Nasser M. Qtaishat

Features of visual function in the naked mole-rat Heterocephalus glaber

The eyes and visual capacity of the naked mole-rat, Heterocephalus glaber, a subterranean rodent, were evaluated using anatomical, biochemical, and functional assays, and compared to other rodents of similar body size (mouse and gerbil). The eye is small compared to mouse, yet possesses cornea, lens, and retina with typical mammalian organization. The optic nerve cross-sectional area and fiber density are ~10% and ~50% that of gerbil, respectively. Levels per unit retinal area of 11-cis and all-trans retinal, derivatives of vitamin A associated with the visual cycle, are comparable to mouse. The corneal electroretinogram (ERG) exhibits early and late negative components that scale with flash strength; raising the body temperature of this poikilothermic animal from 30°C (normal for H. glaber ) to 37°C (normal for mouse) revealed an ERG response with typically mammalian features, but greatly attenuated and with slower kinetics. Leaving the nest chamber was a behavior correlated with light onset displayed preferentially by breeding females. Optical models of five mole-rat eyes suggest reasonable, but variable, image formation at the retina, possibly related to age. Results are consistent with amorphous light detection, possibly useful for circadian entrainment or escape behavior in the event of tunnel breeches.

Retinal abnormalities associated with the G90D mutation in opsin

Several mutations in the opsin gene have been associated with congenital stationary night blindness, considered to be a relatively nonprogressive disorder. In the present study, we examined the structural and functional changes induced by one of these mutations, i.e., substitution of aspartic acid for glycine at position 90 (G90D). Transgenic mice were created in which the ratio of transgenic opsin transcript to endogenous was 0.5:1, 1.7:1, or 2.5:1 and were studied via light and electron microscopy, immunocytochemistry, electroretinography (ERG), and spectrophotometry. Retinas with transgenic opsin levels equivalent to one endogenous allele (G0.5) appeared normal for a period of about 3–4 months, but at later ages there were disorganized, shortened rod outer segments (ROS), and a loss of photoreceptor nuclei. Higher levels of G90D opsin expression produced earlier signs of retinal degeneration and more severe disruption of photoreceptor morphology. Despite these adverse effects, the mutation had a positive effect on the retinas of rhodopsin knockout (R−/−) mice, whose visual cells fail to form ROS and rapidly degenerate. Incorporation of the transgene in the null background (G+/−/R−/− or G+/+/R−/−) led to the development of ROS containing G90D opsin and prolonged survival of photoreceptors. Absorbance spectra measured both in vitro and in situ showed a significant reduction of more than 90% in the amount of light‐sensitive pigment in the retinas of G+/+/R−/− mice, and ERG recordings revealed a >1 log unit loss in sensitivity. However, the histological appearances of the retinas of these mice show no significant loss of photoreceptors and little change in the lengths of their outer segments. These findings suggest that much of the ERG sensitivity loss derives from the reduced quantal absorption that results from a failure of G90D opsin to bind to its chromophore and form a normal complement of light‐sensitive visual pigment. J. Comp. Neurol. 478:149–163, 2004.

Excitation and desensitization of mouse rod photoreceptors in vivo following bright adapting light.

Electroretinographic (ERG) methods were used to determine response properties of mouse rod photoreceptors in vivo following adapting illumination that produced a significant extent of rhodopsin bleaching. Bleaching levels prevailing at ∼10 min and ∼20 min after the adapting exposure were on average 14% and 9%, respectively, based on the analysis of visual cycle retinoids in the eye tissues. Recovery of the rod response to the adapting light was monitored by analysing the ERG a‐wave response to a bright probe flash presented at varying times during dark adaptation. A paired‐flash procedure, in which the probe flash was presented at defined times after a weak test flash of fixed strength, was used to determine sensitivity of the rod response to the test flash. Recovery of the response to the adapting light was 80% complete at 13.5 ± 3.0 min (mean ±s.d.; n= 7) after adapting light offset. The adapting light caused prolonged desensitization of the weak‐flash response derived from paired‐flash data. By comparison with results obtained in the absence of the adapting exposure, desensitization determined with a test‐probe interval of 80 ms was ∼fourfold after 5 min of dark adaptation and ∼twofold after 20 min. The results indicate, for mouse rods in vivo, that the time scale for recovery of weak‐flash sensitivity substantially exceeds that for the recovery of circulating current following significant rhodopsin bleaching. The lingering desensitization may reflect a reduced efficiency of signal transmission in the phototransduction cascade distinct from that due to residual excitation.

CYSTEINE-TERMINATED B-DOMAIN OF STAPHYLOCOCCUS AUREUS PROTEIN A AS A SCAFFOLD FOR TARGETING GABAA RECEPTORS

This study reports the preparation and characterization of cysteine-terminated B-domain (Bd-cys) of Staphylococcus aureus protein A, in combination with immunoglobulin G (IgG) antibodies directed against the ρ1 and α1 subunits of GABA(A) receptors, for localizing reagents of interest to the target receptor. A cysteine residue was inserted at the C terminus of the cysteine-lacking B-domain (Bd) and used for conjugating maleimide-containing compounds. As determined by enzyme-linked immunosorbent assay (ELISA), binding of a Bd-cys-S-fluorescein conjugate to polyclonal guinea pig anti-GABA(A)-ρ1 and rabbit anti-GABA(A)-α1 IgG was similar to that exhibited by full-length protein A. Surface plasmon resonance analysis of the interaction of Bd-cys-S-PEG3400-biotin conjugate (where PEG is polyethylene glycol) with anti-GABA(A)-ρ1 and anti-GABA(A)-α1 yielded K(D) values of 6.4 ± 1.9 and 0.4 ± 0.1 nM, respectively. Fluorescence anisotropy analysis of the binding of Bd-cys-S-fluorescein to the two antibodies yielded EC50 values of 65 and 18 nM, respectively. As determined with biotin-reactive fluorescent reagents, Bd-cys-S-PEG3400-biotin specifically bound to the plasma membrane of Xenopus laevis oocytes that expressed α1β2γ2 or homomeric ρ1 GABA(A) receptors and were pretreated with the corresponding anti-GABA(A) IgG. The IgG-binding specificity and high affinity of Bd-cys conjugates illustrate the potential of these conjugates, in combination with a selected IgG, to localize compounds of interest at specific cell surface proteins.

Activation of membrane receptors by neurotransmitter released from temperature-sensitive hydrogels

The present paper describes the design, construction and testing of a temperature-sensitive N-isopropylacrylamide hydrogel device for studying the controlled presentation of gamma-aminobutyric acid (GABA) to GABA(C) membrane receptors expressed in Xenopus laevis oocytes. Upon temperature lowering, the GABA-loaded hydrogel positioned near the surface of the GABA(C)-expressing oocyte elicits a membrane current response resembling that induced by superfusion of the oocyte with free GABA. The response to cooling is not observed when GABA is omitted from the hydrogel loading solution. In addition, picrotoxin, a known GABA(C) receptor antagonist, inhibits the oocyte membrane current response associated with temperature lowering of GABA-loaded hydrogels. The data indicate that the present system affords a temperature-regulated release of GABA from the hydrogel and a resulting activation of the expressed GABA(C) receptors.

Preservation of Retinoid Influx into Eye Tissues of ABCR-Deficient Mice

Using an in vivo radiolabeling technique, we investigated the movement of retinoid into the retinal pigment epithelium (RPE) of the abcr-/- mouse, which lacks the photoreceptor ABCR protein and is a model for Stargardt disease. Eye tissues and serum obtained from dark-adapted, 5- to 8-month-old abcr-/- and control mice following the intraperitoneal injection of all-trans (3H)retinol were analyzed to determine the inferred influx of retinoid from the serum into the RPE. At 4.5 hr post-injection, the inferred all-trans retinol influx in abcr-/- mice, which possess the leucine 450 variant of RPE65 protein, was 0.011 ± 0.004 nmol (n = 3). This value did not differ significantly from that determined in age-matched controls possessing the methionine 450 variant of RPE65 (0.015 ± 0.003 nmol; n = 3) or from 3-month-old wildtype mice that possess the leucine 450 RPE65 variant (0.020 ± 0.007; n = 4). Thus, the absence of ABCR does not significantly compromise the passage of retinoid from the serum into the RPE under dark-adapted conditions.

Erratum: Retinal abnormalities associated with the G90D mutation in opsin (The Journal of Comparative Neurology (2004) 478 (149-163))

The original article to which this Erratum refers was published in The Journal of Comparative Neurology (2004)478 (2)149–163