Retinal neural transmission represents a key function of the eye. Identifying the molecular components of this vital process is helped by studies of selected human genetic eye disorders. For example, mutations in the calcium channel subunit gene CACNA1F cause incomplete X-linked congenital stationary night blindness (CSNB2 or iCSNB), a human retinal disorder with abnormal electrophysiological response and visual impairments consistent with a retinal neurotransmission defect. To understand the subcellular basis of this retinal disorder, we generated a mouse with a loss-of-function mutation by inserting a self-excising Cre-lox-neo cassette into exon 7 of the murine orthologue, Cacna1f. Electroretinography of the mutant mouse revealed a scotopic a-wave of marginally reduced amplitude compared with the wild-type mouse and absence of the post-receptoral b-wave and oscillatory potentials. Cone ERG responses together with visual evoked potentials and multi-unit activity in the superior colliculus were also absent. Calcium imaging in Fluo-4 loaded retinal slices depolarized with KCl showed 90% less peak signal in the photoreceptor synapses of the Cacna1f mutant than in wild-type mice. The absence of post-receptoral ERG responses and the diminished photoreceptor calcium signals are consistent with a loss of Ca((2+)) channel function in photoreceptors. Immunocytochemistry showed no detectable Ca(v)1.4 protein in the outer plexiform layer of Cacna1f-mutant mice, profound loss of photoreceptor synapses, and abnormal dendritic sprouting of second-order neurons in the photoreceptor layer. Together, these findings in the Cacna1f-mutant mouse reveal that the Ca(v)1.4 calcium channel is vital for the functional assembly and/or maintenance and synaptic functions of photoreceptor ribbon synapses. Moreover, the outcome of this study provides critical clues to the pathophysiology of the human retinal channelopathy of X-linked incomplete CSNB.

Cone photoreceptors were isolated enzymatically and their ionic currents studied by the whole-cell, gigaseal voltage-clamp technique. Five nonsynaptic currents were identified. A prominent, poorly selective cation current, Ih, activated after a delay during hyperpolarizations and then deactivated with a delay on return to potentials >-50 mV. An empirical model for I h gating kinetics is developed with three open and two closed states. Depolarization elicits a small, voltage-gated calcium current (Ic~). Block by nitrendipine, nickel, cadmium, and cobalt, increase of current with barium, lack of rapid inactivation, and relatively high threshold suggest an L-type Ca channel. No evidence was found for low-threshold Ca channels. An anion current Io~c~) was present after pulses that led to a significant inward Ic~ (but not I~) and was not elicited when cobalt was present. Tails of Icltc~ ) were short (100 ms) after short depolarizations and were longer after longer depolarizations. Two TEA-sensitive K currents were also elicited by depolarizations. One, Iv~c~, was calcium sensitive. We looked for modulation of Ih, Ic~, and Ic,cc~) by a number of neurotransmitters. No changes of Ih were seen, but Ic~ and Iatc~) were depressed in a few cones when GABA or adenosine were applied. We discuss how this modulation might contribute to the feedback effects of horizontal cells on cones when surrounding cones are illuminated.

Key points• In mouse models for retinal degeneration, photoreceptor death leads to membrane oscillation in the remnant AII amacrine-ON cone bipolar cell network through an unknown mechanism.• We found such oscillations require voltage-gated Na + channels and gap junctions but not hyperpolarization-activated currents (I h ).• Na + channels are expressed predominantly in AII amacrine cells and I h in ON cone bipolar cells, and appear to interact via gap junctions to shape oscillations.• Similar intrinsic oscillations arose in the wild-type (wt) AII amacrine-ON cone bipolar cell network when photoreceptor inputs to bipolar cells were pharmacologically occluded.• Computational modelling captures experimental findings when a low level of cellular heterogeneity is introduced in the coupled network.• These unique insights into the cellular mechanisms underlying spontaneous activity in the degenerating retina might aid in designing the most effective strategies to restore vision using retinal prosthesis. AbstractIn the rd1 mouse model for retinal degeneration, the loss of photoreceptors results in oscillatory activity (∼10-20 Hz) within the remnant electrically coupled network of retinal ON cone bipolar and AII amacrine cells. We tested the role of hyperpolarization-activated currents (I h ), voltage-gated Na + channels and gap junctions in mediating such oscillatory activity. Blocking I h (1 mM Cs + ) hyperpolarized the network and augmented activity, while antagonizing voltage-dependent Na + channels (1 μM TTX) abolished oscillatory activity in the AII amacrine-ON cone bipolar cell network. Voltage-gated Na + channels were only observed in AII amacrine cells, implicating these cells as major drivers of activity. Pharmacologically uncoupling the network (200 μM meclofenamic acid (MFA)) blocked oscillations in all cells indicating that Na + channels exert their influence over multiple cell types within the network. In wt retina, occluding photoreceptor inputs to bipolar cells (10 μM NBQX and 50 μM L-AP4) resulted in a mild (∼10 mV) hyperpolarization and the induction of oscillatory activity within the AII amacrine-ON cone bipolar cell network. These oscillations had similar properties to those observed in rd1 retina, suggesting that no major degeneration-induced network rewiring is required to trigger spontaneous oscillations. Finally, we constructed a simplified computational model that exhibited Na + channel-dependent network oscillations. In this model, mild heterogeneities in channel densities between individual neurons reproduced our experimental findings. These results indicate that TTX-sensitive Na + channels in AII amacrine cells trigger degeneration-induced network oscillations, which provide a persistent synaptic drive to downstream remnant neurons, thus appearing to replace photoreceptors as the principal drivers of retinal activity.

Cyclic-nucleotide-gated (CNG) channels in outer segments of vertebrate photoreceptors generate electrical signals in response to changes in cyclic GMP concentration during phototransduction 1 . CNG channels also allow the influx of Ca 2+ , which is essential for photoreceptor adaptation 2 . In cone photoreceptors, cGMP triggers an increase in membrane capacitance indicative of exocytosis, suggesting that CNG channels are also involved in synaptic function 3 . Here we examine whether CNG channels reside in cone terminals and whether they regulate neurotransmitter release, specifically in response to nitric oxide (NO), a retrograde transmitter that increases cGMP synthesis and potentiates synaptic transmission in the brain [4][5][6] . Using intact retina, we show that endogenous NO modulates synapses between cones and horizontal cells. In experiments on isolated cones, we show directly that CNG channels occur in clusters and are indirectly activated by S-nitrosocysteine (SNC), an NO donor. Furthermore, both SNC and pCPTcGMP, a membrane-permeant analogue of cGMP, trigger the release of transmitter from the cone terminals. The NO-induced transmitter release is suppressed by guanylate cyclase inhibitors and prevented by direct activation of CNG channels, indicating that their activation is required for NO to elicit release. These results expand our view of CNG channel function to include the regulation of synaptic transmission and mediation of the presynaptic effects of NO.Patch-clamp experiments were performed on acutely dissociated cones from lizard retina, used because they possess large (5-10 μm diameter) presynaptic terminals (labelled T in Fig. 1a). By varying the duration of proteolytic enzyme treatment and trituration, we observed either intact cones or cones devoid of outer segments and/or presynaptic terminals. To investigate whether CNG channels are present in the terminals we applied the whole-cell patch-clamp configuration to cones containing terminals and cones lacking terminals. All the cones selected for use in these experiments were devoid of outer segments, to eliminate their contribution to the whole-cell CNG current. To ensure that the cone terminals were intact and functional, we applied depolarizing voltage pulses to assay for the presence of a voltage-gated Ca 2+ current, which has been previously characterized in cones with healthy terminals 3,7,8 .The results of these experiments are shown in Fig. 1b. All cones exhibiting a voltage-gated Ca 2+ current also exhibited an inward current when the membrane-permeant cGMP analogue pCPT-cGMP (8-para-chlorothio-cGMP) was applied (9 of 9 cells). In contrast, none of the cones without terminals exhibited either the voltage-gated Ca 2+ current or pCPT-cGMP-activated current (0 of 8 cells). To confirm that the pCPT-cGMP-activated current is specifically localized to the terminal, 'whole-terminal' recordings were obtained after isolating the terminal from the inner segment by transecting the axon with a glass probe. Isolated terminals exhibited bot...

Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals. Both conventional and unconventional neurotransmitters, as well as an ephaptic effect, have been proposed, but the intercellular messaging that mediates the inhibitory feedback signal from postsynaptic horizontal cells (HCs) to cones remains unknown. We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones. In isolated, dark-adapted goldfish retina, we assessed feedback in the responses of HCs to light and found that strengthened pH buffering reduced both rollback and the depolarization to red light. In zebrafish retinal slices loaded with Fluo-4, depolarization with elevated K ϩ increased Ca signals in the synaptic terminals of cone photoreceptors. Kainic acid, which depolarizes HCs but has no direct effect on cones, depressed the K ϩ -induced Ca signal, whereas CNQX, which hyperpolarizes HCs, increased the Ca signals, suggesting that polarization of HCs alters inhibitory feedback to cones. We found that these feedback signals were blocked by elevated extracellular pH buffering, as well as amiloride and divalent cations. Voltage clamp of isolated HCs revealed an amiloride-sensitive conductance that could mediate modulation of cleft pH dependent on the membrane potential of these postsynaptic cells.

Synaptic transmission of the light response from photoreceptors to second-order cells of the retina was studied with the whole-cell patch-clamp technique in tiger salamander (Ambystoma tigrinum) retinal slices. Synaptic strength is modulated by extracellular pH in a striking manner: Light-sensitive postsynaptic currents in horizontal and bipolar cells were found to be exponential functions of pH, exhibiting an e-fold increase per 0.23 pH unit over the pH range from 7 to 8. Calcium channel currents in isolated photoreceptors were measured and also exhibited proton sensitivity. External alkalinization from pH 7 to 8 shifted the voltage dependence of channel activation negative by 12 mV. A model of the synaptic transfer function suggested that presynaptic Ca channels could be the primary sites of proton action. Increased Ca influx and transmitter release brought about by alkalinization give rise to larger postsynaptic currents. These results suggest that activity-dependent interstitial pH changes known to occur in the retina, while not alleviating signal clipping at this synapse, may provide an adaptative mechanism controlling gain at the photoreceptor output synapse.Signal transfer across synapses in the nervous system involves events at both pre-and postsynaptic membranes, many of which are targets of transmission-altering interventions. Presynaptic Ca channels are often the targets of neuromodulators, since the activity of these channels controls Ca-dependent neurotransmitter release. Transmitter release at the photoreceptor output synapse is Ca dependent and graded with membrane potential over a narrow range of presynaptic voltages (1).In many cell types, extracellular pH modulates Ca channel gating and permeation significantly in the physiological range (2-7). Our present report shows a strong action of extracellular pH on synaptic output from photoreceptors, and this led us to consider the possibility that proton modulation of Ca channels modulates this synapse. By characterizing photoreceptor Ca channel pH sensitivity and adopting these features in a model of synaptic transfer, our work begins to offer an explanation for the strong anoxia and pH dependence reported previously for the horizontal cell membrane potential (8-11). Our result suggests that small light-induced alkalinizations observed in the retinas of several species (12)(13)(14) alter transmission gain at this synapse. MATERIALS AND METHODSSynaptic Currents in Retinal Slices. Retinal slices (15) were made from dark-adapted eyes of larval tiger salamanders (Ambystoma tigrinum) and cells were whole-cell patchclamped (Axopatch 1D; Axon Instruments, Foster City, CA) under infrared illumination. Photoreceptors, horizontal cells, and bipolar cells could be identified by their position and morphology in the slice. Some cell identifications were made with 1% Lucifer yellow staining via the patch electrode.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement...

Key points• Inhibitory feedback from horizontal cells to photoreceptors regulates synaptic gain and contributes to centre-surround receptive field formation via mechanisms that are not fully understood.• We show that horizontal cell calcium channels and ionotropic GABA receptors mediate the inhibitory feedback, and that the results of their actions are blocked by strong pH buffering with Hepes.• GABA appears to act not upon the photoreceptor but instead upon the horizontal cell itself.The horizontal cell GABA receptors are permeable to chloride and bicarbonate, meaning their activation can produce changes in synaptic cleft pH.• These results suggest that activation of calcium channels in a depolarized horizontal cell releases GABA, which acts in an autaptic manner to increase bicarbonate permeability. The resulting influx of bicarbonate contributes to acidification of the synaptic cleft, inhibiting photoreceptor calcium channels, the hallmark of inhibitory feedback at this synapse.Abstract Horizontal cells send inhibitory feedback to photoreceptors, helping form antagonistic receptive fields in the retina, but the neurotransmitter and the mechanisms underlying this signalling are not known. Since the proteins responsible for conventional Ca 2+ -dependent release of GABAergic synaptic vesicles are present in mammalian horizontal cells, we investigated this conventional mechanism as the means by which horizontal cells inhibit photoreceptors. Using Ca 2+ imaging in rat retinal slices, we confirm that horizontal cell depolarization with kainate inhibits and horizontal cell hyperpolarization with NBQX disinhibits the Ca 2+ signals produced by pH-sensitive activation of voltage-gated calcium channels (Ca channels) in photoreceptors. We show that while 100 μM Co 2+ reduces photoreceptor Ca 2+ signals, it disinhibits them at 10 μM, an effect reminiscent of earlier studies where low [Co 2+ ] eliminated feedback. The low [Co 2+ ] disinhibition is pH sensitive. We localized L-, N-and P/Q-type Ca channels in rat horizontal cells, and showed that both the N-type Ca channel blocker ω-conotoxin GVIA and the P/Q-type Ca channel blocker ω-agatoxin IVA increased Ca 2+ signals in photoreceptors in a pH-sensitive manner. Pronounced actions of GABAergic agents on feedback signals to photoreceptors were observed, and are pH sensitive, but are inconsistent with direct inhibition by GABA of photoreceptor [Ca 2+ ]. Patch-clamp studies revealed that GABA activates a conductance having high bicarbonate permeability in isolated horizontal cells, suggesting that the commonality of pH sensitivity throughout the results could arise from a GABA autofeedback action in horizontal * N.C. Brecha and S. Barnes are equal senior authors. cells. This could change cleft pH with concomitant inhibitory influences on photoreceptor Ca channels.

We show that carbenoxolone, a drug used to block hemichannels in the retina to test the ephaptic model of horizontal cell inhibitory feedback, has strong inhibitory effects on voltage-gated Ca channels. Carbenoxolone (100 microM) reduced photoreceptor-to-horizontal cell synaptic transmission by 92%. Applied to patch-clamped, isolated cone photoreceptors, carbenoxolone inhibited Ca channels with an EC(50) of 48 microM. At 100 microM, it reduced cone Ca channel current by 37%, reduced depolarization-evoked [Ca(2+)] signals in fluo-4 loaded retinal slices by 57% and inhibited Ca channels in Müller cells by 52%. A synaptic transfer model suggests that the degree of block of Ca channels accounts for the reduction in synaptic transmission. These results suggest broad inhibitory actions for carbenoxolone in the retina that must be considered when interpreting its effects on inhibitory feedback.