Joseph Bilotta

The zebrafish as a model visual system.

The zebrafish has bacome an important vertebrate model in developmental neuroscience because it is a useful model for embryology, developmental biology, and genetic analysis. The similarities of its visual system to that of other vertebrates also make this animal a valuable model in vision science. The anatomical, physiological, and behavioral components of zebrafish visual processing have been studied in adult and in developing zebrafish. Its retinal anatomy continues to develop following hatching, providing an opportunity to correlate the development of retinal structure with visual physiology and behavior. In addition, a number of genetic mutations have been developed which are used to examine the contributions of genetics to visual development and function. This article will provide an overview of studies of zebrafish anatomical, physiological and behavioral processing, and the effects if genetic and environmental manipulations on visual development.

Rod contributions to the electroretinogram of the dark‐adapted developing zebrafish

Anatomical studies of the developing zebrafish retina have shown that rods approach maturity at about 15 days postfertilization (dpf). Past work has examined the photopic spectral sensitivity function of the developing zebrafish, but not spectral sensitivity under dark‐adapted conditions. This study examined rod contributions to the dark‐adapted spectral sensitivity function of the ERG b‐wave component in developing zebrafish. ERG responses to stimuli of various wavelengths and irradiances were obtained from dark‐adapted fish at 6–8, 13–15, 21–24, and 27–29 dpf. The results show that dark‐adapted spectral sensitivity varied with age. Spectral sensitivity functions of the 6–8 and 13–15 dpf groups appeared to be cone dominated and contained little or no rod contributions. Spectral sensitivity functions of the 21–24 and 27–29 dpf groups appeared to have both rod and cone contributions. Even at the oldest age group tested, the dark‐adapted spectral sensitivity function did not match the adult function. Thus, consistent with anatomical findings, the rod contributions to the ERG spectral sensitivity function appear to develop with age; however, these contributions are still not adult‐like by 29 dpf, which is contrary to anatomical work. These results illustrate that the zebrafish is an excellent model for visual development.

Effects of embryonic exposure to ethanol on zebrafish visual function

Across a variety of species, including humans, it has been shown that embryos exposed to ethanol display eye abnormalities as well as deficiencies in visual physiology and behavior. The purpose of this study was to examine the effects of embryonic exposure to ethanol on visual function in zebrafish. Visual function was assessed physiologically, via electroretinogram (ERG) recordings, and behaviorally, by measuring visual acuity with the optomotor response. Zebrafish larvae were exposed to 1.5% ethanol at various times during development, including the period of maximal eye development. The results show that ethanol effects on visual function were most pronounced when exposure occurred during eye development. ERG recordings from ethanol-exposed larvae differed from normal subjects both in shape of the response waveform and in visual thresholds under both light and dark adaptation; the differences were more pronounced under lower levels of adaptation. Also, ethanol-exposed larvae displayed lower visual acuity as determined from the optomotor response. These results indicate embryonic ethanol exposure affects visual function particularly when exposure occurs during eye development. In addition, these findings illustrate the usefulness of the zebrafish as a viable animal model for studying Fetal Alcohol Syndrome (FAS).

Cone contributions to the photopic spectral sensitivity of the zebrafish ERG

Microspectrophotometry studies show that zebrafish (Danio rerio) possess four cone photopigments. The purpose of this study was to determine the cone contributions to the zebrafish photopic increment threshold spectral-sensitivity function. Electroretinogram (ERG) b-wave responses to monochromatic lights presented on a broadband or chromatic background were obtained. It was found that under the broadband background condition, the zebrafish spectral-sensitivity function showed several peaks that were narrower in sensitivity compared to the cone spectra. The spectral-sensitivity function was modeled with L - M and M - S opponent interactions and nonopponent S- and U-cone mechanisms. Using chromatic adaptation designed to suppress the contribution of the S-cones, a strong U-cone contribution to the spectral-sensitivity function was revealed, and the contributions of the S-cones to the M - S mechanism were reduced. These results show that the b-wave component of the ERG receives input from all four cone types and appears to reflect color opponent mechanisms. Thus, zebrafish may possess the fundamental properties necessary for color vision.

Assessing appetitive choice discrimination learning in zebrafish.

Within the last decade, the zebrafish (Danio rerio) has emerged as an important vertebrate model in developmental biology and medicine for problems typically associated with humans. However, where behavioral assays are needed, the utility of the zebrafish model has been limited by the narrow range of procedures so far developed to investigate zebrafish learning. The purpose of this study was to further develop and test procedures to study appetitive choice discrimination learning in zebrafish. Zebrafish were conditioned to swim into one of three chambers for food reinforcement. The correct (S+) chamber on a trial was signaled by the presence of a light stimulus in the chamber; the two negative (S-) chambers were dark. Each of the 15 fish tested learned the discrimination to a criterion of 80% correct in both of two consecutive sessions. Tests for stimulus control showed that discriminative behavior was indeed under the control of the S+ discriminandum. These results were discussed in relation to the recent report of zebrafish discrimination learning in a two-alternative task, and the importance of examining individual zebrafish learning curves.

ERG assessment of zebrafish retinal development.

Research has shown that adult zebrafish have a complex visual system, with two possible opponent mechanisms. Anatomically, zebrafish retina develops in a sequential manner and is immature at hatching. The purpose of the present study was to assess zebrafish retinal development using the electroretinogram (ERG). ERG responses to visual stimuli were obtained from 4-5, 6-8, 13-15, and 21-24 days postfertilization (dpf) zebrafish. Individual waveforms were assessed and compared across the four age groups. Spectral-sensitivity functions were calculated for the a- and b-wave components of the ERG response. Results showed that the ERG waveforms and spectral-sensitivity functions varied with age. While the 21-24 dpf subjects had an ERG waveform that was similar to that of adults, the younger subjects did not. Although there were modest differences in the a-wave spectral sensitivity, substantial differences were found in the b-wave spectral sensitivities across the ages. There was a consistent strong response to ultraviolet wavelengths, while across the remaining parts of the spectrum, there was a gradual increase in sensitivity with age. Also, the 21-24 dpf subjects appear to have adult-like U- and S-cone functions, but were missing the L-M and the M-S opponent mechanisms found in the adult. These results support the findings of the anatomical studies and demonstrate that the zebrafish is a useful model for examining the development of retinal function.

Noncell-autonomous photoreceptor degeneration in a zebrafish model of choroideremia

Choroideremia is an X-linked hereditary retinal degeneration resulting from mutations in the Rab escort protein-1 (REP1). The Rep1 protein facilitates posttranslational modification of Rab proteins, which regulate intracellular trafficking in the retinal pigment epithelium (RPE) and photoreceptors and are likely involved in the removal of outer segment disk membranes by the RPE. A critical question for potential treatment of choroideremia is whether photoreceptor degeneration results from autonomous defects in opsin transport within the photoreceptor or as a nonautonomous and secondary consequence of RPE degeneration. To address this question, we have characterized the retinal pathology in zebrafish rep1 mutants, which carry a recessive nonsense mutation in the REP1 gene. Zebrafish rep1 mutants exhibit degeneration of the RPE and photoreceptors and complete loss of visual function as measured by electroretinograms. In the mutant RPE, photoreceptor outer segment material was not effectively eliminated, and large vacuoles were observed. However, opsin trafficking in photoreceptors occurred normally. Mosaic analysis revealed that photoreceptor degeneration was nonautonomous and required contact with the mutant RPE as mutant photoreceptors were rescued in wild-type hosts and wild-type photoreceptors degenerated in mutant hosts. We conclude that mutations in REP1 disrupt cellular processes in the RPE, which causes photoreceptor death as a secondary consequence. These results suggest that therapies that correct the RPE may successfully rescue photoreceptor loss in choroideremia.

Effects of abnormal lighting on the development of zebrafish visual behavior

Studies across several species have demonstrated that the developing visual system is influenced by environmental conditions. This study examined the effects of abnormal lighting environments on zebrafish visual behavior. Zebrafish were raised under different lighting conditions from fertilization to 6 days postfertilization (dpf). These conditions included a normal light/dark cycle, constant dark, constant light, as well as exposure to various lighting cycles with intense light. Visual acuity was tested using the optomotor response to black and white vertical stripes of various widths. The results showed that visual acuity of the normal fish improved with age. In addition, fish raised in constant light had significantly lower visual acuity than fish raised under normal lighting conditions. Subjects raised in constant dark showed somewhat lower visual acuity than normal subjects when tested at 12-14 dpf, but the deficits were not as severe as those found in fish raised in constant light. Intense light rearing did not have any substantial effects on visual development, unless it was constant. In conclusion, under normal conditions, zebrafish visual acuity improves with age, supporting previous work on zebrafish development. In addition, zebrafish visual behavior is altered by abnormal lighting conditions; the most severe decrements occur when raised under constant light.

The effects of temperature on the dark-adapted spectral sensitivity function of the adult zebrafish

In goldfish and other cold-blooded vertebrates, temperature can influence the rhodopsin/porphyropsin contributions to the rod photoreceptors. This study examined the effects of temperature on the spectral sensitivity function of the dark-adapted zebrafish. Zebrafish were housed in either a warm (28-30 degrees C) or cold (22-25 degrees C) tank prior to testing. Fish were dark-adapted for at least 1 h and electroretinogram (ERG) responses to 200 ms stimuli of various wavelengths and irradiances were obtained. Results show that water temperature affected the spectral sensitivity function of the ERG b-wave. Subjects housed in the warm temperatures had a spectral sensitivity consistent with the rhodopsin absorption curve; however, fish housed in the colder temperatures had a spectral sensitivity function that was the result of a rhodopsin/porphyropsin mixture. In addition, ultraviolet cones (lambda max: 362 nm) contributed to the dark-adapted spectral sensitivity function under both temperature conditions. Consistent with the results from other fish, the dark-adapted visual system of the zebrafish can be influenced by water temperature. The results of this study demonstrate the necessity of maintaining a stable environment when examining the contributions of the photoreceptors to the visual response.

Visual processing of the zebrafish optic tectum before and after optic nerve damage

Although the zebrafish has become an important model in visual neuroscience, little has been done to examine the processing of its higher visual centers. The purpose of this work was twofold. The first purpose was to examine the physiology of the zebrafish retinotectal system and its relationship to retinal physiology. Spectral sensitivity functions were derived from visually evoked tectal responses and these functions were compared to the functions of electroretinogram (ERG) responses obtained using the same stimulus conditions. The second purpose was to examine the recovery of visual functioning of the tectum following optic nerve damage. The optic nerves of adult zebrafish were damaged (crushed), and tectal visual processing was assessed following damage. The results showed that the spectral sensitivity functions based on the On-responses of the tectum and ERG were qualitatively similar. The functions based on each response type received similar cone contributions including both nonopponent and opponent contributions. However, the spectral sensitivity functions based on the Off-responses of the tectum and ERG differed. The results also showed that the zebrafish visual system is capable of neural regeneration. By 90 days following an optic nerve crush, the spectral sensitivity function based on the tectal On-response was similar to functions obtained from normal zebrafish. Although the tectal Off-response did recover, the spectral sensitivity based on the Off-response was not the same as the function of normal zebrafish. These results support the notion that different levels of the visual system process information differently and that the zebrafish visual system, like those of other lower vertebrates, is capable of functional regeneration.