Removal of glutamatergic signaling from mRGCs unmasks secondary signaling pathway for pupillary light responses.

<p>(<b>A</b>) Schematic of photoreception in the retina of adult mice. Visual signals from photoreceptors are relayed to mRGCs and other retinal ganglion cells via VGLUT1-dependent signaling. MRGCs signal to brain via VGLUT2-dependent signaling and PACAP. (<b>B</b>) Experimental setup to test the pupillary light reflex (PLR). Adult mice were dark adapted and then placed into a restraining chamber. The consensual PLR was monitored with an infrared camera. (<b>C</b>) Typical PLR responses in control and cKO mice. (<b>D</b>) Mean normalized pupil diameter in control and cKO mice during 1 min before light onset, during 1 min light and during 1 min after the light was turned off. Pupil diameter was measured every 2 sec and then normalized to 10-sec baseline before the light onset. Shaded areas mark one standard deviation from the mean. (<b>E</b>) Percent maximum constriction in response to 1 min light. Pupils constricted on average 80% in control mice (n = 8) but only 50% in cKO mice (n = 15). Error bars mark one standard deviation from the mean. Asterisks (***) indicate <i>P</i><0.001 (Student’s <i>t</i>-test). The incomplete constriction in cKO mice was not due to defects in the iris sphincter muscle or its cholinergic innervation as topical application of pilocarpine constricted the cKO pupils to the control levels (n = 4). (<b>F</b>) Mean time to maximum constriction during 1 min after light onset was 11 sec in control mice (n = 8) and 36 sec in cKO mice (n = 15). Error bars mark one standard deviation from the mean. Asterisks (***) indicate <i>P</i><0.001 (Student’s <i>t</i>-test).