Depletion of cholinergic amacrine cells by a novel immunotoxin does not perturb the formation of segregated on and off cone bipolar cell projections

Cone bipolar cells are the first retinal neurons that respond in a differential manner to light onset and offset. In the mature retina, the terminal arbors of On and Off cone bipolar cells terminate in different sublaminas of the inner plexiform layer (IPL) where they form synapses with the dendrites of On and Off retinal ganglion cells and with the stratified processes of cholinergic amacrine cells. Here we first show that cholinergic processes within the On and Off sublaminas of the IPL are present early in development, being evident in the rat on the day of birth, similar to10 d before the formation of segregated cone bipolar cell axons. This temporal sequence, as well as our previous finding that the segregation of On and Off cone bipolar cell inputs occurs in the absence of retinal ganglion cells, suggested that cholinergic amacrine cells could provide a scaffold for the subsequent in-growth of bipolar cell axons. To test this hypothesis directly, a new cholinergic cell immunotoxin was constructed by conjugating saporin, the ribosome-inactivating protein toxin, to an antibody against the vesicular acetylcholine transporter. A single intraocular injection of the immunotoxin caused a rapid, complete, and selective loss of cholinergic amacrine cells from the developing rat retina. On and Off cone bipolar cells were visualized using an antibody against recoverin, the calcium-binding protein that labels the soma and processes of these interneurons. After complete depletion of cholinergic amacrine cells, cone bipolar cell axon terminals still formed their two characteristic strata within the IPL. These findings demonstrate that the presence of cholinergic amacrine cells is not required for the segregation of recovering positive On and Off cone bipolar cell projections.