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.

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 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.

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.

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 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.

Hair cell interactions in the statocyst of Hermissenda.

Hair cells in the statocyst of Hermissenda crassicornis respond to mechanical stimulation with a short latency (<2 ms) depolarizing generator potential that is followed by hyperpolarization and inhibition of spike activity. Mechanically evoked hyperpolarization and spike inhibition were abolished by cutting the static nerve, repetitive mechanical stimulation, tetrodotoxin (TTX), and Co(++). Since none of these procedures markedly altered the generator potential it was concluded that the hyperpolarization is an inhibitory synaptic potential and not a component of the mechanotransduction process. Intracellular recordings from pairs of hair cells in the same statocyst and in statocysts on opposite sides of the brain revealed that hair cells are connected by chemical and/or electrical synapses. All chemical interactions were inhibitory. Hyperpolarization and spike inhibition result from inhibitory interactions between hair cells in the same and in opposite statocysts.

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.

Selective uptake of lucifer yellow by retinal cells.

When turtle retinae were incubated with the fluorescent dye, lucifer yellow, in the absence of Ca2+, the dye was selectively accumulated by cell bodies located in the inner nuclear layer (INL). The morphological features of the labeled cells suggested that they were bipolar cells. Other fluorescent dyes, Procion yellow and Primulin, were also taken up by somata in the INL, in the absence of external Ca2+, although the identity of the labeled cells was uncertain. As with turtle retina, lucifer yellow was accumulated predominantly by cell bodies in the INL of goldfish, frog, and rat retinae. Lucifer yellow uptake appeared to be independent of synaptic activity since dark-adaptation or aspartate treatment of retinae did not alter the dye uptake. Further, retinae from dystrophic (RCS) rats showed uptake similar to that seen in normal rat retinae. After uptake, most of the dye was found intracellularly as patches or vacuoles in the somata of the labeled cells. Dye uptake was not inhibited by removal of Na+ from the incubation medium. Further, prior treatment with metabolic inhibitors, cyanide and iodoacetate, or cytochalasin B, did not block the dye uptake.

Distribution of membrane proteins in mechanically dissociated retinal rods.

Solitary rods were isolated from frog retinas by mechanical dissociation. Typically, the rods cleave sclerad to the nucleus and consist of outer segments with attached partial inner segments with either tapered or rounded profiles. Light and electron microscopy reveal that the outer and inner segments of rods with tapered inner segments, like rods in the intact retina, are joined by a single connecting cilium. In contrast, the outer and inner segments of rods with rounded inner segments are fused, with no extracellular cleft between the two segments. Opsin distribution was studied in both unfused and fused rods by light and electron microscopic immunocytochemistry. Extensive surface labeling is restricted to the outer segments of tapered rods, as observed in vivo. In contrast, both inner and outer segments of rods with rounded inner segments (fused) label heavily with anti-opsin. Thus opsin, a mobile membrane protein, diffuses from the outer to the inner segment of fused rods. Segregated distribution of opsin in unfused rods suggests that the connecting cilium and/or its associated structures may normally act as a diffusion barrier between the outer and inner segments to mobile membrane proteins such as opsin. Immunofluorescence studies demonstrate that Na+/K+ ATPase is restricted in distribution to the inner segment and calycal processes of both fused and unfused isolated rods, as observed in vivo. Maintenance of its restricted distribution in fused cells indicates that Na+/K+ ATPase is not mobile and may be tethered in the surface membrane of the inner segment.

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.

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.

Multiple light-evoked conductance changes in the photoreceptors of Hermissenda crassicornis.

1. Light responses were recorded from the photoreceptors of Hermissenda crassicornis. The response to a flash is a complex potential change involving an initial depolarization, a hyperpolarization, and a depolarizing tail. None of the phases of the response are due to synaptic interactions.2. Polarization of the membrane by extrinsic current indicates that three separate conductance changes are associated with the response. The initial depolarization and hyperpolarization are accompanied by conductance increases and the tail with a conductance decrease. The initial depolarization has a positive reversal potential and the hyperpolarizing and tail phase have a reversal voltage more negative than resting potential.3. The different processes that give rise to the conductance changes have similar spectral sensitivities but are affected unequally by light adaptation. Strong light adaptation reduced the depolarizing phases more than the hyperpolarizing phase, so that following an adapting stimulus the cell responded to illumination with a pure hyperpolarization (isolated hyperpolarization).4. Removal of external Na(+) ions greatly reduced the initial depolarization. In Na(+)-free sea water the cell responds to dim flashes with a slow depolarization (isolated tail) that involves a conductance decrease, and has the same reversal potential as the hyperpolarizing response recorded from light adapted cells.5. The amplitude of the isolated hyperpolarization and tail varied inversely with the external K(+) concentration.6. It is concluded that in Hermissenda photoreceptors light initiates processes that result in three distinct permeability changes. Following a brief flash there is: a rapid and transient increase in Na(+) permeability that is responsible for the initial depolarization, a less rapid increase in K(+) permeability that is responsible for the hyperpolarizing phase, and a delayed decrease in K(+) permeability that gives rise to the depolarizing tail.

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.

Light-evoked contraction of the photosensitive iris of the frog.

Smooth muscle cells of the frog iris sphincter contain rhodopsin and contract in response to light. The mechanism of light-evoked contraction was studied with particular attention paid to the role of calcium. The contractile proteins of the sphincter smooth muscle cell can be activated by an increase in intracellular calcium. Light-evoked contraction, however, is not accompanied by a measurable change in membrane potential and occurs in the absence of extracellular Na+ and/or Ca2+, as well as in the presence of isotonic KCI. Maximum light-evoked tension is reduced by exposure to Ca2+-free solutions containing EGTA and high K+ and is restored by incubation in solutions containing Ca2+. The restored response, which persists after return to Ca2+-free solution, depends on the concentration of Ca2+ in the incubating solution and on the duration of the incubation. The results support the conclusion that light produces contraction of the iris sphincter by causing the release of Ca2+ from an intracellular storage site.

Patch-clamp recordings of the light-sensitive dark noise in retinal rods from the lizard and frog.

In cell-attached recordings from rods in the intact lizard retina, light decreased a standing inward membrane current with a reversal potential approximately 60 mV more positive than the resting potential. The peak amplitude of saturating responses depended upon the area of recorded membrane and varied from cell to cell over approximately 100-fold range. Small patches of membrane gave variable responses to identical moderately intense flashes. Whole-cell voltage-clamp recordings were obtained on isolated frog rods with intact ellipsoids. Peak whole-cell photocurrent was related to flash intensity by a Michaelis equation with saturating response amplitudes ranging up to 30 pA in 0.1 mM-Ca2+ Ringer solution. In darkness the steady-state current-voltage relation, determined with whole-cell voltage clamp, showed outward rectification. Photocurrent had nearly constant amplitude between -80 and -10 mV, a mean reversal potential of +8 mV and recovered from flashes more slowly at positive holding potentials. Although it was not possible to resolve light-sensitive single-channel current events, power spectral analysis revealed both low- and high-frequency components of the light-sensitive noise in both cell-attached and whole-cell recordings. The low-frequency component was described by the product of two Lorentzians using time constants derived from the kinetics of the dim flash response. The high-frequency component of the light-sensitive noise was described by a single Lorentzian with a half-power frequency of 62 Hz in lizard and 212 Hz in frog. The half-power frequency was not appreciably affected by steady illumination. The Lorentzian nature of the noise suggests that the light-sensitive channel is a pore rather than a shuttle-type carrier. In cell-attached recordings the high-frequency component declined monotonically with increasing light intensity, suggesting that less than one-half of the channels are open in darkness. Furthermore, the ratio of the variance of the high-frequency noise to the mean photocurrent was independent of light intensity. Changing external Ca2+ from 0.1 to 0.5 mM reduced the ratio from 19.7 to 9.0 fA without a significant effect on the cut-off frequency of the noise. The results support the conclusion that the light-sensitive pore is opened by an internal transmitter that acts as an agonist and that both open and closed states of the pore may be blocked by external Ca2+. The conductance of the light-sensitive pore in the absence of external Ca2+ is estimated to be 1.25-2 pS.(ABSTRACT TRUNCATED AT 400 WORDS)