Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial) cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

Self-referencing H-selective electrodes were used to measure extracellular H fluxes from Müller (glial) cells isolated from the tiger salamander retina. A novel chamber enabled stable recordings using H-selective microelectrodes in a self-referencing format using bicarbonate-based buffer solutions. A small basal H flux was observed from the end foot region of quiescent cells bathed in 24 mM bicarbonate-based solutions, and increasing extracellular potassium induced a dose-dependent increase in H flux. Barium at 6 mM also increased H flux. Potassium-induced extracellular acidifications were abolished when bicarbonate was replaced by 1 mM HEPES. The carbonic anhydrase antagonist benzolamide potentiated the potassium-induced extracellular acidification, while 300 μM DIDS, 300 μM SITS, and 30 μM S0859 significantly reduced the response. Potassium-induced extracellular acidifications persisted in solutions lacking extracellular calcium, although potassium-induced changes in intracellular calcium monitored with Oregon Green were abolished. Exchange of external sodium with choline also eliminated the potassium-induced extracellular acidification. Removal of extracellular sodium by itself induced a transient alkalinization, and replacement of sodium induced a transient acidification, both of which were blocked by 300 μM DIDS. Recordings at the apical portion of the cell showed smaller potassium-induced extracellular H fluxes, and removal of the end foot region further decreased the H flux, suggesting that the end foot was the major source of acidifications. These studies demonstrate that self-referencing H-selective electrodes can be used to monitor H fluxes from retinal Müller cells in bicarbonate-based solutions and confirm the presence of a sodium-coupled bicarbonate transporter, the activity of which is largely restricted to the end foot of the cell. The present study uses self-referencing H-selective electrodes for the first time to measure H fluxes from Müller (glial) cells isolated from tiger salamander retina. These studies demonstrate bicarbonate transport as a potent regulator of extracellular levels of acidity around Müller cells and point toward a need for further studies aimed at addressing how such glial cell pH regulatory mechanisms may shape neuronal signaling.

Obtaining a parts list of the sensory components of the retina is vital to understand the effects of light in behavior, health, and disease. Rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) are the best described photoreceptors in the mammalian retina, but recent functional roles have been proposed for retinal neuropsin (Opn5)-an atypical opsin. However, little is known about the pattern of Opn5 expression in the retina. Using cre (Opn5 cre ) and cre-dependent reporters, we uncover patterns of Opn5 expression and find that Opn5 is restricted to retinal ganglion cells (RGCs). Opn5-RGCs are nonhomogenously distributed through the retina, with greater densities of cells located in the dorsotemporal quadrant. In addition to the local topology of these cells, using cre-dependent AAV viral tracing, we surveyed their central targets and found that they are biased towards image-forming and image-stabilizing regions. Finally, molecular and electrophysiological profiling reveal that Opn5-RGCs comprise previously defined RGC types that respond optimally to edges and objectmotion (F-mini-ONs, HD2, HD1, LEDs, ooDSRGCs, etc.). Together, these data describe the second collection of RGCs that express atypical opsins in the mouse, and expand the roles of image-forming cells in retinal physiology and function. INTRODUCTIONRetinal ganglion cells (RGCs) comprise a diverse collection of 45 or more projection neuron types that link retinal events, such as theThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

In early sensory systems, cell-type diversity generally increases from the periphery into the brain, resulting in a greater heterogeneity of responses to the same stimuli. Surround suppression is a canonical visual computation that begins within the retina and is found at varying levels across retinal ganglion cell types. Our results show that divergence in the level of surround suppression occurs subcellularly, at bipolar cell synapses. Using single-cell electrophysiology and serial block-face scanning electron microscopy, we show that two retinal ganglion cell types exhibit very different levels of surround suppression even though they receive input from the same set of bipolar cell types. This divergence of the bipolar cell signal occurs through synapse-specific regulation by amacrine cells at the scale of tens of microns. These findings indicate that each synapse of a single bipolar cell can carry a unique visual signal, expanding the number of possible functional channels at the earliest stages of visual processing.

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Members of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell types (RGCs), the projection sensory neuron conveying visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing Alkaline Phosphatase (AP) and intersectional genetics had identified three types of Pou4f3/Brn3c positive (Brn3c + ) RGCs. Here, we describe a novel Brn3c Cre mouse allele generated by serial Dre to Cre recombination. We use this allele to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus properties of Brn3c + RGC types. Furthermore, we explore Brn3c-expressing brain nuclei. Our analysis reveals a much larger number of Brn3c + RGCs and more diverse set of RGC types than previously reported. The majority of RGCs having expressed Brn3c during development are still Brn3c positive in the adult, and all of them express Brn3a while only about half express Brn3b. Intersection of Brn3b and Brn3c expression highlights an area of increased RGC density, similar to an area centralis, corresponding to part of the binocular field of view of the mouse. Brn3c + neurons and projections are present in multiple brain nuclei. Brn3c + RGC projections can be detected in the Lateral Geniculate Nucleus (LGN), Pretectal Area (PTA) and Superior Colliculus (SC) but also in the thalamic reticular nucleus (TRN), a visual circuit station that was not previously described to receive retinal input. Most Brn3c + neurons of the brain are confined to the pretectum and the dorsal midbrain. Amongst theses we identify a previously unknown Brn3c + subdivision of the deep mesencephalic nucleus (DpMe). Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity and midbrain cytoarchitectonic, and opens the avenue for specific characterization and manipulation of these structures.Retinal Ganglion Cells (RGCs) are projection sensory neurons that convey multiple aspects of visual information from the eye to retinorecipient areas of the brain (Ramón y Cajal, 1972). Work in many species has documented the abundant diversity among RGC types, by characterizing distinctions in dendritic arbors, responses to visual stimuli, projections to a variety of brain nuclei and circuit

Members of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell (RGCs) types, the main transducers of visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing alkaline phosphatase (AP) and intersectional genetics had identified three types of Brn3c positive (Brn3c +) RGCs. Here, we describe a novel Brn3c Cre mouse allele generated by serial Dre to Cre recombination and use it to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus response properties of Brn3c + RGC types. Furthermore, we explore brain nuclei that express Brn3c or receive input from Brn3c + neurons. Our analysis reveals a much larger number of Brn3c + RGCs and more diverse set of RGC types than previously reported. Most RGCs expressing Brn3c during development are still Brn3c positive in the adult, and all express Brn3a while only about half express Brn3b. Genetic Brn3c-Brn3b intersection reveals an area of increased RGC density, extending from dorsotemporal to ventrolateral across the retina and overlapping with the mouse binocular field of view. In addition, we report a Brn3c + RGC projection to the thalamic reticular nucleus, a visual nucleus that was not previously shown to receive retinal input. Furthermore, Brn3c + neurons highlight a previously unknown subdivision of the deep mesencephalic nucleus. Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity, and cytoarchitectonic.

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