Glucagon–Expressing Neurons That Are Intrinsic to the Retina May Regulate the Proliferation of Neural Progenitors in the Postnatal Chicken Eye

Abstract

Abstract: : Purpose: The aims of this study were to: (1) test whether glucagon–expressing amacrine cells express glucagon–like peptide 1 (GLP1), (2) test whether these cells produce neurites that ramify in the circumferential marginal zone (CMZ), and (3) test whether exogenous glucagon and GLP1 influence the proliferation of progenitors in the CMZ of the postnatal chicken retina. Methods: We labeled whole–mounts and frozen sections of the retina with antibodies to glucagon, GLP1, Pax6, neurofilament, and Hu to identify retinal neurons and assayed whether the neurites of these cells project into the CMZ. We made intraocular injections of BrdU and insulin, glucagon, GLP1, or the glucagon antagonist [des–His¹, Glu⁹] glucagon–NH₂ (Phe⁶) to assay for the proliferation of cells in the CMZ. We used standard methods of immunocytochemistry, epifluorescence and confocal microscopy. Results: The glucagon–positive neurons are immunoreactive for GLP1, and neurites positive for these neuropeptides are heavily clustered in the CMZ. Two types of glucagon–immunoreactive cells were identified; a sparsly distributed type of amacrine cell, that has been well–described previously, and a novel subtype of retinal neuron. The novel type of neuron is immunoreactive for glucagon, GLP1, neurofilament, Pax6 and Hu, and is found only in ventral and peripheral regions of the retina. The somata are large (>10 micrometers) and unipolar, located in the proximal inner nuclear layer, and produce an axon that projects several mm within the distal inner plexiform layer into the far peripheral retina. Intraocular injections of glucagon or GLP1 suppress the proliferation of retinal progenitors, while the antagonist had no effect. The increased proliferation of progenitors that is induced by injections of insulin can be suppressed by co–application of glucagon or GLP1. Conclusions: A novel type of axon–producing neuron within the avian retina projects to and may heavily innervate the CMZ. Exogenous glucagon and GLP1 suppress the proliferation of CMZ progenitors and antagonize the increased proliferation that is induced by exogenous insulin. We propose that the proliferation of progenitors at the retinal margin may be regulated by glucagon/GLP1–positive neurons that are intrinsic to the retina.

Keywords: immunohistochemistry • neuropeptides • retina: proximal (bipolar, amacrine, and ganglion cells)