研究目的
Investigating the role of orexins in modulating signal transmission to dopaminergic amacrine cells (DACs) from both outer retinal photoreceptors (rods and cones) and inner retinal photoreceptors (melanopsin-expressing intrinsically photosensitive retinal ganglion cells [ipRGCs]).
研究成果
Our results suggest that orexins could in?uence visual function via the dopaminergic system in the mammalian retina.
研究不足
The study does not attempt to determine which types of upstream neurons are inhibited or excited by orexin-A, as multiple neurons and neural pathways are involved in signal transmission to DACs.
1:Experimental Design and Method Selection
A whole-cell voltage-clamp technique was used to record light-induced responses from genetically labeled DACs in ?at-mount mouse retinas. Rod and cone signaling to DACs was con?rmed pharmacologically (in wild-type retinas), whereas retrograde melanopsin signaling to DACs was isolated either pharmacologically (in wild-type retinas) or by genetic deletion of rod and cone function (in transgenic mice).
2:Sample Selection and Data Sources
Adult male and female mice were used in all experiments. Three lines of transgenic mice were used for the present study: wild-type TH::RFP mice, Opn4-only TH::RFP mice, and opn4-tdTomato mice.
3:List of Experimental Equipment and Materials
Whole-cell currents from DACs were ampli?ed with an Axopatch 200B ampli?er and acquired using a Digidata 1550A digitizer (Molecular Devices, Sunnyvale, CA, USA). Cell-attached activity was recorded from ipRGCs using an HEKA patch-clamp ampli?er and data acquisition system (HEKA, Lambrecht, Germany).
4:Experimental Procedures and Operational Workflow
The retina was dissected under dim red light and was then placed with the photoreceptor side down in a recording chamber mounted on the stage of an upright conventional ?uorescence microscope. Oxygenated extracellular medium continuously perfused the recording chamber, which was kept in darkness for approximately 1 hour prior to recording.
5:Data Analysis Methods
Electrophysiologic data were analyzed of?ine using the Clamp?t 10.4 (Molecular Devices) and SigmaPlot 12.0 (Systat Software, Erkrath, Germany) software packages. The light-induced peak current amplitude of each DAC was measured before and during drug application.
独家科研数据包,助您复现前沿成果,加速创新突破
获取完整内容