A Handful of Details to Ensure the Experimental Reproducibility on the FORCED Running Wheel in Rodents: A Systematic Review

Abstract

Purpose: To identify and characterize numerically and topographically the population of alpha retinal ganglion cells (alpha RGCs) and their subtypes, the sustained-response ON-center alpha RGCs (ONs-alpha RGCs), which correspond to the type 4 intrinsically photosensitive RGCs (M4-ipRGCs), the transient-response ON-center alpha RGCs (ONt-alpha RGCs), the sustained-response OFF-center alpha RGCs (OFFs-alpha RGCs), and the transient-response OFF-center alpha RGCs (OFFt-alpha RGCs) in the adult pigmented mouse retina.Methods: The alpha RGC population and its subtypes were studied in flat-mounted retinas and radial sections immunodetected against non-phosphorylated high molecular weight neurofilament subunit (SMI-32) or osteopontin (OPN), two alpha RGCs pan-markers; Calbindin, expressed in ONs-alpha RGCs, and amacrines; T-box transcription factor T-brain 2 (Tbr2), a key transcriptional regulator for ipRGC development and maintenance, expressed in ipRGCs and GABA-displaced amacrine cells; OPN4, an anti-melanopsin antibody; or Brn3a and Brn3c, markers of RGCs. The total population of RGCs was counted automatically and alpha RGCs and its subtypes were counted manually, and color-coded neighborhood maps were used for their topographical representation.Results: The total mean number of alpha RGCs per retina is 2,252 +/- 306 SMI32(+)alpha RGCs and 2,315 +/- 175 OPN+alpha RGCs (n = 10), representing 5.08% and 5.22% of the total number of RGCs traced from the optic nerve, respectively. alpha RGCs are distributed throughout the retina, showing a higher density in the temporal hemiretina. ONs-alpha RGCs represent approximate to 36% [841 +/- 110 cells (n = 10)] of all alpha RGCs and are located throughout the retina, with the highest density in the temporal region. ONt-alpha RGCs represent approximate to 34% [797 +/- 146 cells (n = 10)] of all alpha RGCs and are mainly located in the central retinal region. OFF-alpha RGCs represent the remaining 32% of total alpha RGCs and are divided equally between OFFs-alpha RGCs and OFFt-alpha RGCs [363 +/- 50 cells (n = 10) and 376 +/- 36 cells (n = 10), respectively]. OFFs-alpha RGCs are mainly located in the supero-temporal peripheral region of the retina and OFFt-alpha RGCs in the mid-peripheral region of the retina, especially in the infero-temporal region.Conclusions: The combination of specific antibodies is a useful tool to identify and study alpha RGCs and their subtypes. alpha RGCs are distributed throughout the retina presenting higher density in the temporal area. The sustained ON and OFF response subtypes are mainly located in the periphery while the transient ON and OFF response subtypes are found in the central regions of the retina.