Identification of Mouse Retinal Ganglion Cell Subtypes Through Fluorescent Tracing

Place a retinal tissue isolated from a transgenic mouse with fluorescently labeled retinal ganglion cells, or RGCs, in a recording chamber.

Visual information is relayed to the RGCs, whose axons transmit it to the brain via the optic nerve.

RGC subtypes are distinguished by the branching patterns of their dendrites, which stratify into sublayers within the inner retinal layer.

Flatten the retinal tissue and secure it using a tissue anchor. 

Transfer the chamber onto a microscope stage and perfuse an oxygenated solution to maintain RGC viability.

Using infrared illumination, identify the fluorescent RGCs.

Position a pipette containing a fluorescent tracer onto an RGC. 

Apply positive pressure to prevent clogging.

Switch to negative pressure, drawing the membrane into the pipette to form a seal. 

Apply suction to rupture the membrane, establishing a whole-cell configuration that facilitates tracer diffusion into the dendrites.

Observe the dendritic morphology and stratification to identify the RGC subtypes.

Wash the retina in the oxygenated extracellular solution and transfer it to a glass bottom recording chamber with a plastic transfer pipette. After that, use forceps to carefully flatten the tissue with the photoreceptor layer facing down. Remove excess fluid using a pipette. And anchor the tissue using a platinum ring with nylon mesh. Then fill the chamber with the oxygenated extracellular solution and mount it onto a microscope stage. Perfuse the tissue with the oxygenated extracellular solution at 2 to 4 milliliters per minute.

To prepare for this procedure, pull some glass micro pipettes for electrophysiological recordings using a micro-pipette puller. Observe the ganglion cell layer using IR-DIC optics. Then identify the GFP plus ganglion cells using epifluorescence at about 480 nanometers. Next, locate the pipette filled with intracellular solution in DIC. Apply slight positive pressure and zero any voltage offsets on the amplifier.

Subsequently, lower the glass micro-pipette on a GFP-positive cell and apply test voltage command steps to monitor the seal resistance. The negative pressure should form a Giga ohm seal between the pipette and the cell membrane. After forming a stable seal, rupture the membrane by applying brief pulses of negative pressure to gain whole cell access. Wait one to two minutes for the dendrites of the cell to fill with fluorescent tracer.