Glutamatergic Neurotransmission from Melanopsin Retinal Ganglion Cells Is Required for Neonatal Photoaversion but Not Adult Pupillary Light Reflex Anton Delwig 1 , Sriparna Majumdar 1 , Kelly Ahern 2 , Matthew M. LaVail 1,2 , Robert Edwards 3,4 , Thomas S. Hnasko 4,5 , David R. Copenhagen 1,3 * 1 Department of Ophthalmology, University of California San Francisco, San Francisco, California, United States of America, 2 Department of Anatomy, University of California San Francisco, San Francisco, California United States of America, 3 Department of Physiology, University of California San Francisco, San Francisco, California, United States of America, 4 Department of Neurology, University of California, San Francisco San Francisco, California, United States of America, 5 Department of Neurosciences, University of California San Diego, San Diego, California, United States of America Abstract Melanopsin-expressing retinal ganglion cells (mRGCs) in the eye play an important role in many light-activated non-image- forming functions including neonatal photoaversion and the adult pupillary light reflex (PLR). MRGCs rely on glutamate and possibly PACAP (pituitary adenylate cyclase-activating polypeptide) to relay visual signals to the brain. However, the role of these neurotransmitters for individual non-image-forming responses remains poorly understood. To clarify the role of glutamatergic signaling from mRGCs in neonatal aversion to light and in adult PLR, we conditionally deleted vesicular glutamate transporter (VGLUT2) selectively from mRGCs in mice. We found that deletion of VGLUT2 in mRGCs abolished negative phototaxis and light-induced distress vocalizations in neonatal mice, underscoring a necessary role for glutamatergic signaling. In adult mice, loss of VGLUT2 in mRGCs resulted in a slow and an incomplete PLR. We conclude that glutamatergic neurotransmission from mRGCs is required for neonatal photoaversion but is complemented by another non- glutamatergic signaling mechanism for the pupillary light reflex in adult mice. We speculate that this complementary signaling might be due to PACAP neurotransmission from mRGCs. Citation: Delwig A, Majumdar S, Ahern K, LaVail MM, Edwards R, et al. (2013) Glutamatergic Neurotransmission from Melanopsin Retinal Ganglion Cells Is Required for Neonatal Photoaversion but Not Adult Pupillary Light Reflex. PLoS ONE 8(12): e83974. doi:10.1371/journal.pone.0083974 Editor: Gianluca Tosini, Morehouse School of Medicine, United States of America Received September 23, 2013; Accepted November 11, 2013; Published December 31, 2013 Copyright: ß 2013 Delwig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding from National Institutes of Health (NIH) EY01869 (DRC); NIH P30 EY002162 (Core Grant); Knights Templar Eye Foundation (AD), http://www. knightstemplar.org/ktef/; That Man May See, UCSF (DRC) http://thatmanmaysee.org/Research to Prevent Blindness (DRC, Department of Ophthalmology, UCSF) https://www.rpbusa.org/rpb/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cope@phy.ucsf.edu Introduction Melanopsin-expressing retinal ganglion cells (mRGCs) in the eye mediate many light-evoked non-image forming functions including neonatal photoaversion [1,2] and the adult pupillary light reflex (PLR) [3,4]. Both glutamatergic and peptidergic neurotransmission mechanisms have been postulated to relay visual signals from mRGCs to their neuronal targets in the brain [5,6]. However, the role of these neurotransmitters for individual non-image forming responses remains poorly understood. Glutamatergic synaptic transmission requires the sequestration of glutamate into presynaptic vesicles. One of three isoforms of the vesicular glutamate transporter, VGLUT1, VGLUT2 or VGLUT3, is essential for filling vesicles in glutamatergic neurons (reviewed in [7]). Individual classes of neuron almost always express a single VGLUT isoform. Retinal ganglion cells (RGCs), the projecting output neurons of the retina, stain exclusively with VGLUT2 antibodies and express VGLUT2 mRNA [8–10]. Prior studies of glutamatergic neurotransmission from retinal ganglion cells to the thalamus and between midbrain neurons in the ventral tegmental area demonstrated that conditional deletion of VGLUT2 abolishes evoked synaptic release of glutamate from these neurons [11,12]. Thus, loss of VGLUT2 expression in mRGCs would be expected to abolish light-activated glutamater- gic signaling from mRGCs. MRGCs also express pituitary adenylate cyclase-activating polypeptide (PACAP), which is present in the retina before birth [5,13] and co-localizes with VGLUT2 in mRGC projections in the brain [6]. PACAP signaling occurs on a slower timescale [14] but, in principle, could mediate many light-elicited non-image forming functions that often occur over extended periods of time (seconds to hours). In neonatal mice, light evokes aversive responses including negative phototaxis and distress ultrasonic vocalizations [1,2]. Until postnatal day 10 (P10), these responses are mediated by mRGCs, the only functional photoreceptors in the eye at this age [1,2]. The extent to which retinofugal signal transmission from mRGCs relies on glutamatergic signaling in young neonates is not known. The pupillary light reflex (PLR) in adult mice is mediated exclusively by signaling from mRGCs. Visual signals for the PLR can originate from intrinsic light activation of mRGCs themselves, or from light-activated rod and cone signals that synaptically drive the mRGCs. The necessity of mRGC-mediated neurotransmission is exemplified by the finding that selective destruction of mRGCs PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e83974