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Chapter 2
Materials and Methods
2.1 Materials
Tiger salamanders (ambystoma tigrinum) were kept on a 12 hour light-dark cycle in a temperature-
controlled environment. Animals were handled according to NIH guidelines, and the Baylor
College of Medicine Committee for Animal Use approved the methods of this study. The
salamanders were first dark adapted for 45 minutes, then anesthetized with MS2222 and
quickly decapitated after they were unresponsive. Eyes were then enucleated and the retinas
were dissected under infra-red light with night vision scopes (BE Meyers, Redmond, WA)
mounted to a stereomicroscope. The dissected whole retina was then fixed to a piece of filter
paper which was first secured to the bottom of the recording chamber with a small amount
of silicon grease and with a small window for transillumination carved into it. The ground
electrode consisted of chlorided silver wire. The chamber was mounted in a Zeiss Universal
Microscope (Carl Zeiss, Oberkochen, Germany) with Hoffman Modulation Contrast inside
a light isolating faraday cage. Perfusion was supplied by gravity feed through a valve mani-
fold (ALA Scientific, Westbury, NY), and solution was recirculated with a peristaltic pump.
Extracellular solutions were bubbled with 100% O2. For experiments measuring light re-
sponses, a deep infrared filter was placed on the microscope condenser and all extraneous