Hemichannel-Mediated and pH-Based Feedback from Horizontal Cells to Cones in the Vertebrate Retina

Abstract

BackgroundRecent studies designed to identify the mechanism by which retinal horizontal cells communicate with cones have implicated two processes. According to one account, horizontal cell hyperpolarization induces an increase in pH within the synaptic cleft that activates the calcium current (Ca2+-current) in cones, enhancing transmitter release. An alternative account suggests that horizontal cell hyperpolarization increases the Ca2+-current to promote transmitter release through a hemichannel-mediated ephaptic mechanism.Methodology/Principal FindingsTo distinguish between these mechanisms, we interfered with the pH regulating systems in the retina and studied the effects on the feedback responses of cones and horizontal cells. We found that the pH buffers HEPES and Tris partially inhibit feedback responses in cones and horizontal cells and lead to intracellular acidification of neurons. Application of 25 mM acetate, which does not change the extracellular pH buffer capacity, does lead to both intracellular acidification and inhibition of feedback. Because intracellular acidification is known to inhibit hemichannels, the key experiment used to test the pH hypothesis, i.e. increasing the extracellular pH buffer capacity, does not discriminate between a pH-based feedback system and a hemichannel-mediated feedback system. To test the pH hypothesis in a manner independent of artificial pH-buffer systems, we studied the effect of interfering with the endogenous pH buffer, the bicarbonate/carbonic anhydrase system. Inhibition of carbonic anhydrase allowed for large changes in pH in the synaptic cleft of bipolar cell terminals and cone terminals, but the predicted enhancement of the cone feedback responses, according to the pH-hypothesis, was not observed. These experiments thus failed to support a proton mediated feedback mechanism. The alternative hypothesis, the hemichannel-mediated ephaptic feedback mechanism, was therefore studied experimentally, and its feasibility was buttressed by means of a quantitative computer model of the cone/horizontal cell synapse.ConclusionWe conclude that the data presented in this paper offers further support for physiologically relevant ephaptic interactions in the retina.

Intracellular pH modulates inner segment calcium homeostasis in vertebrate photoreceptors

“…For example, switching from bicarbonate-based to HEPES-buffered saline dramatically increased the rate of photoreceptor neurotransmitter release and the amplitude of lightinduced responses of horizontal cells in the intact salamander retina (28), suggesting that loss of bicarbonate alkalinizes salamander photoreceptors. In contrast, application of 20 mM HEPES acidified and hyperpolarized horizontal cells and reduced the amplitude of light-induced responses in teleost and rabbit retinas (23,27). While pH i was shown to modulate gap-junctional permeability in horizontal cells (23), the properties of voltage-operated, store-operated, and cytosolic Ca 2ϩ buffering mechanisms in teleost/rabbit photoreceptors remain to be characterized.…”

“…Vertebrate photoreceptors may also express electroneutral Cl Ϫ /HCO 3 Ϫ exchange (28,34,38,59) and electrogenic Na ϩ -HCO 3 Ϫ cotransport and Na ϩ -driven C Ϫ /HCO 3 Ϫ exchange mechanisms (10,83), which could be functionally coupled to carbonic anhydrases within the bicarbonate transport metabolon (62). It is likely that addition of bicarbonate and activation of cytosolic and extracellular carbonic anhydrases has additional effects on photoreceptor pH i and [Ca 2ϩ ] i regulation, outer retinal feedback, and photoreceptor cell death (10,23,83). For example, switching from bicarbonate-based to HEPES-buffered saline dramatically increased the rate of photoreceptor neurotransmitter release and the amplitude of lightinduced responses of horizontal cells in the intact salamander retina (28), suggesting that loss of bicarbonate alkalinizes salamander photoreceptors.…”

“…Continuous acid loading can have marked consequences for photoreceptor light sensitivity and output (31-32, 45, 77). Accordingly, experimental manipulation of extracellular pH (pH o ) affects virtually every level of Ca 2ϩ -dependent process in rods and cones, including membrane surface charge (4), operating range of voltage-gated channels in the inner segment region (3,5,21,26,77), activity of acid-sensing ion channels (22), cGMP-dependent light-regulated channels in the outer segment (38,45), ERG a-wave (38), synaptic transmission (5,37), and synaptic feedback (23,31,77). While pronounced, these effects of pH o do not provide straightforward insights into the signaling function of intracellularly generated protons.…”

Connexin and pannexin signaling pathways, an architectural blueprint for CNS physiology and pathology?

“…In fish retina, HCs and Panx channels have been shown to contribute to a very fast ephaptic and a relatively slow proton-mediated mechanism, respectively (see Fig. 5b for more detailed information concerning the signaling mechanisms) [158][159][160][161][162]. HC/ Panx1 channel-mediated ephaptic transmission was also recently demonstrated in the mouse retina, albeit it is not completely clear whether it involves HCs and/or Panx1 channels [30,163].…”

Translating connexin biology into therapeutics

“…Pannexin channel opening on the other hand may be a regulated response leading to an ATP and UTP "find me" signal for phagocytes during apoptosis [33]. and reptile species [44][45][46][47]. Even here, the relevant connexin isoforms are not present in the mouse and either a different isoform is involved or a different channel type entirely as in the cochlea [48].…”