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Seunghoon Lee, Minggang Chen, Z.Jimmy Zhou; Functional Characterization of Micro-connectomes in the Direction-Selective Circuit by Focal Laser Photolysis and Dual Patch Clamp. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6325.
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To investigate the physiology and functional connectivity at individual synapses in the direction-selective (DS) circuit of the mammalian retina.
Dual patch clamp recordings were made from displaced starburst amacrine (SAC) and On-Off DS ganglion cells (DSGC) in the wholemount rabbit retina, following characterization of DSGC receptive field properties. Caged Ca2+ (DM-NTP) was included in the pipette solution for the SAC. UV laser spots were directed to individual dendritic contact points between SAC and DSGC under two-photon imaging to evoke focal Ca2+ photolysis and local synaptic transmission, which was detected simultaneously under dual patch clamp.
Synaptic transmission was detected at individual synaptic sites between SAC and DSGC upon spot photolysis under the condition in which endogenous Ca2+-dependent synaptic transmission was blocked pharmacologically. GABAergic postsynaptic currents were detected in a subpopulation of dendritic contact sites between a DSGC and a SAC on the null side, but not the preferred side, of the DSGC. Most GABA synapses were found to be associated with SAC varicosities at the cross points between SAC and DSGC dendrites. When GABAergic transmission was detected between co-fasiculating dendritic segments of SAC and DSGC, it seemed that multiple synapses existed in close proximity. In most cases, the GABAergic postsynaptic currents had a unitary amplitude of ~15 pA at a holding potential of -70 mV; however, larger amplitudes (40-50 pA) were occasionally observed at some synapses. GABAergic currents at individual synaptic sites consisted of both transient and sustained components. So far, cholinergic transmission has not been co-detected at sites where GABAergic transmission was detected under the same stimulation condition.
The results suggest that differences in the number, rather than the strength, of GABA synapses underlie the directionally asymmetric inhibition from SACs to DSGCs, and that cholinergic and GABAergic transmissions between SAC and DSGC have different synaptic mechanisms and/or organization. The study demonstrates that spot photolysis combined with dual patch clamp allows functional characterization and mapping of synaptic connectivity at individual synapses in the DS circuitry.
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