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G-protein βγ subunits (Gβγ) interact with presynaptic proteins and regulate neurotransmitter release downstream of Ca influx. To accomplish their roles in sensory signaling, photoreceptor synapses use specialized presynaptic proteins that support neurotransmission at active zone structures known as ribbons. While several G-protein coupled receptors (GPCRs) influence synaptic transmission at ribbon synapses of cones and other retinal neurons, it is unknown whether Gβγ contributes to these effects. We tested whether activation of one particular GPCR, a metabotropic glutamate receptor (mGluR), can reduce cone synaptic transmission via Gβγ in tiger salamander retinas. In recordings from horizontal cells, we found that an mGluR agonist (L-AP4) reduced cone-driven light responses and mEPSC frequency. In paired recordings of cones and horizontal cells, L-AP4 slightly reduced cone I (∼10%) and caused a larger reduction in cone-driven EPSCs (∼30%). Proximity ligation assay revealed direct interactions between SNAP-25 and Gβγ subunits in retinal synaptic layers. Pretreatment with the SNAP-25 cleaving protease BoNT/A inhibited L-AP4 effects on synaptic transmission, as did introduction of a peptide derived from the SNAP-25 C terminus. Introducing Gβγ subunits directly into cones reduced EPSC amplitude. This effect was inhibited by BoNT/A, supporting a role for Gβγ/SNAP-25 interactions. However, the mGluR-dependent reduction in I was not mimicked by Gβγ, indicating that this effect was independent of Gβγ. The finding that synaptic transmission at cone ribbon synapses is regulated by Gβγ/SNAP-25 interactions indicates that these mechanisms are shared by conventional and ribbon-type synapses. Gβγ liberated from other photoreceptor GPCRs is also likely to regulate synaptic transmission. Dynamic regulation of synaptic transmission by presynaptic G-protein coupled receptors shapes information flow through neural circuits. At the first synapse in the visual system, presynaptic metabotropic glutamate receptors (mGluRs) regulate cone photoreceptor synaptic transmission, although the mechanisms and functional impact of this are unclear. We show that mGluRs regulate light response encoding across the cone synapse, accomplished in part by triggering G-protein βγ subunits (Gβγ) interactions with SNAP-25, a core component of the synaptic vesicle fusion machinery. In addition to revealing a role in visual processing, this provides the first demonstration that Gβγ/SNAP-25 interactions regulate synaptic function at a ribbon-type synapse, contributing to an emerging picture of the ubiquity of Gβγ/SNARE interactions in regulating synaptic transmission throughout the nervous system.
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Horizontal cells are interneurons that synapse with photoreceptors in the outer retina. Their genesis during development is subject to regulation by transcription factors in a hierarchical manner. Previously, we showed that Onecut 1 (Oc1), an atypical homeodomain transcription factor, is expressed in developing horizontal cells (HCs) and retinal ganglion cells (RGCs) in the mouse retina. Herein, by knocking out Oc1 specifically in the developing retina, we show that the majority (∼80%) of HCs fail to form during early retinal development, implying that Oc1 is essential for HC genesis. However, no other retinal cell types, including RGCs, were affected in the Oc1 knock-out. Analysis of the genetic relationship between Oc1 and other transcription factor genes required for HC development revealed that Oc1 functions downstream of FoxN4, in parallel with Ptf1a, but upstream of Lim1 and Prox1. By in utero electroporation, we found that Oc1 and Ptf1a together are not only essential, but also sufficient for determination of HC fate. In addition, the synaptic connections in the outer plexiform layer are defective in Oc1-null mice, and photoreceptors undergo age-dependent degeneration, indicating that HCs are not only an integral part of the retinal circuitry, but also are essential for the survival of photoreceptors. In sum, these results demonstrate that Oc1 is a critical determinant of HC fate, and reveal that HCs are essential for photoreceptor viability, retinal integrity, and normal visual function.
Connexin channels mediate electrical synaptic transmission when assembled as cell-to-cell pores at gap junctions and can mediate transmembrane currents when expressed in plasma membranes as hemichannels. They are widely expressed in the vertebrate retina where in electrical synapses they are critical for transmission of visual signals. While the roles of connexins in electrical synapses are well-studied, the function and roles of connexin hemichannels in the nervous system are less well understood. Genetic deletion in zebrafish of connexin (Cx) 55.5 alters horizontal cell feedback to cones, spectral responses, and visual behavior. Here, we have characterized the properties of hemichannel currents in zebrafish retinal horizontal cells and examined the roles of two connexin isoforms, Cx55.5 and Cx52.6, that are coexpressed in these cells. We report that zebrafish horizontal cells express hemichannel currents that conduct inward current at physiological negative potentials and Ca(2+) levels. Manipulation of Cx55.5 and Cx52.6 gene expression in horizontal cells of adult zebrafish revealed that both Cx55.5 and Cx52.6 contribute to hemichannel currents; however, Cx55.5 expression is necessary for high-amplitude currents. Similarly, coexpression of Cx55.5 with Cx52.6 in oocytes increased hemichannel currents in a supra-additive manner. Taken together these results demonstrate that zebrafish horizontal cell hemichannel currents exhibit the functional characteristics necessary to contribute to synaptic feedback at the first visual synapse, that both Cx55.5 and Cx52.6 contribute to hemichannel currents, and that Cx55.5 may have an additional regulatory function enhancing the amplitude of hemichannel currents.
Hemi-gap-junction (HGJ) channels of retinal horizontal cells (HCs) function as transmembrane ion channels that are modulated by voltage and calcium. As an endogenous retinal neuromodulator, zinc, which is coreleased with glutamate at photoreceptor synapses, plays an important role in shaping visual signals by acting on postsynaptic HCs in vivo. To understand more fully the regulation and function of HC HGJ channels, we examined the effect of Zn(2+) on HGJ channel currents in bass retinal HCs. Hemichannel currents elicited by depolarization in Ca(2+)-free medium and in 1 mM Ca(2+) medium were significantly inhibited by extracellular Zn(2+). The inhibition by Zn(2+) of hemichannel currents was dose dependent with a half-maximum inhibitory concentration of 37 microM. Compared with other divalent cations, Zn(2+) exhibited higher inhibitory potency, with the order being Zn(2+) > Cd(2+) approximately Co(2+) > Ca(2+) > Ba(2+) > Mg(2+). Zn(2+) and Ca(2+) were found to modulate HGJ channels independently in additivity experiments. Modification of histidine residues with N-bromosuccinimide suppressed the inhibitory action of Zn(2+), whereas modification of cysteine residues had no significant effect on Zn(2+) inhibition. Taken together, these results suggest that zinc acts on HGJ channels in a calcium-independent way and that histidine residues on the extracellular domain of HGJ channels mediate the inhibitory action of zinc.
The vertebrate neural retina comprises six classes of neurons and one class of glial cells, all derived from a population of multipotent progenitors. There is little information on the molecular mechanisms governing the specification of cell type identity from multipotent progenitors in the developing retina. We report that Ptf1a, a basic-helix-loop-helix (bHLH) transcription factor, is transiently expressed by post-mitotic precursors in the developing mouse retina. Recombination-based lineage tracing analysis in vivo revealed that Ptf1a expression marks retinal precursors with competence to exclusively produce horizontal and amacrine neurons. Inactivation of Ptf1a leads to a fate-switch in these precursors that causes them to adopt a ganglion cell fate. This mis-specification of neurons results in a complete loss of horizontal cells, a profound decrease of amacrine cells and an increase in ganglion cells. Furthermore, we identify Ptf1a as a primary downstream target for Foxn4, a forkhead transcription factor involved in the genesis of horizontal and amacrine neurons. These data, together with the previous findings on Foxn4, provide a model in which the Foxn4-Ptf1a pathway plays a central role in directing the differentiation of retinal progenitors towards horizontal and amacrine cell fates.
Extracellular Ca2+ and Zn2+ influence many aspects of retinal function. Here, we examined the effect of external Ca2+ and Zn2+ on potassium channels of retinal horizontal cells. When extracellular Ca2+ was lowered from 3 mM to 0.3 mM, horizontal cell transient outward currents elicited by voltage steps from resting membrane potential (-70 mV) were decreased by approximately 50%, whereas the sustained currents remained unchanged. This effect was due to a hyperpolarizing shift in the steady-state inactivation curve of A-type K+ currents when extracellular Ca2+ concentration was lowered. The mean half inactivation potential of the steady-state inactivation curves was hyperpolarized from -56.3 +/- 4.7 mV in 3 mM Ca2+ to -76.4 +/- 3.9 mV in 0.3 mM Ca2+. Neither the state-steady activation curve nor the kinetics of inactivation was significantly changed in low extracellular Ca2+. The addition of 30 microM Zn2+ restored peak outward currents in 0.3 mM Ca2+. The half inactivation voltages were depolarized from -70 +/- 2.8 mV in 0.3 mM Ca2+ to -56 +/- 2.6 mV in 0.3 mM Ca2+ plus 30 microM Zn2+. Taken together, the results indicate that external Ca2+ and Zn2+ maintain the activity of A-type potassium channels in retinal horizontal cells by influencing the voltage dependence of steady-state inactivation.