Abstract
Purpose: :
The photopigment melanopsin confers direct photosensitivity upon a small subset of retinal output neurons, the intrinsically photosensitive retinal ganglion cells (ipRGCs). Recent evidence has revealed five structurally distinct subtypes of ipRGCs, M1 through M5. The most recently identified subtypes, M4 and M5, may influence the cortical visual system. Both stratify in the ON sublayer of the IPL, but M4 cells have much larger dendritic fields than M5 cells. Beyond this, little is known about their structure or light responses. Our goal was to provide a more complete description these ipRGC subtypes.
Methods: :
We used Opn4cre/+;Z/EG mice, in which all ipRGCs express green fluorescent protein (GFP), to target them for intracellular dye-filling or electrophysiological recording in wholemounts. Cells were filled with Lucifer Yellow and/or Neurobiotin. Retinas were immunostained for choline acetyltransferase (ChAT) and imaged by epifluorescence and confocal microscopy. Light responses were recorded under whole-cell voltage or current clamp in control medium (Ames) to reveal synaptically-driven light responses and under synaptic blockade to isolate intrinsic light responses.
Results: :
M4 cells (n=10) had the largest somas (19±2µm mean±SD) and largest dendritic fields (353±73µm) of all ipRGCs and were more highly branched than M1 or M2 cells (33±7 branch points). By contrast, M5 cells (n=9) had smallish somas (16±4µm) and small bushy dendritic fields (220±41µm diameter; 39±11 branch points). M4 dendrites stratified within the inner half of the On-ChAT IPL stratum and slightly below it. This is slightly distal to where M2 cells stratify, and very different from the stratification patterns of M1 and M3 subtypes. M4 cells had ON type synaptically driven light responses that covered a wide dynamic range (1010-1015 photons•cm-2•s-1). They were dominated by S-cone input with a weaker contribution from M-cones; there was no obvious chromatic opponency. The intrinsic light responses of M4 cells were of lower amplitude (0-12 pA) and higher threshold (1015-1016 photons•cm-2•s-1) than in M1 and M2 cells, but comparable in their slow kinetics and spectral tuning, with greatest sensitivity at 480 nm as expected for mediation by melanopsin.
Conclusions: :
M4 and M5 cells are distinguishable from other ipRGC subtypes by virtue of their unique anatomical and physiological properties. The lower sensitivity of M4 (and perhaps M5) cells relative to other subtypes presumably reflects their lower levels of melanopsin expression.
Keywords: ganglion cells • electrophysiology: non-clinical • microscopy: light/fluorescence/immunohistochemistry