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.

Small alterations in extracellular H+ can profoundly alter neurotransmitter release by neurons. We examined mechanisms by which extracellular ATP induces an extracellular H+ flux from Müller glial cells, which surround synaptic connections throughout the vertebrate retina. Müller glia were isolated from tiger salamander retinae and H+ fluxes examined using self-referencing H+-selective microelectrodes. Experiments were performed in 1 mM HEPES with no bicarbonate present. Replacement of extracellular sodium by choline decreased H+ efflux induced by 10 µM ATP by 75%. ATP-induced H+ efflux was also reduced by Na+/H+ exchange inhibitors. Amiloride reduced H+ efflux initiated by 10 µM ATP by 60%, while 10 µM cariporide decreased by 37%, and 25 µM zoniporide reduced H+ flux by 32%. ATP-induced H+ fluxes were not significantly altered by the K+/H+ pump blockers SCH28080 or TAK438, and replacement of all extracellular chloride with gluconate was without effect on H+ fluxes. Recordings of ATP-induced H+ efflux from cells simultaneously whole-cell voltage-clamped revealed no effect of membrane potential from -70 mV to 0 mV. Restoration of extracellular potassium after cells were bathed in 0 mM potassium produced a transient alteration in ATP-dependent H+ efflux. The transient response to extracellular potassium occurred only when extracellular sodium was present and was abolished by 1 mM ouabain, suggesting alterations in sodium gradients mediated by Na+/K ATPase activity. Our data indicate that the majority of H+ efflux elicited by extracellular ATP from isolated Müller cells is mediated by Na+/H+ exchange.

Long-range axonal projections provide the foundation for functional connectivity between brain regions and are critical in the modulation of behavior. Descending projections from medial prefrontal cortex (mPFC) to various target regions regulate critical behavioral functions including decision making, social behavior and mood. While specific mPFC projections have distinct behavioral roles, individual mPFC projection neurons can also innervate multiple target regions. Yet how mPFC projection neurons divide their axons across the brain is poorly understood. In this study, we mapped the axon collaterals of mPFC neurons that project to nucleus accumbens (NAc), ventral tegmental area (VTA), or contralateral mPFC (cmPFC) in mice. We used tissue clearing and light sheet fluorescence microscopy to visualize the 3-D structure of axonal arbors across the intact brain. While machine learning can automate analysis of axons in images of cleared tissue, it is challenging to train a model that generalizes to all axonal structures because the appearance of axons varies by target region. In this study, we present DeepTraCE (Deep learning-based image Tracing with Combined-model Enhancement), a new strategy for axon segmentation and quantification in images of cleared tissue. DeepTraCE is based on the deep-learning framework TRAILMAP; it achieves highly accurate axon detection by combining multiple machine learning models that are each applied to different brain regions. Using DeepTraCE, we find that cmPFC, NAc, and VTA-projecting mPFC neurons represent largely separable classes with unique axon collaterals in cortical, olfactory, and thalamic regions, respectively.