Steven Barnes

Proton-Mediated Feedback Inhibition of Presynaptic Calcium Channels at the Cone Photoreceptor Synapse

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+ 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+-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.

Calcium Channels at the Photoreceptor Synapse

Presynaptic Ca2+ channels mediate early stages of visual information processing in photoreceptors by facilitating the release of neurotransmitter and by receiving modulatory input that alters transmission. Two types of L-type Ca2+ channels, composed of alpha1F and alpha1D subunits and having similar biophysical andpharmacological properties, appear to form the principle voltage-dependent Ca2+ influx pathways in rods and cones, respectively. The role played by these channels in neurotransmitter release at these graded potential, non-spiking synapses, has been well described. The channels mediate sustained glutamate release in darkness where the cells rest at potentials near -40 mV, and signal increases in light intensity as the cells hyperpolarize negative to this value. Synaptic modulation and integration mediated by these channels has not yet been as fully described but appears to involve GABA, nitric oxide (NO), glutamate, and dopamine. Ca2+ permeable cyclic nucleotide gated (CNG) channels appear to have supporting roles at the photoreceptor output synapse and may transduce NO signals from other cells by either directly permitting Ca2+ influx or by providing depolarizing influences that gate voltage dependent Ca2+ channels.

Intrinsic oscillatory activity arising within the electrically coupled AII amacrine–ON cone bipolar cell network is driven by voltage-gated Na+ channels

•  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 (Ih). •  Na+ channels are expressed predominantly in AII amacrine cells and Ih 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.

Calcium channels in rat horizontal cells regulate feedback inhibition of photoreceptors through an unconventional GABA‐ and pH‐sensitive mechanism

•  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.

Adenosine inhibits calcium channel currents via A1 receptors on salamander retinal ganglion cells in a mini‐slice preparation

The effects of adenosine on high‐voltage‐activated calcium channel currents in tiger salamander retinal ganglion cells were investigated in a mini‐slice preparation. Adenosine produced a concentration‐dependent decrease in the amplitude of calcium channel current with a maximum inhibition of 26%. The effects of adenosine on calcium channel current were both time‐ and voltage‐dependent. In cells dialyzed with GTP‐γ‐s, adenosine caused a sustained and irreversible inhibition of calcium channel current, suggesting involvement of a GTP‐binding protein. The inhibitory effect of adenosine on calcium channel current was blocked by the A1 antagonist 8‐cyclopentyltheophylline (DPCPX, 1–10 µm), but not by the A2 antagonist 3‐7‐dimethyl‐1‐propargylxanthine (DMPX, 10 µm), and was mimicked by the A1 agonist N 6‐cyclohexyladenosine (CHA, 1 µm) but not by the A2 agonist 5′‐(N‐cyclopropyl) carbox‐amidoadenosine (CPCA, 1 µm). Adenosines inhibition of calcium channel current was not affected by the L‐type calcium channel blocker nifedipine (5 µm). However, adenosines inhibition of calcium channel current was reduced to approximately 10% after application of ω‐conotoxin GVIA (1 µm), suggesting that adenosine inhibits N‐type calcium channels. These results show that adenosine acts on an A1 adenosine receptor subtype via a G protein‐coupled pathway to inhibit the component of calcium channel current carried in N‐type calcium channels.

Regulation of α1G T-type calcium channel gene (CACNA1G) expression during neuronal differentiation

Down‐regulation of T‐type Ca channel current and mRNA occurs following differentiation of Y79 retinoblastoma cells. To understand how the decrease in expression is linked to cell differentiation, we examined transcriptional regulation of the Cav3.1 Ca channel gene, CACNA1G. We identified two putative promoters (A and B) in 1.3 kb of cloned genomic DNA. Reverse transcriptase‐polymerase chain reaction and 5′ rapid amplification of cDNA ends‐polymerase chain reaction analyses demonstrated that two transcripts with different 5′ untranslated regions are generated by different transcription start sites, with promoter A favoured in undifferentiated cells and promoter B favoured in differentiated cells. Functional analyses of the promoter sequence revealed that both promoters are active. Enhancer and repressor sequences were identified upstream of promoter A and B, respectively. These results suggest that the down‐regulation of α1G mRNA in differentiated Y79 cells is mediated primarily by decreased activity of promoter A, which could occur in conjunction with repression of the activity of promoter B. The decrease in T‐type Ca channel expression in Y79 cells may be an essential signal affecting phenotypic maturation and expression of other ion channel subtypes in the differentiated cells.

Stable Mossy Fiber Long-Term Potentiation Requires Calcium Influx at the Granule Cell Soma, Protein Synthesis, and Microtubule-Dependent Axonal Transport

The synapses formed by the mossy fiber (MF) axons of hippocampal dentate gyrus granule neurons onto CA3 pyramidal neurons exhibit an intriguing form of experience-dependent synaptic plasticity that is induced and expressed presynaptically. In contrast to most other CNS synapses, long-term potentiation (LTP) at the MF–CA3 synapse is readily induced even during blockade of postsynaptic glutamate receptors. Furthermore, blocking voltage-gated Ca2+ channels prevents MF-LTP, supporting an involvement of presynaptic Ca2+ signaling via voltage-gated Ca2+ channels in MF-LTP induction. We examined the contribution of activity in both dentate granule cell somata and MF terminals to MF-LTP. We found that the induction of stable MF-LTP requires tetanization-induced action potentials not only at MF boutons, but also at dentate granule cell somata. Similarly, blocking Ca2+ influx via voltage-gated Ca2+ channels only at the granule cell soma was sufficient to disrupt MF-LTP. Finally, blocking protein synthesis or blocking fast axonal transport mechanisms via disruption of axonal tubulin filaments resulted in decremental MF-LTP. Collectively, these data suggest that—in addition to Ca2+ influx at the MF terminals—induction of MF synaptic plasticity requires action potential-dependent Ca2+ signaling at granule cell somata, protein synthesis, and fast axonal transport along MFs. A parsimonious interpretation of these results is that somatic activity triggers protein synthesis at the soma; newly synthesized proteins are then transported to MF terminals, where they contribute to the stabilization of MF-LTP. Finally, the present data imply that synaptic plasticity at the MF–CA3 synapse can be affected by local modulation of somatic and presynaptic Ca2+ channel activity.

Calcium-activated chloride channels in the retina

This review examines the function of calcium-activated chloride currents (ICl(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, ICl(Ca) has been most prominently studied in cones, where it activates in response to depolarization-evoked Ca2+ 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 ICl(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.

Inhibitory action of diltiazem on voltage-gated calcium channels in cone photoreceptors

The benzothiazepine, diltiazem, is commonly used as an inhibitor of vascular L-type Ca channels, and is a clinically important anti-anginal and antihypertensive medication. In the retina, diltiazem also inhibits cyclic-nucleotide gated (CNG) channels, including the cGMP-gated channels in photoreceptors, and has been suggested to be a neuroprotectant in an animal model of retinitis pigmentosa, a degenerative disease of photoreceptors. In contrast to CNG channels, the actions of diltiazem on photoreceptor Ca channels have not been studied. We show that D-cis-diltiazem can block Ca channels in cone photoreceptors and that the potency and efficacy of cone photoreceptor Ca channel inhibition by this drug is unconventional. Over the concentration range of 5-500 microM diltiazem, the dose response curve was biphasic with a high affinity saturation level of approximately 30% block in the 20-50 microM range (IC(50)=4.9 microM) and a low affinity saturation block (near 100%) with concentrations up to 500 microM (IC(50)=100.4 microM). The degree of block was found to be equivalent when Bay K 8644 was used to increase Ca channel current, indicating that the levels of block do not result from multiple Ca channel subtypes having differing sensitivities to diltiazem. Calcium imaging showed that the relatively low efficacy of the high-affinity Ca channel block was not due to the species of charge-carrying divalent cation nor that it was associated with dialysis of cellular contents. These data contribute to an emerging perspective that the photoreceptor Ca channel has properties unique from other L-type channels, an important consideration should these channels become a target for testing putative neuroprotective therapies.

Targeted Deletion of Vesicular GABA Transporter from Retinal Horizontal Cells Eliminates Feedback Modulation of Photoreceptor Calcium Channels.

Abstract The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGATflox/flox mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT−/− mice were the same as Cx57-VGAT+/+ controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT−/− mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT−/− mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors.