Cone degeneration following rod ablation in a reversible model of retinal degeneration.

PURPOSE: Amphibian retinas regenerate after injury, making them ideal for studying the mechanisms of retinal regeneration, but this leaves their value as models of retinal degeneration in question. The authors asked whether the initial cellular changes after rod loss in the regenerative model Xenopus laevis mimic those observed in nonregenerative models. They also asked whether rod loss was reversible. METHODS: The authors generated transgenic X. laevis expressing the Escherichia coli enzyme nitroreductase (NTR) under the control of the rod-specific rhodopsin (XOP) promoter. NTR converts the antibiotic metronidazole (Mtz) into an interstrand DNA cross-linker. A visually mediated behavioral assay and immunohistochemistry were used to determine the effects of Mtz on the vision and retinas of XOPNTR F1 tadpoles. RESULTS: NTR expression was detected only in the rods of XOPNTR tadpoles. Mtz treatment resulted in rapid vision loss and near complete ablation of rod photoreceptors by day 12. Müller glial cell hypertrophy and progressive cone degeneration followed rod cell ablation. When animals were allowed to recover, new rods were born and formed outer segments. CONCLUSIONS: The initial secondary cellular changes detected in the rodless tadpole retina mimic those observed in other models of retinal degeneration. The rapid and synchronous rod loss in XOPNTR animals suggested this model may prove useful in the study of retinal degeneration. Moreover, the regenerative capacity of the Xenopus retina makes these animals a valuable tool for identifying the cellular and molecular mechanisms at work in lower vertebrates with the remarkable capacity of retinal regeneration.

Light responses in rods of vitamin A-deprived Xenopus.

PURPOSE: Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A-deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration. METHODS: The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry. RESULTS: The vitamin A-deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A-deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds(-1)) was twofold greater than the maximum levels attainable by control rods ( approximately 7 seconds(-1)). CONCLUSIONS: Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.

Developmental regulation of calcium-dependent feedback in Xenopus rods.

The kinetics of activation and inactivation in the phototransduction pathway of developing Xenopus rods were studied. The gain of the activation steps in transduction (amplification) increased and photoresponses became more rapid as the rods matured from the larval to the adult stage. The time to peak was significantly shorter in adults (1.3 s) than tadpoles (2 s). Moreover, adult rods recovered twice as fast from saturating flashes than did larval rods without changes of the dominant time constant (2.5 s). Guanylate cyclase (GC) activity, determined using IBMX steps, increased in adult rods from approximately 1.1 s(-1) to 3.7 s(-1) 5 s after a saturating flash delivering 6,000 photoisomerizations. In larval rods, it increased from 1.8 s(-1) to 4.0 s(-1) 9 s after an equivalent flash. However, the ratio of amplification to the measured dark phosphodiesterase activity was constant. Guanylate cyclase-activating protein (GCAP1) levels and normalized Na+/Ca2+, K+ exchanger currents were increased in adults compared with tadpoles. Together, these results are consistent with the acceleration of the recovery phase in adult rods via developmental regulation of calcium homeostasis. Despite these large changes, the single photon response amplitude was approximately 0.6 pA throughout development. Reduction of calcium feedback with BAPTA increased adult single photon response amplitudes threefold and reduced its cutoff frequency to that observed with tadpole rods. Linear mathematical modeling suggests that calcium-dependent feedback can account for the observed differences in the power spectra of larval and adult rods. We conclude that larval Xenopus maximize sensitivity at the expense of slower response kinetics while adults maximize response kinetics at the expense of sensitivity.

Circadian modulation of temporal properties of the rod pathway in larval Xenopus.

Circadian clocks are integral components of visual systems. They help adjust an animal's vision to diurnal changes in ambient illumination. To understand how circadian clocks may adapt visual sensitivity, we investigated the spatial and temporal properties of optomotor responses of young Xenopus laevis tadpoles (Nieuwkoop and Faber, developmental stage 48) using a modified 2-alternative preferential-viewing method. We maintained animals in constant darkness and measured temporal sensitivity during their subjective day and night. We found that their behavioral responses can be explained in terms of 2 mechanisms with different temporal properties. The more sensitive mechanism operates at low temporal frequencies and intermediate wavelengths (lambdamax = 520 nm), properties consistent with rod signals. Threshold for this mechanism is approximately 0.04 photoisomerizations rod(-1) s(-1), consistent with single-photon detection. A less-sensitive mechanism responds to higher temporal frequencies (cutoff = 12 Hz) and has broad spectral sensitivity (370-720 nm), consistent with multiple classes of cone signals. This cone mechanism does not change, but the cutoff frequency of the more sensitive rod mechanism shifts from 0.35 Hz at night to 1.1 Hz during the subjective day, thereby enhancing the animal's sensitivity to dim rapidly changing stimuli. This day-night shift in rod temporal cutoff frequency cycles in complete darkness, characteristic of an endogenous circadian rhythm. The temporal properties of the behaviorally measured rod mechanism correspond closely with those of the electrophysiologically measured retinal response, indicating that the rod signals are modulated at the level of the outer retina.

Constrained optimization of Drude's equations eliminates effects of confounding molecules for the polarimetric measurement of glucose.

Common confounding factors for polarimetric concentration measurements include additional optical rotations from unknown optically active molecules, linear birefringence of the medium, and path length variability. We show that by approximating Drude's equation and taking several measurements from the same sample at different wavelengths, the error due to confounding rotations in the measurements can theoretically be canceled. The analysis is developed with regard to glucose sensing in aqueous humor. First, we show that the optical rotatory dispersions of the known molecules in bovine aqueous humor could be represented by Drude's equations. Then, the total optical rotation is approximated by a function combining Drude's equations for the major contributors in the sample, i.e., glucose, glutamine, fructose, and phenylalanine. The concentration-related unknown coefficients in the approximating function are found by constrained nonlinear optimization of the function at different wavelengths. This technique is tested on a published data set and four alterations of those data: (1) concentrations randomly varied within narrow limits, (2) similar to alteration 1 but with significantly elevated glucose concentration, (3) similar to alteration 1 but with significantly decreased glucose concentration, and (4) concentrations randomly varied within wider limits than alteration 1. The method produces very accurate glucose-concentration estimates in all of these data sets. The relative error was smaller than 1% in all except the low-glucose sample (1.4%). This method may prove useful in noninvasive glucose measurement in humans.