Depletion of Cholinergic Amacrine Cells by a Novel Immunotoxin Does Not Perturb the Formation of Segregated On and Off Cone Bipolar Cell Projections

Abstract

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, approximately 10 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 recoverin-positive On and Off cone bipolar cell projections.

Recruitment of the Rod Pathway by Cones in the Absence of Rods

“…The pattern and level of stratification of the axonal arbors of bipolar cells in the inner plexiform layer, which reflect their functional diversity, are not influenced by major changes in retinal architecture: the neonatal elimination of ganglion cells, which are their major target (Gunhan-Agar et al, 2000), the selective removal of cholinergic amacrine cells (Gunhan et al, 2002), the formation of supernumerary neurons in the inner nuclear layer (Strettoi and Volpini, 2002), or an imbalance in afferent activity (Bodnarenko et al, 1995), do affect the number of bipolar cells but not the connectivity in the ipl. Our data demonstrate that the lamination of rod bipolar cells, and probably the wiring of their axons, are not affected by the absence of their normal presynaptic partners.…”

The role of nAChR‐mediated spontaneous retinal activity in visual system development

“…A recent study in rats has also demonstrated that bipolar cell stratification is normal in the absence of RGCs (Gunhan-Agar et al, 2000). In addition, in rats treated with an immunotoxin that selectively eliminates starburst amacrine cells, bipolar cell stratification appears normal (Gunhan et al, 2002) [ Fig. 3(B)].…”

“…Normal P12 retina is shown on left P12 retina where cholinergic cells were depleted at P2 is shown on right. (Adapted from Gunhan et al, 2002). Muir-Robinson et al, 2002).…”

Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina

“…With no absolute requirement for their major synaptic partners, how do amacrine cells achieve their stratification pattern? This question has remained elusive, as available immunocytochemical markers only label differentiated amacrine cells after they are stratified (Bansal et al, 2000;Gunhan et al, 2002;Reese et al, 2001;Stacy and Wong, 2003). It has therefore not been possible to determine whether amacrine cell neurites remodel within the IPL before occupying a sublamina, or whether they specifically target a sublamina, responding to cues already present in the nascent IPL.…”