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

Background: Recent 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 withinthe 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 ahemichannel-mediated ephaptic mechanism.Methodology/Principal Findings: To 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 ofneurons. 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 discriminatebetween 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 synapticcleft 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 cellsynapse.Conclusion: We conclude that the data presented in this paper offers further support for physiologically relevant ephaptic interactions in the retina

Mechanisms of lateral-inhibitory feedback from horizontal cells to cone photoreceptors at the first synapse of the retina

Polarization of the horizontal cell (HC) membrane potential causes changes in the synaptic cleft pH that result in inhibitory feedback from HCs to cone photoreceptors (PRs). HCs average signals from many PRs and so negative feedback onto PR terminals from HCs subtracts the average luminance of the visual scene from the light responses of an individual cone. This feedback operates by changing the voltage-dependence and amplitude of the L-type Ca2+ current (ICa) that regulates synaptic release. Feedback regulation of PR Ca2+ channels involves protons but the mechanism by which this pH change occurs is unclear. We investigated three possible sources for protons in the cone synaptic cleft: 1) extracellular carbonic anhydrase (CA), 2) protons released into the cleft upon exocytosis of synaptic vesicles, and 3) sodium-hydrogen exchangers (NHEs). Using electrophysiological measurements of HC to cone feedback, we found that CA and vesicular protons are not major sources of protons for feedback. Feedback was eliminated by removal of extracellular Na+ and significantly inhibited by an NHE antagonist, cariporide, implicating NHEs as a significant source of protons. While NHEs are a major proton source, they are not known to be voltage-sensitive and thus unlikely to be responsible for changes in extracellular proton levels caused by changes in HC membrane potential. Instead we found that removal of bicarbonate and inhibition of bicarbonate transporters with 500 μM DIDS both eliminated feedback, suggesting that HC polarization changes extracellular pH by altering bicarbonate transport. To test whether an ephaptic mechanism is involved in mediating feedback, we used paired whole cell recordings to hyperpolarize the HC while cone ICa was active and then measured the kinetics of feedback-induced changes in the cone membrane current. The time constants of the resulting feedback current were slower than the measurement time resolution and not instantaneous as predicted by an ephaptic mechanism

MAGUK SCAFFOLDS ORGANIZE A KEY SYNAPTIC COMPLEX IN HORIZONTAL CELL PROCESSES CONTACTING PHOTORECEPTORS

Synaptic processes and plasticity of synapses are mediated by large suites of proteins. In most cases, many of these proteins are tethered together by synaptic scaffold proteins. Scaffold proteins have a large number and typically a variety of protein interaction domains that allow many different proteins to be assembled into functional complexes. As each scaffold protein has a different set of protein interaction domains and a unique set of interacting partners, the presence of synaptic scaffolds can provide insight into the molecular mechanisms that regulate synaptic processes. In studies of rabbit retina, we found SAP102 and Chapsyn110 selectively localized in the tips of B-type horizontal cell processes where they contact cone and rod photoreceptors. We further identified some known SAP102 binding partners, kainate receptor GluR6/7 and inward rectifier potassium channel Kir2.1, closely associated with SAP102 in the processes of invaginating HCs. In contrast, in the mouse retina we identified Chapsyn110 as the major scaffold in the tips of horizontal cells contacting photoreceptors. Kir2.1 was found to be assembled with SAP102 into a complex with GluR6/7 in photoreceptor invaginations in Rabbit. GluR6/7 and Kir2.1 presumably are involved in synaptic processes that govern cell-to-cell communication, and could both contribute in different ways to synaptic currents that mediate feedback signaling. Notably, we failed to find evidence for the presence of Cx57 or Cx59, but Pannexin1 immunolabeling was positive in the OPL of mouse retina suggesting that it could play a role in ephaptic and pH mediated signaling. Polyamines regulate many ion channels including Kir2.1. During the day polyamine immunolabeling was unexpectedly high in photoreceptor terminals compared to other areas of the retina. If polyamines are released, they may regulate the activity of Kir2.1 channels located in the tips of HCs. Alternatively, the presence of polyamines may potentiate GluR6/7 by reducing the transition to desensitized state causing an increase in channel conductance. The presence of SAP102 and Chapsyn110 and their binding partners in both cone and rod invaginating synapses suggests that whatever mechanism is supported by this protein complex is present in both types of photoreceptors

Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

BACKGROUND: Syntaxins 1 through 4 are SNAP receptor (SNARE) proteins that mediate vesicular trafficking to the plasma membrane. In retina, syntaxins 1 and 3 are expressed at conventional and ribbon synapses, respectively, suggesting that synaptic trafficking functions differ among syntaxin isoforms. To better understand syntaxins in synaptic signaling and trafficking, we further examined the cell- and synapse-specific expression of syntaxins 1 through 4 in the mouse retina by immunolabeling and confocal microscopy. RESULTS: Each isoform was expressed in the retina and showed a unique distribution in the synaptic layers of the retina, with little or no colocalization of isoforms. Syntaxin 1 was present in amacrine cell bodies and processes and conventional presynaptic terminals in the inner plexiform layer (IPL). Syntaxin 2 was present in amacrine cells and their processes in the IPL, but showed little colocalization with syntaxin 1 or other presynaptic markers. Syntaxin 3 was found in glutamatergic photoreceptor and bipolar cell ribbon synapses, but was absent from putative conventional glutamatergic amacrine cell synapses. Syntaxin 4 was localized to horizontal cell processes in the ribbon synaptic complexes of photoreceptor terminals and in puncta in the IPL that contacted dopaminergic and CD15-positive amacrine cells. Syntaxins 2 and 4 often were apposed to synaptic active zones labeled for bassoon. CONCLUSION: These results indicate that each syntaxin isoform has unique, non-redundant functions in synaptic signaling and trafficking. Syntaxins 1 and 3 mediate presynaptic transmitter release from conventional and ribbon synapses, respectively. Syntaxins 2 and 4 are not presynaptic and likely mediate post-synaptic trafficking

Identification of feedback mechanisms from horizontal cells to cone photoreceptors in the mouse retina using two‐photon calcium imaging and pharmacology

In neurons, transmitter release from axon terminals is directly linked to the calcium level (Thoreson, 2007; Jackman et al., 2009). Thus, one key mechanism to control transmitter release is to modulate presynaptic calcium by synaptic feedback (reviewed in Kamermans and Fahrenfort, 2004). “Traditional” GABAergic feedback but also more unconventional mechanisms like ephaptic and pH‐mediated feedback are found in many parts of the central nervous system (reviewed in Voronin, 2000; Chesler, 2003). However, little is known if these mechanisms operate in parallel to control transmitter release – that is, form a complex feedback system –, and if so, to what extent they fulfil distinct functions. An excellent system to study such feedback mechanisms is the photoreceptor synapse in the retina. This study investigated how the glutamatergic output of cone photoreceptors (cones) in the mouse retina is shaped by different feedback mechanisms from postsynaptic GABAergic horizontal cells using a combination of two‐photon calcium imaging and pharmacology at the level of individual cone axon terminals. I provide evidence that ephaptic feedback sets the cone output gain by defining the basal calcium level, a mechanism that may be crucial for adapting cones to the ambient light level. In contrast, pH‐mediated feedback did not modulate the cone basal calcium level, but affected the size and shape of light‐evoked cone calcium signals in a contrast‐dependent way: low contrast light responses were amplified, whereas high contrast light responses were reduced. Finally, I provide functional evidence that GABA shapes light‐evoked calcium signals in cones. Because we could not localize ionotropic GABA receptors on cone axon terminals using electron microscopy, this suggests that GABA may act through GABA auto‐receptors on horizontal cells, thereby possibly modulating ephaptic and/or pH‐mediated feedback. Taken together, the results of my thesis suggest that at the cone synapse, ephaptic and pH‐mediated feedback may fulfil distinct functions to adjust the output of cones to changing ambient light levels and stimulus contrasts, and the efficacy of these feedback mechanisms is likely modulated by GABA release in the outer retina. Such an intricate feedback system at the first synapse of our visual system could be important for reliable information transfer from one neuron to the next. It is possible that similarly complex synapses with different feedback mechanisms also play a role in other parts of the nervous system

Synaptic Transmission from Horizontal Cells to Cones Is Impaired by Loss of Connexin Hemichannels

In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was strongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an important step in resolving a long-standing debate about the unusual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina

A Positive Feedback Synapse from Retinal Horizontal Cells to Cone Photoreceptors

Cone photoreceptors and horizontal cells (HCs) have a reciprocal synapse that underlies lateral inhibition and establishes the antagonistic center-surround organization of the visual system. Cones transmit to HCs through an excitatory synapse and HCs feed back to cones through an inhibitory synapse. Here we report that HCs also transmit to cone terminals a positive feedback signal that elevates intracellular Ca2+ and accelerates neurotransmitter release. Positive and negative feedback are both initiated by AMPA receptors on HCs, but positive feedback appears to be mediated by a change in HC Ca2+, whereas negative feedback is mediated by a change in HC membrane potential. Local uncaging of AMPA receptor agonists suggests that positive feedback is spatially constrained to active HC-cone synapses, whereas the negative feedback signal spreads through HCs to affect release from surrounding cones. By locally offsetting the effects of negative feedback, positive feedback may amplify photoreceptor synaptic release without sacrificing HC-mediated contrast enhancement

Properties of Synaptic Transmission from Rods and Cones in The Outer Plexiform Layer of The Vertebrate Retina

Photoreceptors are the first neurons in the visual system. They transduce changes in light intensity into graded changes in membrane potential that are then transformed into chemical signals by regulating the release of glutamate-filled synaptic vesicles. Rod and cone photoreceptors release glutamate continuously in darkness and release slows in light. To help track rapid changes in light intensity, photoreceptors are capable of both rapid exocytosis and rapid endocytosis of synaptic vesicles. Endocytosis is needed for recycling synaptic vesicles but also appears to be important for removing proteins and lipids from active zones to restore release site function after prior vesicle fusion. Synaptic exocytosis from vertebrate photoreceptors involves synaptic ribbons that cluster vesicles near the presynaptic membrane. We hypothesized that such clustering increases the likelihood that exocytosis at one ribbon release site may disrupt release at an adjacent site. Consistent with this, studies described in Chapter 2 showed that endocytosis is needed to rapidly restore release site competence at photoreceptor ribbon synapses. We combined optical and electrophysiological techniques to show that endocytosis is important for restoring late steps in the vesicle fusion process but does not appear to be needed for vesicles to dock successfully at the membrane. Release site clearance by endocytosis is thus essential for continuous release in photoreceptors. We explore mechanisms that contribute to efficient synaptic vesicle exocytosis and endocytosis in Chapter 3. Exocytosis and endocytosis of synaptic vesicles can be coupled in two general ways. In the full-collapse model, the vesicle membrane merges completely with the plasma membrane and so vesicles must be fully reconstructed before they can be retrieved by endocytosis. In the kiss-and-run model, a vesicle briefly contacts the plasma membrane through a small fusion pore that permits release of small molecules but the vesicle does not flatten into the plasma membrane. The vesicle with its complement of proteins is quickly recycled to the cytoplasm after closure of the fusion pore during kiss-and-run. Using a combination of techniques including total internal reflectance fluorescence microscopy (TIRFM), confocal microscopy, electron microscopy, and membrane capacitance measurements, we found that kiss-and-run exocytosis and endocytosis contributes to more than 50% of the release events in photoreceptors. In addition to speeding endocytosis, kiss-and-run fusion may limit disruption of release site structure during fusion, providing an efficient mechanism to facilitate sustained release. HCs not only receive excitatory feedforward signals from photoreceptors, but also send inhibitory feedback signals back to photoreceptors. At normal physiological membrane potentials in cones, inhibitory feedback from HCs to cones increases the activity of L-type voltage-gated Ca2+ channels producing inward feedback currents that increase the synaptic release of glutamate. In the final chapter of this thesis, we describe studies using paired whole cell recordings to determine if, in addition to Ca2+ currents, other currents also contribute to these inward feedback currents in cones. We found that feedback currents in cones involve a smaller than expected contribution from Ca2+-activated Cl- currents and a larger than expected contribution from Cl- currents associated with glutamate transporter activity in cones