The PDE6 mutation in the rd10 retinal degeneration mouse model causes protein mislocalization and instability and promotes cell death through increased ion influx.

The retinal degeneration model rd10 contains a missense mutation of the catalytic PDE6 ß subunit, which hydrolyzes cGMP in response to light. This model produces cell death more slowly than others caused by PDE6 loss of function, making it of particular interest for studying potential therapeutics. We used morphology, biochemistry, and single-cell physiology to examine the mechanism of rd10 degeneration. Our results show that the mutation produces no alteration of Pde6b RNA but does dramatically decrease maximal and basal PDE6 activity, apparently caused by a decrease in protein stability and transport. The enzymatic properties of the remaining mutant PDE6 appear to be nearly normal. We demonstrate that an increase in free cGMP, which would result from decreased PDE6 activity and serve to increase opening of the cGMP-gated channels and calcium influx, is an underlying cause of cell death: degeneration of rd10/Cngb1-/- double mutants is slower than the parent rd10 line. Paradoxically, degeneration in rd10/Cngb1-/- is also slower than in Cngb1-/- This rescue is correlated with a lowering of cGMP content in Cngb1-/- retinas and suggests that it may be caused by mislocalization of active PDE6. Single-cell recordings from rd10 rods show that the rates of rise and decay of the response are significantly slower; simulations indicate that these changes are primarily the result of the decrease in PDE6 concentration and rod collecting area. Together, these results provide insights into the complex mechanisms that underlie rd10-mediated retinal degeneration and a cautionary note for analysis of therapeutic interventions.

Role of recoverin in rod photoreceptor light adaptation.

KEY POINTS: Recoverin is a small molecular-weight, calcium-binding protein in rod outer segments that can modulate the rate of rhodopsin phosphorylation. We describe two additional and perhaps more important functions during photoreceptor light adaptation. Recoverin influences the rate of change of adaptation. In wild-type rods, sensitivity and response integration time adapt with similar time constants of 150-200 ms. In Rv-/- rods lacking recoverin, sensitivity declines faster and integration time is already shorter and not significantly altered. During steady light exposure, rod circulating current slowly increases during a time course of tens of seconds, gradually extending the operating range of the rod. In Rv-/- rods, this mechanism is deleted, steady-state currents are already larger and rods saturate at brighter intensities. We propose that recoverin modulates spontaneous and light-activated phophodiesterase-6, the phototransduction effector enzyme, to increase sensitivity in dim light but improve responsiveness to change in brighter illumination. ABSTRACT: Recoverin is a small molecular-weight, calcium-binding protein in rod outer segments that binds to G-protein receptor kinase 1 and can alter the rate of rhodopsin phosphorylation. A change in phosphorylation should change the lifetime of light-activated rhodopsin and the gain of phototransduction, but deletion of recoverin has little effect on the sensitivity of rods either in the dark or in dim-to-moderate background light. We describe two additional functions perhaps of greater physiological significance. (i) When the ambient intensity increases, sensitivity and integration time decrease in wild-type (WT) rods with similar time constants of 150-200 ms. Recoverin is part of the mechanism controlling this process because, in Rv-/- rods lacking recoverin, sensitivity declines more rapidly and integration time is already shorter and not further altered. (ii) During steady light exposure, WT rod circulating current slowly increases during a time course of tens of seconds, gradually extending the operating range of the rod. In Rv-/- rods, this mechanism is also deleted, steady-state currents are already larger and rods saturate at brighter intensities. We argue that neither (i) nor (ii) can be caused by modulation of rhodopsin phosphorylation but may instead be produced by direct modulation of phophodiesterase-6 (PDE6), the phototransduction effector enzyme. We propose that recoverin in dark-adapted rods keeps the integration time long and the spontaneous PDE6 rate relatively high to improve sensitivity. In background light, the integration time is decreased to facilitate detection of change and motion and the spontaneous PDE6 rate decreases to augment the rod working range.

Detection of single photons by toad and mouse rods.

Amphibian and mammalian rods can both detect single photons of light even though they differ greatly in physical dimensions, mammalian rods being much smaller in diameter than amphibian rods. To understand the changes in physiology and biochemistry required by such large differences in outer segment geometry, we developed a computational approach, taking into account the spatial organization of the outer segment divided into compartments, together with molecular dynamics simulations of the signaling cascade. We generated simulations of the single-photon response together with intrinsic background fluctuations in toad and mouse rods. Combining this computational approach with electrophysiological data from mouse rods, we determined key biochemical parameters. On average around one phosphodiesterase (PDE) molecule is spontaneously active per mouse compartment, similar to the value for toad, which is unexpected due to the much smaller diameter in mouse. A larger number of spontaneously active PDEs decreases dark noise, thereby improving detection of single photons; it also increases cGMP turnover, which accelerates the decay of the light response. These constraints explain the higher PDE density in mammalian compared with amphibian rods that compensates for the much smaller diameter of mammalian disks. We further find that the rate of cGMP hydrolysis by light-activated PDE is diffusion limited, which is not the case for spontaneously activated PDE. As a consequence, in the small outer segment of a mouse rod only a few activated PDEs are sufficient to generate a signal that overcomes noise, which permits a shorter lifetime of activated rhodopsin and greater temporal resolution.

Modulation of mouse rod photoreceptor responses by Grb14 protein.

Previous experiments have indicated that growth factor receptor-bound protein 14 (Grb14) may modulate rod photoreceptor cGMP-gated channels by decreasing channel affinity for cGMP; however, the function of Grb14 in rod physiology is not known. In this study, we examined the role of Grb14 by recording electrical responses from rods in which the gene for the Grb14 protein had been deleted. Suction-electrode recordings from single mouse rods showed that responses of dark-adapted Grb14(-/-) mice to brief flashes decayed more rapidly than strain-controlled wild type (WT) rods, with decreased values of both integration time and the exponential time course of decay (τREC). This result is consistent with an increase in channel affinity for cGMP produced by deletion of Grb14. However, Grb14(-/-) mouse rods also showed little change in dark current and a large and significant decrease in the limiting time constant τD, which are not consistent with an effect on channel affinity but seem rather to indicate modulation of the rate of inactivation of cyclic nucleotide phosphodiesterase 6 (PDE6). Grb14 has been reported to translocate from the inner to the outer segment in bright light, but we saw effects on response time course even in dark-adapted rods, although the effects were somewhat greater after rods had been adapted by exposure to bleaching illumination. Our results indicate that the mechanism of Grb14 action may be more complex than previously realized.

Modulation of mouse rod response decay by rhodopsin kinase and recoverin.

Light isomerizes 11-cis-retinal in a retinal rod and produces an active form of rhodopsin (Rh*) that binds to the G-protein transducin and activates the phototransduction cascade. Rh* is turned off by phosphorylation by rhodopsin kinase [G-protein-coupled receptor kinase 1 (GRK1)] and subsequent binding of arrestin. To evaluate the role of GRK1 in rod light response decay, we have generated the transgenic mouse RKS561L in which GRK1, which is normally present at only 2-3% of rhodopsin, is overexpressed by ∼12-fold. Overexpression of GRK1 increases the rate of Rh* phosphorylation and reduces the exponential decay constant of the response (τ(REC)) and the limiting time constant (τ(D)) both by ∼30%; these decreases are highly significant. Similar decreases are produced in Rv(-/-) rods, in which the GRK1-binding protein recoverin has been genetically deleted. These changes in response decay are produced by acceleration of light-activated phosphodiesterase (PDE*) decay rather than Rh* decay, because light-activated PDE* decay remains rate limiting for response decay in both RKS561L and Rv(-/-) rods. A model incorporating an effect of GRK1 on light-activated PDE* decay rate can satisfactorily account for the changes in response amplitude and waveform. Modulation of response decay in background light is nearly eliminated by deletion of recoverin. Our experiments indicate that rhodopsin kinase and recoverin, in addition to their well-known role in regulating the turning off of Rh*, can also modulate the decay of light-activated PDE*, and the effects of these proteins on light-activated PDE* decay may be responsible for the quickening of response recovery in background light.

Loss of caveolin-1 impairs retinal function due to disturbance of subretinal microenvironment.

Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K(+) affinity of the RPE-expressed α1-Na(+)/K(+)-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis.

Spontaneous activity of opsin apoprotein is a cause of Leber congenital amaurosis.

Mutations in Rpe65 disrupt synthesis of the opsin chromophore ligand 11-cis-retinal and cause Leber congenital amaurosis (LCA), a severe, early-onset retinal dystrophy. To test whether light-independent signaling by unliganded opsin causes the degeneration, we used Rpe65-null mice, a model of LCA. Dark-adapted Rpe65-/- mice behaved as if light adapted, exhibiting reduced circulating current, accelerated response turn-off, and diminished intracellular calcium. A genetic block of transducin signaling completely rescued degeneration irrespective of an elevated level of retinyl ester. These studies clearly show that activation of sensory transduction by unliganded opsin, and not the accumulation of retinyl esters, causes light-independent retinal degeneration in LCA. A similar mechanism may also be responsible for degeneration induced by vitamin A deprivation.

Channel modulation and the mechanism of light adaptation in mouse rods.

Vertebrate photoreceptors are thought to adapt to light by a change in Ca(2+), which is postulated to mediate modulation of (1) excited rhodopsin (Rh*) by Ca(2+)-dependent binding of recoverin, (2) guanylyl cyclase activity via Ca(2+)-dependent GCAP proteins, and (3) cyclic nucleotide-gated channels by binding of Ca(2+)-calmodulin. Previous experiments genetically deleted recoverin and the GCAPs and showed that significant regulation of sensitivity survives removal of (1) and (2). We genetically deleted the channel Ca(2+)-calmodulin binding site in the mouse Mus musculus and found that removal of (3) alters response waveform, but removal of (3) or of (2) and (3) together still leaves much of adaptation intact. These experiments demonstrate that an important additional mechanism is required, which other experiments indicate may be regulation of phosphodiesterase 6 (PDE6). We therefore constructed a kinetic model in which light produces a Ca(2+)-mediated decrease in PDE6 decay rate, with the novel feature that both spontaneously activated and light-activated PDE6 are modulated. This model, together with Ca(2+)-dependent acceleration of guanylyl cyclase, can successfully account for changes in sensitivity and response waveform in background light.

Background light produces a recoverin-dependent modulation of activated-rhodopsin lifetime in mouse rods.

The Ca(2+)-binding protein recoverin is thought to regulate rhodopsin kinase and to modulate the lifetime of the photoexcited state of rhodopsin (Rh*), the visual pigment of vertebrate rods. Recoverin has been postulated to inhibit the kinase in darkness, when Ca(2+) is high, and to be released from the disk membrane in light when Ca(2+) is low, accelerating rhodopsin phosphorylation and shortening the lifetime of Rh*. This proposal has remained controversial, in part because the normally rapid turnoff of Rh* has made Rh* modulation difficult to study in an intact rod. To circumvent this problem, we have made mice that underexpress rhodopsin kinase so that Rh* turnoff is rate limiting for the decay of the rod light response. We show that background light speeds the decay of Rh* turnoff, and that this no longer occurs in mice that have had recoverin knocked out. This is the first demonstration in an intact rod that light accelerates Rh* inactivation and that the Ca(2+)-binding protein recoverin may be required for the light-dependent modulation of Rh* lifetime.

Night blindness and the mechanism of constitutive signaling of mutant G90D rhodopsin.

The G90D rhodopsin mutation is known to produce congenital night blindness in humans. This mutation produces a similar condition in mice, because rods of animals heterozygous (D+) or homozygous (D+/+) for this mutation have decreased dark current and sensitivity, reduced Ca(2+), and accelerated values of tau(REC) and tau(D), similar to light-adapted wild-type (WT) rods. Our experiments indicate that G90D pigment activates the cascade, producing an equivalent background light of approximately 130 Rh* rod(-1) for D+ and 890 Rh* rod(-1) for D+/+. The active species of the G90D pigment could be unregenerated G90D opsin or G90D rhodopsin, either spontaneously activated (as Rh*) or in some other form. Addition of 11-cis-retinal in lipid vesicles, which produces regeneration of both WT and G90D opsin in intact rods and ROS membranes, had no effect on the waveform or sensitivity of dark-adapted G90D responses, indicating that the active species is not G90D opsin. The noise spectra of dark-adapted G90D and WT rods are similar, and the G90D noise variance is much less than of a WT rod exposed to background light of about the same intensity as the G90D equivalent light, indicating that Rh* is not the active species. We hypothesize that G90D rhodopsin undergoes spontaneous changes in molecular conformation which activate the transduction cascade with low gain. Our experiments provide the first indication that a mutant form of the rhodopsin molecule bound to its 11-cis-chromophore can stimulate the visual cascade spontaneously at a rate large enough to produce visual dysfunction.

Modulation of phosphodiesterase6 turnoff during background illumination in mouse rod photoreceptors.

In rod photoreceptors of wild-type mice, background light produces an acceleration of the decay of responses to brief flashes, accompanied by a decrease in the rate-limiting time constant for response decay. In rods in which phosphodiesterase gamma (PDEgamma) lacks one of its sites of phosphorylation (T35A rods), both the waveform of response decay and the rate-limiting time constant are nearly unaffected by backgrounds. These effects are not the result of the removal of the phosphorylation site per se, because rods lacking both of the phosphorylation sites of PDEgamma (T22A/T35A rods) adapt to light in a nearly normal manner. Because PDEgamma is one of the proteins of the GTPase activating protein (GAP) complex, our experiments argue for a novel mechanism of photoreceptor light adaptation produced by modulation of GAP-dependent hydrolysis of transducin alpha GTP. In PDEgamma T35A rods, a change in the conformation of the PDEgamma subunit may hinder or mask this mechanism, which in mammals appears to be primarily responsible for the quickening of the temporal resolution of the rod response in backgrounds. Modulation of PDE turnoff also helps to prevent premature saturation of the rod in bright backgrounds, thus making an important contribution to light adaptation. Our experiments provide evidence for modulation of GAP protein-dependent response turnoff, which may also play a role in controlling signal duration at hormone receptors and synapses in the CNS.

Constitutive excitation by Gly90Asp rhodopsin rescues rods from degeneration caused by elevated production of cGMP in the dark.

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca(2+) sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca(2+) in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca(2+) measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca(2+) sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca(2+) concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca(2+) can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

GAP-independent termination of photoreceptor light response by excess gamma subunit of the cGMP-phosphodiesterase.

We have generated a mouse with rod photoreceptors overexpressing the gamma inhibitory subunit (PDE6gamma) of the photoreceptor G-protein effector cGMP phosphodiesterase (PDE6). PDE6gamma overexpression decreases the rate of rise of the rod response at dim intensities, indicating a reduction in the gain of transduction that may be the result of cytoplasmic PDE6gamma binding to activated transducin alpha GTP (Talpha-GTP) before the Talpha-GTP binds to endogenous PDE6gamma. Excess PDE6gamma also produces a marked acceleration in the falling phase of the light response and more rapid recovery of sensitivity and circulating current after prolonged light exposure. These effects are not mediated by accelerating GTP hydrolysis through the GAP (GTPase activating protein) complex, because the decay of the light response is also accelerated in rods that overexpress PDE6gamma but lack RGS9. Our results show that the PDE6gamma binding sites of PDE6 alpha and beta are accessible to excess (presumably cytoplasmic) PDE6gamma in the light, once endogenous PDE6gamma has been displaced from its binding site by Talpha-GTP. They also suggest that in the presence of Talpha-GTP, the PDE6gamma remains attached to the rest of the PDE6 molecule, but after conversion of Talpha-GTP to Talpha-GDP, the PDE6gamma may dissociate from the PDE6 and exchange with a cytoplasmic pool. This pool may exist even in wild-type rods and may explain the decay of rod photoresponses in the presence of nonhydrolyzable analogs of GTP.

Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness.

Mutations in the beta-subunit of cGMP-phosphodiesterase (PDE6beta) can lead to either progressive retinal disease, such as human retinitis pigmentosa (RP), or stationary disease, such as congenital stationary night blindness (CSNB). Individuals with CSNB in the Rambusch pedigree were found to carry the H258N allele of PDE6B (MIM# 180072); a similar mutation was not found in RP patients. This report describes an individual carrying the H258N allele, who presented with generalized retinal dysfunction affecting the rod system and a locus of dysfunction at the rod-bipolar interface. Also described are preclinical studies in which transgenic mice with the H258N allele were generated to study the pathophysiological mechanisms of CSNB. While Pde6b(rd1)/Pde6b(rd1) mice have severe photoreceptor degeneration, as in human RP, the H258N transgene rescued these cells. The cGMP-PDE6 activity of dark-adapted H258N mice showed an approximate three-fold increase in the rate of retinal cGMP hydrolysis: from 130.1 nmol x min(-1) x nmol(-1) rhodopsin in wild-type controls to 319.2 nmol x min(-1) x nmol(-1) rhodopsin in mutants, consistent with the hypothesis that inhibition of the PDE6beta activity by the regulatory PDE6gamma subunit is blocked by this mutation. In the albino (B6CBA x FVB) F2 hybrid background, electroretinograms (ERG) from H258N mice were similar to those obtained from affected Rambusch family members, as well as humans with the most common form of CSNB (X-linked), demonstrating a selective loss of the b-wave with relatively normal a-waves. When the H258N allele was introduced into the DBA background, there was no evidence of selective reduction in b-wave amplitudes; rather a- and b-wave amplitudes were both reduced. Thus, factors other than the PDE6B mutation itself could contribute to the variance of an electrophysiological response. Therefore, caution is advisable when interpreting physiological phenotypes associated with the same allele on different genetic backgrounds. Nevertheless, such animals should be of considerable value in further studies of the molecular pathology of CSNB.

Effects of low AIPL1 expression on phototransduction in rods.

PURPOSE: To investigate the impact of aryl hydrocarbon receptor-interacting protein-like (AIPL)-1 on photoreception in rods. METHODS: Photoresponses of mouse rods expressing lowered amounts of AIPL1 were studied by single-cell and electroretinogram (ERG) recordings. Phototransduction protein levels and enzymatic activities were determined in biochemical assays. Ca2+ dynamics were probed with a fluorescent dye. Comparisons were made to rods expressing mutant Y99C guanylate cyclase activating protein (GCAP)-1, to understand which effects arose from elevated dark levels of cGMP and Ca2+. RESULTS: Except for PDE, transduction protein levels were normal in low-AIPL1 retinas, as were guanylate cyclase (GC), rhodopsin kinase (RK), and normalized phosphodiesterase (PDE) activities. Y99C and low-AIPL1 rods were more sensitive to flashes than normal, but flash responses of low-AIPL1 rods showed an abnormal delay, reduced rate of increase, and longer recovery not present in Y99C rod responses. In addition, low-AIPL1 rods but not Y99C rods failed to reach the normal light-induced minimum in Ca2+ concentration. CONCLUSIONS: Reduced AIPL1 delayed the photoresponse, decreased its amplification constant, slowed a rate-limiting step in its recovery, and limited the light-induced decrease in Ca2+. Not all changes were attributable to decreased PDE or to elevated cGMP and Ca2+ in darkness. Therefore, AIPL1 directly or indirectly affects more than one component of phototransduction.

The Y99C mutation in guanylyl cyclase-activating protein 1 increases intracellular Ca2+ and causes photoreceptor degeneration in transgenic mice.

Guanylyl cyclase-activating proteins (GCAPs) are Ca2+-binding proteins that activate guanylyl cyclase when free Ca2+ concentrations in retinal rods and cones fall after illumination and inhibit the cyclase when free Ca2+ reaches its resting level in the dark. Several forms of retinal dystrophy are caused by mutations in GUCA1A, the gene coding for GCAP1. To investigate the cellular mechanisms affected by the diseased state, we created transgenic mice that express GCAP1 with a Tyr99Cys substitution (Y99C GCAP1) found in human patients with a late-onset retinal dystrophy (Payne et al., 1998). Y99C GCAP1 shifted the Ca2+ sensitivity of the guanylyl cyclase in photoreceptors, keeping it partially active at 250 nM free Ca2+, the normal resting Ca2+ concentration in darkness. The enhanced activity of the cyclase in the dark increased cyclic nucleotide-gated channel activity and elevated the rod outer segment Ca2+ concentration in darkness, measured by using fluo-5F and laser spot microscopy. In different lines of transgenic mice the magnitude of this effect rose with the Y99C GCAP1 expression. Surprisingly, there was little change in the rod photoresponse, indicating that dynamic Ca2+-dependent regulation of cGMP synthesis was preserved. However, the photoreceptors in these mice degenerated, and the rate of the cell loss increased with the level of the transgene expression, unlike in transgenic mice that overexpressed normal GCAP1. These results provide the first direct evidence that a mutation linked to congenital blindness increases Ca2+ in the outer segment, which may trigger the apoptotic process.

Rhodopsin kinase and recoverin modulate phosphodiesterase during mouse photoreceptor light adaptation.

Light stimulates rhodopsin in a retinal rod to activate the G protein transducin, which binds to phosphodiesterase (PDE), relieving PDE inhibition and decreasing guanosine 3',5'-cyclic monophosphate (cGMP) concentration. The decrease in cGMP closes outer segment channels, producing the rod electrical response. Prolonged exposure to light decreases sensitivity and accelerates response kinetics in a process known as light adaptation, mediated at least in part by a decrease in outer segment Ca(2+). Recent evidence indicates that one of the mechanisms of adaptation in mammalian rods is down-regulation of PDE. To investigate the effect of light and a possible role of rhodopsin kinase (G protein-coupled receptor kinase 1 [GRK1]) and the GRK1-regulating protein recoverin on PDE modulation, we used transgenic mice with decreased expression of GTPase-accelerating proteins (GAPs) and, consequently, a less rapid decay of the light response. This slowed decay made the effects of genetic manipulation of GRK1 and recoverin easier to observe and interpret. We monitored the decay of the light response and of light-activated PDE by measuring the exponential response decay time (τREC) and the limiting time constant (τD), the latter of which directly reflects light-activated PDE decay under the conditions of our experiments. We found that, in GAP-underexpressing rods, steady background light decreased both τREC and τD, and the decrease in τD was nearly linear with the decrease in amplitude of the outer segment current. Background light had little effect on τREC or τD if the gene for recoverin was deleted. Moreover, in GAP-underexpressing rods, increased GRK1 expression or deletion of recoverin produced large and highly significant accelerations of τREC and τD. The simplest explanation of our results is that Ca(2+)-dependent regulation of GRK1 by recoverin modulates the decay of light-activated PDE, and that this modulation is responsible for acceleration of response decay and the increase in temporal resolution of rods in background light.

Effect of knocking down the insulin receptor on mouse rod responses.

Previous experiments have shown that the insulin receptor (IR) is expressed in mammalian rods and contributes to the protection of photoreceptors during bright-light exposure. The role of the insulin receptor in the production of the light response is however unknown. We have used suction-electrode recording to examine the responses of rods after conditionally knocking down the insulin receptor. Our results show that these IR knock-down rods have an accelerated decay of the light response and a small decrease in sensitivity by comparison to littermate WT rods. Our results indicate that the insulin receptor may have some role in controlling the rate of rod response decay, but they exclude a major role of the insulin receptor pathway in phototransduction.

Dose-response effects of chronic lithium regimens on spatial memory in the black molly fish.

Lithium is widely used in the management of bipolar disorder, yet memory impairment is a serious side effect. To assess the effects of lithium on spatial working and reference memories, we have employed a plus maze utilizing spontaneous alternation (SA) and place-learning paradigms in two experiments with the black molly fish. Four treatment groups were gavaged with 20 microl of a 10, 100, or 1000 mM lithium chloride (LiCl) solution or ddH(2)O vehicle every 12 h for 22 to 24 days. On Day 15, subjects began an 8-day SA task or a 10-day place-learning task. Results indicate that there is a significant difference in SA performance among the treatment groups for Days 1, 2, and 3. Results of the place-learning task indicate that the 1 M dose group needed significantly more trials to reach criterion and made significantly fewer correct first choices than the other dose groups. Capillary ion analysis determinations of plasma and brain lithium levels illustrate linear dose-response relationships to doses administered. Regression analyses indicate that there is a relationship between SA performance and plasma/brain lithium levels during the initial part of testing. Collectively, the results indicate that chronic lithium administration impairs spatial working and reference memories.

Effect of the ILE86TER mutation in the γ subunit of cGMP phosphodiesterase (PDE6) on rod photoreceptor signaling.

The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and ß subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g(tm1)/Pde6g(tm1) mice that do not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile(86) and Ile(87) residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and ß by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100µM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile(86) and Ile(87) residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.