Response Properties of a Newly Identified Tristratified Narrow Field Amacrine Cell in the Mouse Retina.

Amacrine cells were targeted for whole cell recording using two-photon fluorescence microscopy in a transgenic mouse line in which the promoter for dopamine receptor 2 drove expression of green fluorescent protein in a narrow field tristratified amacrine cell (TNAC) that had not been studied previously. Light evoked a multiphasic response that was the sum of hyperpolarizing and depolarization synaptic inputs consistent with distinct dendritic ramifications in the off and on sublamina of the inner plexiform layer. The amplitude and waveform of the response, which consisted of an initial brief hyperpolarization at light onset followed by recovery to a plateau potential close to dark resting potential and a hyperpolarizing response at the light offset varied little over an intensity range from 0.4 to ~10^6 Rh*/rod/s. This suggests that the cell functions as a differentiator that generates an output signal (a transient reduction in inhibitory input to downstream retina neurons) that is proportional to the derivative of light input independent of its intensity. The underlying circuitry appears to consist of rod and cone driven on and off bipolar cells that provide direct excitatory input to the cell as well as to GABAergic amacrine cells that are synaptically coupled to TNAC. Canonical reagents that blocked excitatory (glutamatergic) and inhibitory (GABA and glycine) synaptic transmission had effects on responses to scotopic stimuli consistent with the rod driven component of the proposed circuit. However, responses evoked by photopic stimuli were paradoxical and could not be interpreted on the basis of conventional thinking about the neuropharmacology of synaptic interactions in the retina.

Inhibitory inputs tune the light response properties of dopaminergic amacrine cells in mouse retina.

Dopamine (DA) is a neuromodulator that in the retina adjusts the circuitry for visual processing in dim and bright light conditions. It is synthesized and released from retinal interneurons called dopaminergic amacrine cells (DACs), whose basic physiology is not yet been fully characterized. To investigate their cellular and input properties as well as light responses, DACs were targeted for whole cell recording in isolated retina using two-photon fluorescence microscopy in a mouse line where the dopamine receptor 2 promoter drives green fluorescent protein (GFP) expression. Differences in membrane properties gave rise to cell-to-cell variation in the pattern of resting spontaneous spike activity ranging from silent to rhythmic to periodic burst discharge. All recorded DACs were light sensitive and generated responses that varied with intensity. The threshold response to light onset was a hyperpolarizing potential change initiated by rod photoreceptors that was blocked by strychnine, indicating a glycinergic amacrine input onto DACs at light onset. With increasing light intensity, the ON response acquired an excitatory component that grew to dominate the response to the strongest stimuli. Responses to bright light (photopic) stimuli also included an inhibitory OFF response mediated by GABAergic amacrine cells driven by the cone OFF pathway. DACs expressed GABA (GABA(A)α1 and GABA(A)α3) and glycine (α2) receptor clusters on soma, axon, and dendrites consistent with the light response being shaped by dual inhibitory inputs that may serve to tune spike discharge for optimal DA release.

Deletion of PrBP/delta impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments.

The mouse Pde6d gene encodes a ubiquitous prenyl binding protein, termed PrBP/delta, of largely unknown physiological function. PrBP/delta was originally identified as a putative rod cGMP phosphodiesterase (PDE6) subunit in the retina, where it is relatively abundant. To investigate the consequences of Pde6d deletion in retina, we generated a Pde6d(-/-) mouse by targeted recombination. Although manifesting reduced body weight, the Pde6d(-/-) mouse was viable and fertile and its retina developed normally. Immunocytochemistry showed that farnesylated rhodopsin kinase (GRK1) and prenylated rod PDE6 catalytic subunits partially mislocalized in Pde6d(-/-) rods, whereas rhodopsin was unaffected. In Pde6d(-/-) rod single-cell recordings, sensitivity to single photons was increased and saturating flash responses were prolonged. Pde6d(-/-) scotopic paired-flash electroretinograms indicated a delay in recovery of the dark state, likely due to reduced levels of GRK1 in rod outer segments. In Pde6d(-/-) cone outer segments, GRK1 and cone PDE6alpha' were present at very low levels and the photopic b-wave amplitudes were reduced by 70%. Thus the absence of PrBP/delta in retina impairs transport of prenylated proteins, particularly GRK1 and cone PDE, to rod and cone outer segments, resulting in altered photoreceptor physiology and a phenotype of a slowly progressing rod/cone dystrophy.

Engineering aspects of enzymatic signal transduction: photoreceptors in the retina.

Identifying the basic module of enzymatic amplification as an irreversible cycle of messenger activation/deactivation by a "push-pull" pair of opposing enzymes, we analyze it in terms of gain, bandwidth, noise, and power consumption. The enzymatic signal transduction cascade is viewed as an information channel, the design of which is governed by the statistical properties of the input and the noise and dynamic range constraints of the output. With the example of vertebrate phototransduction cascade we demonstrate that all of the relevant engineering parameters are controlled by enzyme concentrations and, from functional considerations, derive bounds on the required protein numbers. Conversely, the ability of enzymatic networks to change their response characteristics by varying only the abundance of different enzymes illustrates how functional diversity may be built from nearly conserved molecular components.

Cyclic AMP has no effect on the generation, recovery, or background adaptation of light responses in functionally intact rod outer segments: with implications about the function of phosducin.

In retinal rods, light exposure decreases the total outer segment content of both cGMP and cAMP by about 50%. The functional role of the light-evoked change in cAMP is not known. It is postulated to trigger changes in the phosphorylation state of phosducin, a phosphoprotein that is phosphorylated in the dark by cAMP-dependent protein kinase (PKA) and dephosphorylated by basal phosphatase activity when PKA is inhibited by the light-evoked drop in cAMP. In biochemical studies, dephosphorylated phosducin binds to free beta gamma dimer of transducin (Tbeta gamma) and prevents the regeneration of heterotrimeric transducin by blocking the re-association of the beta gamma and alpha subunits. Phosducin's interaction with Tbeta gamma is blocked when it is phosphorylated on a single residue by PKA. To evaluate the effect of the light-evoked fall in cAMP, functionally intact isolated lizard rod outer segments were dialyzed in whole-cell voltage clamp with a standard internal solution and electrical light responses were recorded with and without adding cAMP to the dialysis solution. Since the total outer segment content of cAMP in darkness is approximately 5 microM, internal dialysis with solution containing a much higher concentration (100 microM) of cAMP (or 8-bromo-cAMP) will overcome the effects of a light-evoked decrease in its concentration by keeping cAMP-dependent processes fully activated. Neither cyclic nucleotide had any influence on the generation, light sensitivity, recovery, or background adaptation of the flash response. These results also argue against the participation of phosducin in the sequence of events that are responsible for these aspects of rod function. This does not exclude the possibility of phosducin being involved in adaptation caused by higher light levels than used in the present study, that is, bleaching adaptation, or in light-dependent processes other than phototransduction.

Longitudinal spread of second messenger signals in isolated rod outer segments of lizards.

1. In vertebrate rods activation of the phototransduction cascade by light triggers changes in the concentrations of at least two diffusible intracellular second messengers (cGMP and Ca2+) whose actions depend on how far they spread from their site of production or entry. To address questions about their spatial spread, cell-attached patch current recording and fluorescence imaging of Calcium Green-dextran were used to measure the longitudinal spread of cGMP and Ca2+, respectively, in functionally intact isolated Gecko gecko lizard rod outer segments under whole-cell voltage clamp. 2. The light-evoked changes in cGMP and Ca2+ concentrations decayed with distance from a site of steady focal activation by two-photon absorption of 1064 nm light with similar decay lengths of approximately 3.5 microm. 3. These results can be understood on the basis of a quantitative model of coupled diffusible intracellular messengers, which is likely to have broad relevance for second messenger signalling pathways in general. 4. The decay length for the spread of adaptation from a site of steady local illumination was about 8 microm, i.e. substantially longer than the decay lengths measured for the spread of cGMP and Ca2+. There are a number of factors, however, that could broaden the apparent relationship between functional changes in the light response and the concentration of a diffusible messenger. For these reasons the measured decay length is an upper limit estimate of the spread of adaptation and does not rule out the possibility that Ca2+ and/or cGMP carry the adaptation signal.

Optical recording of light-evoked calcium signals in the functionally intact retina.

Using two-photon excitation of fluorescent indicator dyes, we measured calcium concentration transients in retinal ganglion and amacrine cells without destroying the light sensitivity of the retina by maximally activating or bleaching the photoreceptors. This allowed an immediate assessment of the cellular morphology and study of the calcium signals evoked by visual stimuli. Calcium dynamics in individual dendritic processes could be examined for extensive periods without deterioration and with little apparent phototoxicity at excitation wavelengths of from 930 to 990 nm. Light-evoked increases in calcium were resolved in ganglion- and amacrine-cell neurites, making it possible to use optical recording to study the relationship between calcium signaling and retinal function.

Effect of rhodopsin C-terminal peptide on photoresponses in functionally intact rod outer segments.

The protein-protein interactions that underlie shut-off of the light-activated rhodopsin were studied using synthetic peptides derived from C-terminal region of the rhodopsin. The photoresponses were recorded in whole-cell voltage clamp from rod outer segments (ROS) that were internally dialyzed with an intracellular solution containing the synthetic peptides. This was the first time that synthetic peptides have been used in functionally intact ROS. None of the tested peptides promoted the shut-off of the photolyzed rhodopsin (R) by stimulating the binding of an activated arrestin to non-phosphorylated R, contrary to what was expected from in vitro experiments (Puig et al. FEBS Lett. 362: 185-188, 1995).

Protein kinase C and IP3 in photoresponses of functionally intact rod outer segments: constraints about their role.

Protein kinase C and polyphosphoinositide metabolism are reported to affect light-activated processes in cell free systems. To investigate their role in phototransduction under more physiological conditions the effects of nonhydrolyzable inositol trisphosphate (IP3) analogs as well as of protein kinase C and phospholipase C inhibitors on the characteristics of the electrical light response were studied. Rod outer segments were dialyzed in whole-cell voltage clamp and photoresponses in the presence and absence of the tested compounds were compared. None of the compounds influenced the light responses suggesting that neither IP3 nor protein kinase C participate in the phototransduction cascade. A number of different proposals about the participation of protein kinase C and inositol trisphosphate (IP3) in the phototransduction process based on a wide variety of in vitro experiments should therefore be reevaluated.

Arrestin with a single amino acid substitution quenches light-activated rhodopsin in a phosphorylation-independent fashion.

Arrestins are members of a superfamily of regulatory proteins that participate in the termination of G protein-mediated signal transduction. In the phototransduction cascade of vertebrate rods, which serves as a prototypical G protein-mediated signaling pathway, the binding of visual arrestin is stimulated by phosphorylation of the C-terminus of photoactivated rhodopsin (Rh*). Arrestin is very selective toward light-activated phosphorhodopsin (P-Rh*). Previously we reported that a single amino acid substitution in arrestin, Arg175Gln, results in a dramatic increase in arrestin binding to Rh* [Gurevich, V. V., & Benovic, J. L. (1995) J. Biol. Chem. 270, 6010-6016]. Here we demonstrate that a similar mutant, arrestin(R175E), binds to light-activated rhodopsin independent of phosphorylation. Arrestin(R175E) binds with high affinity not only to P-Rh* and Rh* but also to light-activated truncated rhodopsin in which the C-terminus phosphorylation sites have been proteolytically removed. In an in vitro assay that monitored rhodopsin-dependent activation of cGMP phosphodiesterase (PDE), wild type arrestin quenched PDE response only when ATP was present to support rhodopsin phosphorylation. In contrast, as little as 30 nM arrestin(R175E) effectively quenched PDE activation in the absence of ATP. Arrestin(R175E) had no effect when the lifetime of Rh* no longer contributed to the time course of PDE activity, suggesting that it disrupts signal transduction at the level of rhodopsin-transducin interaction.

The mechanisms of vertebrate light adaptation: speeded recovery versus slowed activation.

Light adaptation in vertebrate photoreceptors is commonly attributed to a feedback mechanism that reduces the amplitude of the receptor potential by speeding the inactivation of the transduction cascade and hastening the recovery process. Recent studies have challenged this model and suggest instead that desensitization originates mainly from changes in the activation phase rather than the recovery phase of the response. This has important implications for understanding the molecular mechanisms that underlie the control of sensitivity in this G-protein-coupled, signal-transduction pathway.

Ca2+ dependence of dark- and light-adapted flash responses in rod photoreceptors.

Light adaptation is thought to be orchestrated by a Ca2+ feedback signal that desensitizes the response by speeding recovery. To evaluate the role of Ca2+ in adaptation, we compared the effect of lowered Ca2+ on response properties in darkness and during adaptation. Internal Ca2+ was reduced from its normal resting dark level (535 nM) by either background illumination or exposure to Ringer's solution containing low Ca2+ and/or cyclic GMP-gated channel blockers in darkness. Ca2+ reductions in light decreased the activation gain of the transduction process and speeded recovery kinetics, while equivalent Ca2+ reductions in darkness caused similar gain reduction without accelerating recovery. This indicates that adaptational changes in the response are not due purely to feedback effects on recovery.

The calcium feedback signal in the phototransduction cascade of vertebrate rods.

Intracellular free Ca (Cai) was measured in functionally intact rod outer segments in darkness and during light responses using the fluorescent Ca indicator Indo-dextran. In darkness, Cai was 554 +/- 25 nM (n = 28) for -85 +/- 2 pA of circulating dark current (Id) and declined in saturating light to a minimum value of approximately 50 nM with a time course that paralleled the fall in Na:Ca,K exchange current. During a subsaturating flash response that reduced Id by 70%, Cai fell to a minimum of approximately 325 nM and recovered incompletely to a plateau of approximately 450 nM that lasted approximately 15 s after full recovery of Id. During a 60 s step that caused approximately 7-fold reduction in sensitivity of superimposed flash responses, Cai reached a steady-state level of approximately 252 nM.

Purification and physiological evaluation of a guanylate cyclase activating protein from retinal rods.

In retinal rods light triggers a cascade of enzymatic reactions that increases cGMP hydrolysis and generates an electrical signal by causing closure of cGMP-gated ion channels in the photoreceptor outer segment. This leads to a decrease in internal Ca, which activates guanylate cyclase and promotes photoresponse recovery by stimulating the resynthesis of cGMP. We report here that the activation of guanylate cyclase by low Ca is mediated by an approximately 20-kDa protein purified from bovine rod outer segments by using DEAE-Sepharose, hydroxylapatite, and reverse-phase chromatographies. In a reconstituted system, this protein restores the Ca-sensitive regulation of guanylate cyclase and when dialyzed into functionally intact lizard rod outer segment decreases the sensitivity, time to peak, and recovery time of the flash response.

Visual transduction in dialysed detached rod outer segments from lizard retina.

1. Properties of a new preparation for studying the physiology and biochemistry of phototransduction in retinal rods are described. Whole-cell voltage clamp was used to record the generation, maintenance and light-sensitivity of dark current in rod outer segments that had been isolated from the rest of the receptor cell by detachment at the connecting cilium. 2. Detached outer segments dialysed with standard internal solution supplemented with physiological amounts of ATP (5 mM) and GTP (1 mM) developed a standing inward dark current that was the sum of three components: approximately 91% light-sensitive current, approximately 6% Na(+)-Ca2+,K+ exchange current and approximately 3% leakage current. Light-sensitive dark current (mean amplitude approximately -63 pA) was suppressed transiently by brief flashes in an intensity-dependent manner. Light responses had the same kinetics, sensitivity and intensity-response relationship as those recorded from intact rods. 3. Dialysed outer segments differed from intact rods in that intense flashes evoked saturating responses that recovered incompletely to a plateau of reduced dark current caused by incomplete inactivation of the transduction cascade. Light sensitivity was reduced for a short time following an intense flash and then recovered despite persistent reduction of dark current. This suggests that there is no fixed relationship between dark current amplitude and light sensitivity. 4. Light-sensitive dark current faded rapidly when outer segments were not supplied with nucleotides. Outer segments dialysed with solution that contained cyclic GMP, but no ATP or GTP, supported dark current at a level that increased with [cyclic GMP]. When basal phosphodiesterase (PDE) activity is inhibited, 8 microM cyclic GMP supports a dark current of approximately 70 pA. 5. Light sensitivity decreased during recordings made with solution that contained only cyclic GMP, consistent with the inhibition of G protein activation by loss of GTP. After thorough nucleoside triphosphate depletion, however, intense illumination evoked a transient increase rather than a decrease in dark current, i.e. an inverted light response. This result suggests that isomerized rhodopsin may generate a signal that causes either inhibition of basal PDE activity or release of bound cyclic GMP. 6. Sustained Na(+)-Ca2+,K+ exchange current was recorded during steady illumination when Ca2+, but not when Mg2+, was added to the dialysis solution. Exchange current increased with the amount of added Ca2+ and saturated at approximately 18 pA when the dialysis solution contained > or = 10 mM Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of recoverin-like calcium-binding proteins on the photoresponse of retinal rods.

The rod photoresponse is triggered by an enzyme cascade that stimulates cGMP hydrolysis. The resulting fall in cGMP leads to a decrease in Ca2+, which promotes photoresponse recovery by activating guanylate cyclase, causing cGMP resynthesis. In vitro biochemical studies suggest that Ca2+ activation of guanylate cyclase is medicated by recoverin, a 26 kd Ca(2+)-binding protein. To evaluate this, exogenous bovine recoverin and two other homologous Ca(2+)-binding proteins from chicken and Gecko retina were dialyzed into functionally intact Gecko rods using whole-cell recording. All three proteins prolonged the rising phase of the photoresponse without affecting the kinetics of response recovery. These results suggest that recoverin-like proteins affect termination of the transduction cascade, rather than mediate Ca(2+)-sensitive activation of guanylate cyclase.

Some unresolved issues in the physiology and biochemistry of phototransduction.

A number of recent review articles have discussed what is known about the events responsible for generating the electrical light response in vertebrate photoreceptors. The similarity of the material covered and the unanimity of the conclusions drawn have given rise to the popular, but false, impression that visual transduction is understood fully. The purpose of the present review is to dispell this notion by focusing on some of the unresolved issues.

The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction.

The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.