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X. Castell, W. Denk, T. Euler; Dendritic ‘Hotspots’ of Visually-Evoked Ca2+ Signaling in Starburst Amacrine Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5965.
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© ARVO (1962-2015); The Authors (2016-present)
Forty years after the discovery of retinal direction selectivity (DS) the underlying biophysical mechanisms are still not fully understood. As synaptic input to retinal DS ganglion cells (DSGC) is directionally-tuned, motion direction must be presynaptically computed, likely in starburst amacrine cells (SAC), which generate DS Ca2+ signals in their dendrites (Euler et al., 2002, Nature 418:845-852). In these experiments SACs were filled with Ca2+ indicator using sharp microelectrodes. However, during patch-clamp experiments in many cases visually-evoked Ca2+ signals vanished but not DS electrical responses (Hausselt et al., 2004, SfN #299.4). After removal of the patch electrode visually-evoked Ca2+ signals often recover. This suggests the involvement of a dialysis-sensitive signaling mechanism as Ca2+ release from internal stores, which have been shown to participate in transmitter release from cultured retinal amacrine cells (Warrier et al., 2005, J Neurophysiol 94:4196-4208).We aim at determining the distribution of visually-evoked Ca2+ signals along SACs dendritic branches and whether internal Ca2+ stores are involved.
Displaced (ON) SACs in whole-mounted rabbit retina were briefly filled with Ca2+ indicator via microelectrodes, which then were retracted. Dendritic Ca2+ responses to visual stimuli were recorded using 2-photon imaging. Inhibitors were applied with the perfusion medium or locally via pipettes.
The strongest Ca2+ responses were localized to varicosities on the SAC distal dendrites(consistent with earlier observations), which correspond to the output region. The Ca2+ signal distribution was heterogeneous: some branchlets (‘hotspots’) responded strongly to the stimuli, while neighboring ones stayed silent. Moreover, some hotspots were DS while neighboring ones were not. In hotspots Ca2+ signals could be observed during the entire recording time (more than 3 hours). Application of a combination of intracellular-Ca2+-signaling-pathways inhibitors (cyclopiazonic acid, 2-APB, and Ryanodine, inhibiting SERCA pumps, IP3- and Ryanodine-receptors respectively) strongly reduced or abolished visually evoked Ca2+ signals.
Our pharmacological data suggest the involvement of internal Ca2+- stores in the generation of visually-evoked Ca2+ responses in SACs possibly via a Ca2+-induced-Ca2+-release mechanism. Internal Ca2+- stores could support a functional compartmentalization of dendritic Ca2+ signals; this compartmentalization may be reflected by the Ca2+ activity ‘hotspots’ we observed in SAC dendrites.
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