Folia Pharmacol. Jpn. 121 1. NMDA IP spine branch - - - e-mail: tnakamu@ims.u-tokyo.ac.jp branch main shaft branch NMDA bmain shaft IP -induced calcium release c 2. 1 CCD CCD S N Olympus, BX WI
Fig. 1 Spatial segregation of synaptically evoked calcium signaling in proximal dendrite in hippocampal CA neuron. Arrow heads indicate the times at which the images a -c were obtained. Fig. 3 A Saturation of calcium indicators and B fluorescence signals obtained with bis-fura- and magfura-. Fig. 4 Calcium transients in growth cone of cultured neuron. Arrow heads indicate the times at which the images a -c were obtained.
Fig. 2 Optical setup for real-time imaging of single neurons using bis fura-. Olympus, LUMPlan xw IR, xw IR IR-CCD CCD HiSCA C- Photometrics AT Narishige, ITS 2 whole-cell clamp mode break-in - Mdendrite patch
focus x binning CCD S N Photometrics AT CCD x. x. 3 ROI: region of interest fura- nm ROI baseline baseline F F ROI ROI New York Medical College ProfessorW.N.Ross WDI custom-made AQUA-COSMOS-HiSCA
4 A Kd fura- bis-fura- M BmGluR bis-fura- phototoxicity M bis-fura- 5 OSP-EXA, Olympus; C fura- ms Oregon Green -BAPTA- SBFI DM -nm 3. AM AM AM bath application AM fura- BX WILUMPlanFl xw ORCA-ER
AQUA-COSMOS NIKON TE, SuperFluor x C AQUA-COSMOS HiSCA fura- nm UV FM - FM - B CA CA AM 4. spine spine spine CCD spine 1 spine spine M Oregon Green BAPTA- and MAlexaFluo nm Line Line X-Y
Fig. 5 Morphology of the living neuron expressing GFP in a slice preparation. spine Line-scan Line-scan 2 Oregon Green BAPTA- Noran Odyssey spine Line-scan CSU 3 green fluorescent protein GFP GFP - GFP CCD BX WILUMPlanFL xw IR MaiTai nm FV in vivo Nakamura T, Barbara JG, Nakamura K and Ross WN: Synergistic release of Ca from IP -sensitive stores evoked by synaptic activation of mglurs paired with backpropagating action potentials. Neuron 24,- Nakamura T, Nakamura K, Lasser-Ross N, Barbara JG, Sandler VM and Ross WN: Inositol,, -trisphosphate IP -mediated Ca release evoked by metabotropic agonists and backpropagating action potentials in hippocampal CA pyramidal neurons. J Neurosci 20,- Nakamura T, Lasser-Ross N, Nakamura K and Ross WN: Spatial segregation and interaction of calcium signalling mechanisms in rat hippocampal CA pyramidal neurons. J Physiol 543, - Miyakawa H, Ross WN, Jaffe D, Callaway JC, Lasser-Ross N, Lisman JE and Johnston D: Synaptically activated increases in Ca concentration in hippocampal CA pyramidal cells are primarily due to voltage-gated Ca channels. Neuron 9, - in situ 6, - 58, - GFP
Lasser-Ross N, Miyakawa H, Lev-Ram V, Young SR and Ross WN: High time resolution fluorescence imaging with a CCD camera. J Neurosci Methods 36,- 121, - Larkum, et al: Synaptically activated Ca waves in layer and layer rat neocortical pyramidal neurons. J Physiol Lond in press Helmchen F, Imoto K and Sakmann B: Ca buffering and action potential-evoked Ca signaling in dendrites of pyramidal neurons. Biophys J 70, - Sandler VM and Barbara JG: Calcium-induced calcium release contributes to action potential-evoked calcium transients in hippocampal CA pyramidal neurons. J Neurosci 19,- Gomez TM, Snow DM and Letourneau PC: Characterization of spontaneous calcium transients in nerve growth cones and their effect on growth cone migration. Neuron 14,- Renger JJ, Egles C and Liu G: A developmental switch in neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation. Neuron 29,- Womack MD, Walker JW and Khodakhah K: Impaired calcium release in cerebellar Purkinje neurons maintained in culture. J Gen Physiol 115, - Sabatini BL, Oertner TG and Svoboda K: The life cycle of Ca ions in dendritic spines. Neuron 33, - Mainen ZF, Malinow R and Svoboda K: Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399, - Finch EA and Augustine GJ: Local calcium signalingbyinositol-,, -trisphosphate in Purkinje cell dendrites. Nature 396,- Takechi H, Eilers J and Konnerth A: A new class of synaptic response involving calcium release in dendritic spines. Nature 396,- Koizumi S, Bootman MD, Bobanovic LK, Schell MJ, Berridge MJ and Lipp P: Characterization of elementary Ca release signals in NGF-differentiated PC cells and hippocampal neurons. Neuron 22, - Emptage N, Bliss TV and Fine A: Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines. Neuron 22,- Bonhoeffer T and Yuste R: Spine motility: phenomenology, mechanisms, and function. Neuron 35, - Trachtenberg JT, Chen BE, Knott GW, Feng G, Sanes JR, Welker E and Svoboda K: Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, - Abstract Real-time imaging of local Ca signaling in single neurons. Takeshi NAKAMURA Ca Oscillation Project, Japan Science and Technology Corporation, - - Shirokanedai, Minato-ku, Tokyo -, Japan. Folia Pharmacol. Jpn. Nippon Yakurigaku Zasshi 121, In addition to the multiple mechanisms of the intracellular calcium mobilizing pathways, neurons possess multiple functional compartments such as the soma, the axon, the dendrites, and the spines. In this article, technical procedures and tips are described to measure local calcium signaling in response to neuronal excitation in single neurons. Keywords: high speed Ca imaging; patch clamp; two-photon microscopy - New York Medical College, Department of Physiology