Opponent and nonopponent contributions to the zebrafish electroretinogram using heterochromatic flicker photometry.

While some lower vertebrates, such as zebrafish, do not appear to possess anatomically separate pathways of processing visual information (such as M-pathways and P-pathways), it is believed that separate processing of the visual stimulus (such as luminance and chromatic processing) is a basic requirement of vertebrate vision. In this study, spectral sensitivity functions were obtained from electroretinogram responses to heterochromatic flicker photometry stimuli at several flicker rates, including a low flicker rate (2 Hz), in an attempt to predominantly stimulate chromatic processes and a high flicker rate (16 Hz), in an attempt to predominantly stimulate luminance processes. In addition, chromatic adaptation was used to isolate and examine the temporal properties of the different cone-type contributions to the electroretinogram response. Spectral sensitivity functions based on responses to heterochromatic stimuli of a low flicker rate appeared to receive both opponent and nonopponent contributions; however, when the stimulus flicker rate was high, spectral sensitivity appeared to be a function of only nonopponent mechanisms. Also, the differences in cone contributions to the spectral sensitivity functions across the different flicker rates appear to be related to the temporal properties of the cone contributions to the electroretinogram response.

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.

Constant dark-rearing effects on visual adaptation of the zebrafish ERG.

Anatomical and electrophysiological studies have shown that zebrafish retinal neurons develop in a sequential manner. Several studies have examined the impact of restricted rearing environments on zebrafish visual development, but the results have been mixed. The purpose of this study was to examine the development of light adaptation properties of the zebrafish electroretinogram (ERG), and examine the effects of constant dark rearing on retinal development. Subjects were zebrafish, Danio rerio, reared under normal lighting conditions and zebrafish reared in constant dark from fertilization to 6 days postfertilization (dpf). Increment threshold functions were obtained from a- and b-wave ERG responses from normally reared subjects at different ages and from animals exposed to early constant dark rearing. Dark-reared subjects were tested immediately following constant dark exposure (6-9 dpf) and after exposure to normal cyclic lighting (11-13 dpf). Adult zebrafish were significantly more sensitive at lower background illuminations than were larvae zebrafish. Also, constant dark rearing had a differential effect on the a- and b-wave response measures. Constant dark rearing raised b-wave threshold uniformly across background illuminations, while only producing higher a-wave thresholds at low levels of illumination. These results are consistent with findings in studies on zebrafish retinal development, and may help explain some of the discrepancies across studies examining the effects of restricted rearing.

The zebrafish as a model visual system.

The zebrafish has become 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.

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 temporal course of suppression during binocular rivalry.

Orthogonally oriented sinusoidal luminance gratings were dichoptically presented to the observers' left and right eyes. During the subsequent binocular rivalry, a small target was briefly presented (4AFC) to probe the strength of interocular suppression at various temporal latencies. Both stationary and moving rivalrous patterns were investigated. The purpose of experiment 1 was to compare the temporal characteristics of stationary and motion rivalry (0 and 1.2 deg s-1), while that of experiment 2 was to examine rivalry suppression for higher speeds (2 and 4 deg s-1). In all cases, it was found that the strength of suppression remained essentially constant throughout a single phase of binocular rivalry. The results of the investigation also revealed that moving rivalrous patterns lead to greater magnitudes of interocular suppression than static patterns. Despite these differences in the strength of suppression, the results of both experiments show that the temporal characteristics of motion and static rivalry are essentially identical.

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.

Effects of abnormal light-rearing conditions on retinal physiology in larvae zebrafish.

PURPOSE: Anatomic studies have found that zebrafish retinal neurons develop in a sequential fashion. In addition, exposure to abnormal light-rearing conditions produces deficits in visual behavior of larvae zebrafish, even though there appears to be little effect of the light-rearing conditions on the gross morphology of the retina. The purpose of this study was to assess the effects of abnormal light-rearing conditions on larvae zebrafish retinal physiology. METHODS: Larvae zebrafish (Danio rerio) were exposed to constant light (LL), constant dark (DD), or normal cyclic light (LD) from fertilization to 6 days postfertilization (dpf). After 6 days, the animals were placed into normal cyclic light and tested at 6 to 8, 13 to 15, and 21 to 24 dpf. Electroretinogram (ERG) responses to visual stimuli, consisting of various wavelengths and irradiances, were recorded. Comparisons were made across the three age groups and the three light-rearing conditions. RESULTS: Deficits from the light-rearing conditions were seen immediately after exposure (6 8 dpf). The LL-condition subjects showed the greatest deficit in the UV and short-wavelength areas and the DD-condition subjects showed a slight deficit across the entire spectrum. At 13 to 15 dpf, the LL and DD groups showed an increase in sensitivity and by 21 to 24 dpf, the groups no longer differed from controls. CONCLUSIONS: Abnormal lighting environments can adversely influence the physiological development of the larvae zebrafish retina. The pattern of damage that was seen in zebrafish is similar to that found in other vertebrates, including higher vertebrates. However, unlike higher vertebrates, the zebrafish appears to be capable of regeneration. This suggests that the zebrafish would be a viable model for light environment effects and neural regeneration.

Establishing and maintaining a low-cost zebrafish breeding and behavioral research facility.

The purpose of this paper is to describe how to establish and maintain a low-cost zebrafish facility that can be used for both teaching and research purposes. The cost of the facility ranges from about $250 to $1,000, depending on the size of the facility and the experiments that will be conducted. The facility plan presented here also has the advantage that it can be disassembled so that it need not function year-round. A detailed list of supplies and materials is provided, along with detailed instructions on how to set up and maintain the adult and breeding portions of the facility. Finally, there is discussion of some of the general uses of a zebrafish facility, including a variety of procedures that are currently being used in behavioral research with this species.

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.

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.

Effects of mean luminance on goldfish temporal contrast sensitivity.

The goldfish is an important animal model for retinal processing and for understanding the relationship between retinal structure and function. The purpose of this study was to examine the temporal processing of the visual system of this species. Goldfish were classically conditioned to suppress respiration upon presentation of a sinusoidally flickering stimulus. Temporal contrast sensitivity functions (T-CSFs) were determined by measuring contrast threshold at a variety of temporal frequencies across different mean luminances. Goldfish T-CSFs were found to be similar in shape to those of humans. In addition, as the mean luminance of the stimulus decreased, temporal resolution decreased. This implies that the animal's ability to detect high flicker frequencies decreases as the level of light adaptation decreases, as does that of humans. The results support the notion that temporal processing is similar across vertebrate species, and therefore that the goldfish is a useful model for studying temporal processing in the vertebrate retina.

The contributions of ON- and OFF-pathways to contrast sensitivity and spatial resolution in goldfish.

DL-2-amino-4-phosphonobutyric acid (APB) reduces the sensitivity of ON- and OFF-responses in goldfish retina, although the ON-responses are reduced significantly more than the OFF-responses. This paper describes the effects of APB on behavioral sensitivity of goldfish to spatial sinusoidal gratings. Fish were classically conditioned to suppress respiration upon presentation of gratings drifting at 1 Hz; contrast thresholds were measured by an observer-based two-alternative forced-choice procedure. Thresholds were repeated following intraocular injections of APB or physiological saline. Saline had no effect, but APB dramatically reduced contrast sensitivity and shifted contrast sensitivity functions to lower spatial frequencies. The results suggest that both ON- and OFF-pathways are necessary for normal spatial vision and that the effects of APB are consistent with the disruption of both ON- and OFF-pathways.

A pharmacodynamic model to investigate the structure-activity profile of a series of novel opioid analgesics.

A simple mathematical model of analgesia in the rat is developed and utilized to determine quantitative structure-activity relationships for a series of novel 4-anilidopiperidine opioids. The compounds tested (selected alkyl carboxyethyl esters attached at the one position of the piperidine ring) were designed for rapid inactivation by blood and tissue esterases. Model parameters included potency and rate constants for loss of pharmacodynamic effect by hydrolysis dependent and independent processes. A significant correlation is observed between duration of pharmacological effect in vivo and the rate constant for hydrolysis in human blood (r = 0.89). In vivo potency shows a moderate correlation with log P2 (r = -0.77). The validity of the model is shown by comparing model-based parameters which characterize potency and duration of effect in vivo with graphically derived parameters. Significant correlations are observed between model and graphically based estimates of potency (r = 0.75) and between model and graphically based estimates of duration of effect (r = 0.70). This model has potential application in studies of other classes of compounds in which hydrolytic cleavage limits duration of pharmacologic effect.

Refractory pouchitis: does it reflect underlying Crohn's disease?

Typical 'pouchitis' is a well recognised complication of ileal pouches in ulcerative colitis. Infrequently, a refractory pouchitis (RP) presents with certain clinical, endoscopic, and pathological features resembling Crohn's disease and is often ascribed to misdiagnosis of the initial colitis. To test that hypothesis and to identify risk factors for RP, this study reviewed cases of presumed ulcerative colitis with ileal pouches constructed at The Mount Sinai Hospital between 1973 and 1986. Twenty four cases with RP (16 Kock pouches and eight pelvic pouches) and 21 controls were compared for eight clinical variables. The original colectomy slides from 15 RP and 18 control cases were reviewed blindly, classified into five histological categories (corresponding to definite ulcerative colitis, definite Crohn's disease, and three indeterminate groups), and scored for 23 histological features. There were no significant clinical differences between RP and control cases except for more frequent extraintestinal manifestations (38% v 5%) and male preponderance (79% v 43%) in RP. There were also no significant differences between the distributions of RP cases and controls among the five histological categories or in the 23 histological features studied. Refractory pouchitis therefore does not seem to reflect underlying Crohn's disease, but may be linked to immunological mechanisms that are manifested clinically as extraintestinal complications.

Goldfish ganglion cells with unusual receptive field properties.

Goldfish retinal ganglion cells with unusual response properties are described. Each cell was classified as either Y-like or W-like, based upon its responses to sinusoidally modulated contrast-reversal gratings presented at various positions across the cell's receptive field. The unusual responses of the cells (which distinguish them from typical Y-like and W-like cells) occurred when sinusoidal gratings were drifted across the receptive field at a constant temporal rate. These cells responded at double the stimulus temporal rate to low-spatial-frequency gratings; a Fourier decomposition of the response revealed a large second harmonic component. However, to high-spatial-frequency stimuli, the response modulated at the temporal frequency of the stimulus; the Fourier fundamental component dominated the response. To examine the underlying receptive field mechanisms of these cells, each cell's response was analyzed using several different response measures. The results suggest that the receptive field properties of these unusual cells differ from the typical center/surround organization and confirm recent findings that the receptive fields of goldfish ganglion cells consist of inhomogeneities and subareas.

Opioid receptor activity of GI 87084B, a novel ultra-short acting analgesic, in isolated tissues.

GI 87084B (3-[4-methoxycarbonyl-4-[(1-oxopropyl) phenylamino]1-piperidine]propanoic acid, methyl ester, hydrochloride) was found to be a potent opioid agonist in the guinea pig ileum (EC50 = 2.4 +/- 0.6 nM), the rat vas deferens (EC50 = 387 +/- 44 nM) and the mouse vas deferens (EC50 = 39.5 +/- 7.4 nM). In the guinea pig ileum, GI 87084B, was roughly equivalent in potency to fentanyl (EC50 = 1.8 +/- 0.4 nM). GI 87084B was more potent in this tissue than alfentanil (EC50 = 20.1 +/- 1.2 nM) and less potent than sufentanil (EC50 = 0.3 +/- 0.09 nM). Schild analyses of antagonism of GI 87084B by naloxone yielded pKB values of 8.2 and slopes indistinguishable from unity in the guinea pig ileum and the mouse vas deferens. Insurmountable antagonism of GI 87084B by naloxone was observed in the rat vas deferens. However, an empirical measure of antagonist potency could be made: apparent pA2 = 8.1. The agonist dissociation constant (KA) for GI 87084B (220 +/- 90 nM) was determined by receptor alkylation with beta-chlornaltrexamine in the guinea pig ileum. Calculation of receptor occupancy suggested poor receptor-effector coupling and limited receptor reserve in the rat vas deferens, which could explain the insurmountable antagonism seen with higher concentrations of naloxone. These data suggest that GI 87084B acted through the mu class of opioid receptors to inhibit contraction induced by field stimulation in these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Design, synthesis, and pharmacological evaluation of ultrashort- to long-acting opioid analgetics.

In an effort to discover a potent ultrashort-acting mu opioid analgetic that is capable of metabolizing to an inactive species independent of hepatic function, several classes of 4-anilidopiperidine analgetics were synthesized and evaluated. One series of compounds displayed potent mu opioid agonist activity with a high degree of analgesic efficacy and an ultrashort to long duration of action. These analgetics, 4-(methoxycarbonyl)-4-[(1-oxopropyl)phenylamino]-1-piperidinepropanoi c acid alkyl esters, were evaluated in vitro in the guinea pig ileum for mu opioid activity, in vivo in the rat tail withdrawal assay for analgesic efficacy and duration of action, and in vitro in human whole blood for their ability to be metabolized in blood. Compounds in this series were all shown to be potent mu agonists in vitro, but depending upon the alkyl ester substitution the potency and duration of action in vivo varied substantially. The discrepancies between the in vitro and in vivo activities and variations in duration of action are probably due to different rates of ester hydrolysis by blood esterase(s). The SAR with respect to analgesic activity and duration of action as a function of the various esters synthesized is discussed. It was also demonstrated that the duration of action for the ultrashort-acting analgetic, 8, does not change upon prolonged infusion or administration of multiple bolus injections.

DL-2-amino-4-phosphonobutyric acid does not eliminate "ON" responses in the visual system of goldfish.

DL-2-Amino-4-phosphonobutyric acid (APB) suppresses activity in retinal ON pathways. It is generally assumed that loss of the ON pathway would result in loss of ON responses in the visual system. We tested this assumption by recording activity from the optic nerves of intact goldfish (Carassius auratus) before and after intraocular injection of APB. Whole-nerve responses to increments and decrements of light were compared to electroretinogram responses and to tectal evoked potentials. APB severely reduced the amplitude of the electroretinogram b-wave but left ON and OFF responses from the optic nerve and tectum intact, although decreased in sensitivity. We conclude that APB does not completely eliminate ON responses in the visual system, at least in goldfish. The selectivity and effectiveness of APB must be evaluated in other species before this agent can be relied upon as a useful tool in understanding the roles of ON and OFF pathways in visual function.