) 13:00-13:10 13:10-13:40 in vivo 13:40 14:10 1 2 2 12 in vivo 14:10 14:50 Takahiro Takano ()University of Rochester Medical Center Cerebrovascular tone control by astrocytes in vivo 15:00-15:30 "" 15:30 16:00 16:00 16:30 16:40 17:10 17:10 17:40 () 17:40 18:10 D- 19:00-20:00 20:00-21:00 Takahiro TakanoUniversity of Rochester Medical Center In vivo in vitro () 21:00 310
9:00-10:00 () ATP 10:10 10:25 10:25 10:40 ATP 10:40 10:55 10:55 11:10 11:20 11:35 11:35 11:50 11:50 12:05 311
15 915 1 20 15 16 612Hz REM (sharp waves) str. radiatum 130200Hz 100m 25m multiunit activitysingle unit activity S100 312 2 in vivo in vivo2
2 1 1 Ca2 09mm EYFP Cerebral blood flow is dynamically coupled to the regional neuronal activity, but the mechanism by which local microcirculation is controlled remains unclear. Astrocytes have tight association with neurons and their endfeet cover vasculature in brain. Recent studies suggest that astrocytes can change vessel tone, but observations in slice preparations have been contradictory. Here we show that cortical astrocytes in vivo possess a powerful mechanism for rapid vasodilation, using two-photon microscope to visualize perfusion in single vessel in exposed cortex of live animals. Cranial window was made at somatosensory cortex of adult mice and blood vessels were visualized by systemic injection of fluorescent dye conjugated with dextran. Concentration of calcium in astrocytic endfeet surrounding penetrating arteries was monitored after loading with the calcium indicator dye. To selectively stimulate astrocytic endfeet, we triggered calcium increases by photolysis of DMNP-EDTA AM. UV photolysis resulted in rapid increase of calcium in astrocytic endfeet and arterial dilation. Vasodilation occurred with a latency of only 1-2 seconds. The effect is limited to arteries, and stimulating astrocytic Ca2+ signaling in vascular endfeet surrounding veins or capillaries did not trigger the changes in vessel diameters. Inhibiting cyclooxygenase-1 activity by either indomethacin or SC-560 blocked the photolysis-induced hyperemia, while antagonists of other vasomodulators failed to influence it. These observations implicate that astrocytes is in control of local microcirculation, and suggest that one of their physiological roles is to mediate vasodilation in response to increased neural activity. exo uteroenhanced yellow fluorescent protein in vivo 4 3 313
CA1CA3 rhodamine-dextrangfp 1 (5)2 Rac1dominant negative Ephrin-A3/EphA4 EphA4/Fcephrin- A3/Fc EphA4/Fcephrin-A3/Fc 1000585 4 314
1 2 3 chromogranin A, organic cation transporter 3 (OCT3), myelin oligodendrocyte glycoproteinmog, SRY (sex determining region Y)-box 10 SOX10, Transferrin, CNP, OLIG2 SNPSNPHapMap tagsnp tagsnp chromogranin AOCT3 D-D-NMDA NMDA D-D- D- D- NMDANR2BmRNA NMDA D- NMDA D-L- L- D- D- D-NMDA D-in vivo1) 2) 3)4) D- Na+ D- D-Na+ asc-1d- dsm-1dsr- 2 D- D- D- D- NMDA D-D- D- D-NMDA D- 315
316 University of Rochester Medical Centerin vivo in vitro () ( ) ATP ATP 2 1 10 ps VSOR 2 100 ps ATPATP VSOR0.6 nm 1.3 nm VSORClC-3 pl-v VSOR 0.35 nmatp0.6 nm DACVDAC ATP VSOR Pglutamate/PCl=0.2VSOR Pglutamate/PCl=0.15 VSOR
OGD ATP ATP 4 MATP VSOR ATP4-PATP/PCl 0.1 ATP Cl - ATP 99% 2003 3343 432400 - AMPA AMPA Ca2+ AMPA AMPA Transmembrane AMPA receptor Regulatory Proteins (TARPs) AMPATARPsTARPs TARPs AMPA 317
TARPsγ-4 (γ-5) AMPAγ-4(γ-5) TARPs ATP ATP P2X Ca2+ J Physiol 530:469-, 2001; J Neurosci 24:3125-, 2004 GFAP ATP DMNPE-caged ATP 3 µm laser beam uncage"flash and Flush" 200-1000 ms GFAP-GFPCa2+ Ca2+ Ca2+ IP3IP3 5-phosphatase Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Runx2, Osterix, Sox9, RANKRANKL 15 8ddYRunx2, Osterix, Sox9, RANKRANKLRT-PCR Runx2, Osterix Sox9mRNA 318
C γpkc δpkc γpkc 2 PKC γpkc-ko δpkc-ko PKC Tet-Op system transgenic mice γpkc-gfpδpkc-gfp γpkcδpkc-gfp PKC - Opalin 17 shocked 17 2 1 319