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.

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.

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.

Non-invasive recordings of the retinal activity have an important role to play in the diagnosis of retinal pathologies. The detection of diseases that involve retinal ganglion cells (RGCs), such as optic atrophy and glaucoma, may be improved by isolating the RGC contribution from the multifocal electroretinogram (mfERG). In this study, mfERGs were performed on 20 pigs, 1-6 weeks following unilateral retrobulbar optic nerve section (ONS). The stimuli were 103 non-scaled high-contrast hexagons from which summed and individual mfERG responses were obtained in experimental and control fellow eyes under conditions of ketamine (n = 11) or isoflurane anaesthesia (n = 9). The effect of intravitreal injection of tetrodotoxin (TTX; n = 6) was also investigated. The summed mfERG responses showed a first positive peak (P1) with a short latency (21 ms) followed by two smaller peaks (P2 and P3) of longer latency (46 and 65 ms, respectively). While P2 and P3 amplitude were highly correlated with the time post-optic nerve section (ONS) (P2: r 2 = 0.669; P = 0.007; P3: r 2 = 0.651; P = 0.005), P1 was not (r 2 = 0.193; P = 0.38). P1 and P2 showed no implicit time variation as a function of retinal location, while P3 implicit time varied along the axis of the visual streak, generating a naso-temporal asymmetry. However, the P3 implicit time did not vary consistently with distance away from the optic nerve head. Intravitreal injections of TTX reduced P2 and P3 in the control eyes, consistent with the effect of ONS, and also induced a series of regular oscillations lasting up to 200 ms post stimulus. Under isoflurane anaesthesia, all components of the mfERG ifn experimental and control eyes were, at all time points post-ONS, of similar amplitude and without naso-temporal asymmetry, suggesting a reduced participation of RGCs under these anaesthesic conditions. These data clearly demonstrate that it is possible to isolate the RGC contribution from non-invasive multifocal electroretinography. Traditionally, non-invasive clinical electrophysiological techniques for retinal assessment were considered insensitive to the relatively small number of retinal ganglion cells (RGCs) present in the retina. Recently, the photopic negative response and the scotopic threshold response were isolated from the Ganzfeld electroretinogram (ERG). These responses were shown to be affected in experimental models of optic neuropathy (Sieving et al. 1986;Viswanathan et al. 1999;Bui & Fortune, 2003), in patients with glaucoma (Colotto et al. 2000;Cursiefen et al. 2000;Drasdo et al. 2001;Viswanathan et al. 2001) and by intravitreal injection of the voltage-gated sodium channel blocking agent tetrodotoxin (TTX). This finding suggests these responses might be at least partially generated by RGCs. Pattern stimuli have been used conventionally to elicit proportionally larger RGC responses. The potential generated by this technique called pattern electroretinography (PERG) was shown to be sensitive to optic nerve section (ONS) in rat (Berardi et al. 1990), cat (Mafe...

This review examines the function of calcium-activated chloride currents (I(Cl(Ca))) in the retina with an emphasis on their physiological role in photoreceptors. Although found in a variety of neurons and glial cells of the retina, I(Cl(Ca)) has been most prominently studied in cones, where it activates in response to depolarization-evoked Ca(2+) influx. The slow and complex gating kinetics of the chloride current have been considered to reflect the changing submembrane concentration of intracellular calcium. It is likely that the role of I(Cl(Ca)) is to stabilize the membrane potential of cones during synaptic activity and presynaptic Ca channel modulation. Several candidates in the molecular identification of the channel have been put forward but the issue remains unresolved.