, an AMPAR subunit, by inserting into its extracellular N-terminus an enzymatic tag SNAP, which covalently binds benzylguanine-substituted organic-dyes. We then used a novel membrane-impermeable SNAP ligand with improved fluorescence properties (Komatsu et al. JACS, 2011), and
e320
Abstracts / Neuroscience Research 71S (2011) e108–e415
imaged SNAP-GluA1 transfected in cultured hippocampal neurons. A sizable portion of surface GluA1 subunits were labeled within 10–30 min with a low background. GluA1 puncta were distributed throughout the dendrites, especially at spines, and largely overlapped with an excitatory presynaptic marker, VGluT1. Live imaging revealed active GluA1 puncta movement along the dendritic shafts and across spines during basal activity. This new FAPL labeling method paves the way to fast pulse-chase labeling of newly membrane-inserted receptors and will help distinguish AMPARs exocytosis from lateral diffusion events in and out of the synapses, both at rest and during plasticity. doi:10.1016/j.neures.2011.07.1395
P4-b10 GABA conditioning regulated by the transporters in cultured cerebellar glial cells Yui Kurimoto 1 , Yu Yonezawa 1 , Kazuto Kobayashi 2 , Naohiro Hozumi 1 , Chitoshi Takayama 3 , Atsuo Fukuda 4 , Seiji Yamamoto 4 , Sachiko Yoshida 1 1
Dep. Env. & Life Sci., Toyohashi University Tech., Toyohashi, Japan 2 Honda Electronics Co., Ltd., Toyohashi 3 Dep. of Anat. 2, Univ. of the Ryukyu Fac. of Med. Okinawa 4 Hamamatsu Univ. Sch. of Med. Hamamatsu ␥-Amino butyric acid (GABA) is known to play important roles as a modulator in the survival and development of cerebellar cortex. To assist an observation of GABAergic neurons, we produced the vesicular GABA transporter (VGAT)-Venus (VGAT-Venus) transgenic rats. Recently, we have observed distribution and elimination of VGAT in culture of dissociated glial cells and organotypic culture of neonatal cerebellar cortex. This ectopic VGAT expression was also observed even in the undifferentiated dissociated glial culture, which was remained for several weeks. Differentiated fibrous glial cells in vitro, however, exhibited weak Venus fluorescence. To induce differentiation for short time, we applied dibutyryl cyclic AMP (dBcAMP) to cultured glia. Cultures are promoted phosporilation by cAMP. To investigate the source of GABA, we observed GABA release from dissociated glial culture without GABAergic neurons using the enzyme-linked photo assay technique, and confirmed small amount of GABA was released from glial cells. Undifferentiated and VGAT expressed glial cells released more GABA than differentiated fibrous glial cells. Glial differentiation could be also observed using the highresolution impedance microscopy. We would report an interaction between GABA release and transporter expression in glial cells, and discuss physiological mechanism of glial GABA regulation. doi:10.1016/j.neures.2011.07.1396
P4-b11 Molecular sequence and distribution of vesicular glutamate transporter 2 mRNA in the brain of the zebra finch (Taeniopygia guttata) Mohammad Rabiul Karim , Shouichiro Saito, Yasuro Atoji Laboratory of Veterinary Anatomy, Gifu University, Gifu, Japan Among songbirds, the zebra finch is an excellent model system for investigating the neural mechanisms underlying complex behaviors such as vocal communication and learning. The glutamatergic transmission is mediated mainly by the vesicular glutamate transporters (VGLUTs) and VGLUT1 and 2 have been considered the most specific marker for glutamatergic neurons. Here we determined the complementary DNA (cDNA) sequences of zebra finch VGLUT2 (zVGLUT2) gene and mapped the expression pattern of the corresponding mRNA within the zebra finch brain. We identified a full-length cDNA sequence of zVGLUT2 gene and the predicted zVGLUT2 protein showed 94% identity with human VGLUT2 and 98% identity with pigeon and chicken VGLUT2 protein. In situ hybridization autoradiograms showed differential expression patterns for VGLUT2 mRNA in the zebra finch brain. In the telencephalon, zVGLUT2 mRNA was expressed exclusively in the pallium but the subpallium was totally devoid of zVGLUT2 mRNA including area X. In the diencephalon, zVGLUT2 mRNA was more densely distributed in the thalamus than in the hypothalamus. In the mesencephalon and rhombencephalon, rich zVGLUT2 mRNA expression was observed in the optic tectum, and granular layer of the cerebellum. These results suggested that cDNA sequence of zVGLUT2 is comparable to those of VGLUT2 in birds and mammals and the distribution of zVGLUT2 mRNA resembles that of VGLUT2 in pigeon. doi:10.1016/j.neures.2011.07.1397
P4-b12 Modulating effect of Bacopa monniera against monocrotophos induced cholinergic and dopaminergic dysfunctions in rats Rajesh S. Yadav , Rajendra K. Shukla, Madhu L. Sankhwar, Aditya B. Pant, Vinay K. Khanna Development Toxicology Division, Indian Institute of Toxicology Research, Lucknow, India Human exposure to monocrotophos (MCP) is quite imminent and associated with neurological disorders involving cognitive deficits. In view of increasing human health risk following exposure to MCP, studies have been carried out to investigate the neuroprotective effect of Bacopa monniera (Brahmi) in MCP induced neurobehavioral toxicity in rats. A significant impairment in motor activity and rota-rod performance (64%) associated with decreased binding of dopamine-(D2) receptors in corpus striatum (20%) was observed in rats treated with monocrotophos (1.0 mg/kg body weight, p.o., 28 days) as compared to controls. These rats also exhibited a significant decrease in grip strength (19%) and impaired learning activity (49%) associated with decreased binding of muscarinic-cholinergic receptors (49%) and activity of acetylcholinesterase (48%) in hippocampus as compared to controls. A significant protection in motor activity, rota-rod performance, grip strength (21%) and learning activity (53%) was observed in rats simultaneously treated with monocrotophos and brahmi (50 mg/kg, body weight, p.o., 28 days) as compared to rat treated with MCP alone. Binding of dopamine-(D2) receptor in corpus striatum (22%) and muscuranic-cholinergic receptor in hippocampus (28%) was also found to be increased in rats simultaneously treated with MCP and brahmi as compared to those treated with MCP alone. An increase in the activity of acetylcholinesterase in hippocampus was observed in rats as compared to controls. The result of the present study indicates that treatment with Bacopa monniera significantly modulates the cholinergic and dopaminergic functionsand imparts its protective efficacy against MCP induced neurotoxicity in rats. Research fund: Supra Institution Project (SIP - 008). doi:10.1016/j.neures.2011.07.1398
P4-b13 Abnormality in the SNARE-related complex in the syntaxin-1A (R151G) knock-in mice Yumi Watanabe 1,2,3 , Michitoshi Watanabe 1 , Kenji Sakimura 4 , Michihiro Igarashi 1,3 1
Div. of Mol. Cell. Biol., Grad. Sch. of Med. and Dent. Sci., Niigata Univ., Japan JSPS Research Fellow 3 Transdisc. Res. Prog., Niigata Univ., Japan 4 Dept. of Cell. Neurobiol., Brain Res. Inst., Niigata Univ., Japan
2
We previously found that the autophosphorylated CaMKII interacts with syntaxin-1A (Stx1A), a t-SNARE operating the vesicle docking and fusion, in the presence of Ca2+ , and demonstrated at the cellular level that this binding modulates the frequency of exocytosis. This interaction is strongly dependent upon R151 of Stx1A, a highly conserved residue localized in the linker domain of Stx1A, and Stx1A (R151G) completely lost the CaMKIIbinding activity in in vitro experiments. To understand the significance of this interaction more in detail, we produced the knock-in (KI) mice having Stx1A (R151G) of the C57/BL6-strain. As we showed in the former JNS meeting, the immunoprecipitation study using the synaptosomal fraction revealed the significantly reduced interaction between Stx1A and CaMKII in the homozygous KI mice. We also examined the effect of this mutation upon exocytosis and synaptic vesicle recycling using the hippocampal neurons of the KI mice: FM 4-64 uptake during high K+ exposure was significantly decreased in the KI-mice derived neurons, although the KI-mice-derived neurons developed without apparent abnormality in the presynaptic terminal. Furthermore, immunofluorescent experiments showed the weaker fluorescent intensity against anti-Stx1A specific antibody repeatedly in the synaptic area of KImouse derived neurons stimulated with high K+ . These results indicate that the R151G mutation is physiologically involved in the dynamics of the Stx1A-containing protein complex formation. In the current presentation, we examined the Stx1A associated protein between R151G mutant and the wild-type both in synaptosome and in in vitro reconstitution assay. Both experiments revealed the reduced association of complexin to Stx1A (R151G). We also studied the difference of native syntaxin-1A-associated complex in the mutant. The observed abnormality in the SNARE-associated complex partly, if not all, account the defects in the exocytosis observed in the KI-mice. Research fund: KAKENHI (17023019). doi:10.1016/j.neures.2011.07.1399