In the past two years the field of fish vision research has lost three central figures, Bill McFarland, Bill Muntz, and Joe Bilotta. This volume of Visual Neuroscience is dedicated to them. Since the inception of this volume, Henk Spekreijse and Adam Locket, who made significant contributions to fish vision, have also passed away; they are also remembered.

Dr. Joseph Bilotta, an eminent scientist in the field of fish visual neurophysiology and psychophysics, died suddenly and unexpectedly on January 2, 2006. A native of Niagara Falls, NY, Joe was born October 21, 1955. His passion for learning took him on a journey from an Associate Degree in Mathematics in 1975 from Niagara County Community College to a Ph.D. in Experimental Psychology conferred by the City University of New York in 1987 under the mentorship of Dr. Israel Abramov. Joe continued his training at Vanderbilt University, working as a post-doctoral fellow in the laboratory of Dr. Maureen Powers. In 1991, Joe joined the faculty of Western Kentucky University as an assistant professor of Psychology, and quickly moved through the ranks, becoming full professor in 2001.

On August 31, 2004 William N. “MAC” McFarland died in Mt. Vernon, Washington just 11 days shy of his 79th birthday. He was into his second post-retirement professorship (from Cornell and USC) at the Friday Harbor Labs of the University of Washington. Rather than the usual CV with a list of awards and accomplishments, of which Mac had many, I would like to posit the following question, “Why should Mac be honored in this issue?” To those of us who knew and worked with him, the fact that he was “Mac” says it all. However, to those who did not know him, more justification is needed.

Whereas I only worked with Bill for three years when he supervised my PhD at the University of Stirling in the mid 1970s, his influence on me has lasted a lifetime. This piece, rather than summarizing the details of his considerable contributions to science, is a personal tribute to one of the most significant people in my life.

The isolated retina of the goldfish has proven a valuable resource for studying the variability of firing of retinal ganglion cells. Three major areas of study are considered here: the variability of maintained discharges, the correlated firing of neighboring ganglion cells, and the variability of responses to light. The sources of variability, its relationship to retinal processing, and its possible functional role in perception are examined through these three aspects of variability. The results are related to similar studies in mammals (mainly cats). This retrospective is biased toward my studies over 30 years.

To determine whether regenerating neural pathways can support visual behavior, adult goldfish (Carassius auratus) were injected intraocularly with ouabain and tested for the presence of reflexive visual behaviors (dorsal light reflex and optokinetic nystagmus) and the ability to respond to visual stimuli in a classical conditioning paradigm. All visual behaviors were absent or greatly diminished until 8 to 10 weeks, when retinal layering had returned. At 10 weeks post-ouabain, reflexive behaviors to supra-threshold stimuli were near normal; however the ability to detect supra-threshold stimuli in the conditioning paradigm did not recover until 13 weeks. Absolute dark-adapted threshold and light-adapted spectral sensitivity measured at 13 to 17 weeks were abnormal: Dark-adapted threshold was elevated by 1.5 log units and light-adapted spectral sensitivity was markedly narrower than normal. No responses to 50% contrast sinusoidal gratings could be obtained through ouabain-treated eyes using the classical conditioning technique, even though responses through the untreated eye remained. Results demonstrate that: (a) visually mediated behaviors return in goldfish with ouabain-treated retinas; (b) the time course of recovery of reflexive responses in luminance and spatial domains parallels return of ERG function and of tectal activity; and (c) visual function that is mediated by regenerating retina appears not to be as sensitive as vision via normally developed retinal pathways.

A functional role for retinal endocannabinoids has not been determined. We characterized retrograde suppression of membrane currents of goldfish cones in a retinal slice. Whole-cell recordings were obtained from cone inner segments under voltage clamp. IK(V) was elicited by a depolarizing pulse to +54 mV from a holding potential of −70 mV. A fifty-millisecond puff of saline with 70 mM KCl or Group I mGluR agonist DHPG was applied through a pipette directly at a mixed rod/cone (Mb) bipolar cell body. The amplitude of IK(V) decreased 25% compared to the pre-puff control. Retrograde suppression of IK(V) was blocked by CB1 receptor antagonist, SR141716A. The FAAH inhibitor URB597 had no effect on the suppression of IK(V), whereas nimesulide, a COX-2 inhibitor, prolonged the effects of the K+ puff 10-fold. Orlistat, a blocker of 2-AG synthesis, blocked the effect of the K+ puff. Group I mGluR activation of Gq/11 was demonstrated in that a puff with DHPG decreased IK(V) of cones by 32%, an effect blocked by SR141716A. The effect of DHPG was not blocked by the mGluR5 antagonist MPEP, indicating involvement of mGluR1. The suppressive effect of the K+ puff vanished in a Ca2+-free, 2 mM Co2+ saline. TMB-8 or ryanodine, blocked the effect of DHPG, but not that of the K+ puff, showing that calcium influx or release from intracellular stores could mediate retrograde release. We suggest that retrograde suppression of cone IK(V) is mediated by Ca2+-dependent release of 2-AG from Mb bipolar cell dendrites by separate mechanisms: (1) voltage-dependent, mimicked by the K+ puff, that may be activated by the depolarizing ON response to light; (2) voltage-independent, occurring under ambient illumination, mediated by tonic mGluR1 activation. The negative feedback of this latter mechanism could regulate tonic glutamate release from cones within narrow limits, regardless of ambient illumination.

The retina of salmonid fishes has two types of cone photoreceptors: single and double cones. At the nuclear level, these cones are distributed in a square mosaic such that the double cones form the sides of the square and the single cones occupy positions at the centre and at the corners of the square. Double cones consist of two members, one having visual pigment protein maximally sensitive to green light (RH2 opsin), the other maximally sensitive to red light (LWS opsin). Single cones can have opsins maximally sensitive to ultraviolet (UV) or blue light (SWS1 and SWS2 opsins, respectively). In Pacific salmonids, all single cones express UV opsin at hatching. Around the time of yolk sac absorption, single cones start switching opsin expression from UV to blue, in an event that proceeds from the ventral to the dorsal retina. This transformation is accompanied by a loss of single corner cones such that the large juvenile shows corner cones and UV opsin expression in the dorsal retina only. Previous research has shown that adult Pacific salmon have corner cones over large areas of retina suggesting that these cones may be regenerated and that they may express UV opsin. Here we used in-situ hybridization with cRNA probes and RT-PCR to show that: (1) all single cones in non-growth zone areas of the retina express blue opsin and (2) double cone opsin expression alternates around the square mosaic unit. Our results indicate that single cone driven UV sensitivity in adult salmon must emanate from stimulation of growth zone areas.

Color constancy is one of the most impressive features of color vision systems. Although the phenomenon has been studied for decades, its underlying neuronal mechanism remains unresolved. Literature indicates an early, possibly retinal mechanism and a late, possibly cortical mechanism. The early mechanism seems to involve chromatic spatial integration and performs the critical calculations for color constancy. The late mechanism seems to make the color manifest. We briefly review the current evidence for each mechanism. We discuss in more detail a model for the early mechanism that is based on direct measurements of goldfish outer retinal processing and induces color constancy and color contrast. In this study we extrapolate this model to primate retina, illustrating that it is highly likely that a similar mechanism is also present in primates. The logical consequence of our experimental work in goldfish and our model is that the wiring of the cone/horizontal cell system sets the reference point for color vision (i.e., it sets the white point for that animal).

Goldfish (Carassius auratus) were trained to discriminate triangles and squares using a two choice procedure. In the first experiment, three goldfish were trained with food reward on a black outline triangle on a white background, while a black outline square was shown for comparison. In transfer tests, a Kanizsa triangle and a Kanizsa square were presented, perceived by humans as an illusory triangle- or square-shaped surface of slightly higher brightness than the background. The choice behavior in this situation indicates that goldfish are able to discriminate between both figures in almost the same way as in the training situation. In control experiments goldfish did not discriminate between shapes in which humans do not perceive the illusion. A series of generalization experiments was performed indicating the similarity between the tested shapes and the training triangle. From all these findings we conclude that goldfish are able to perceive an illusory triangle or square within the Kanizsa figures. In a second experiment, four goldfish were trained on a white outline triangle versus a white outline square, both on black background with white diagonal lines. In transfer tests in which the shapes were replaced by gaps within the white diagonal lines, goldfish were clearly able to discriminate between the two patterns based on the illusory contours. This was not the case in tranfer tests with phase shifted abutting lines.

Regeneration of the teleost retina following surgical extirpation of 25% to 100% of the neural retina was investigated in goldfish (Carrasius auratus) and sunfish (Lepomis cyanellus). The retina will regenerate following removal of up to 95% of the neural retina, however complete extirpation prevented regeneration. Visual sensitivity was assessed by examining components of the electroretinogram (ERG) and the dorsal light reflex (DLR) during regeneration. B-wave amplitudes in the experimental eyes increased throughout the study and central connections were reestablished as indicated by the progressive improvement in the dorsal light reflex. The recovery of visual function was closely correlated with retinal regeneration. Visual recovery progressed more slowly than following complete cytotoxic destruction of the mature retina (Mensinger & Powers, 1999) because the surgery removed a large number of the pluripotent cell population and restricted the number and distribution of regenerating foci.

Simple (dorsal light reflex) and complex (predator-prey interactions) visually mediated behaviors were used concurrently with morphological examination to assess restoration of visual function following optic nerve crush in bluegill (Lepomis macrochirus) × pumpkinseed (Lepomis gibbosus) hybrid sunfish. Regenerating optic nerve axons projected into the stratum opticum-stratum fibrosum et griseum superficiale by week 2, the stratum griseum centrale by week 4, and stratum album centrale by week 6. Initial projections into the laminae were diffuse and less stratified compared to controls. By week 12, the projection pattern of regenerating nerve fibers closely resembled the innervation of normal tecta. Visual improvements were correlated with increasing projections into the tectum. The dorsal light reflex improved from a 45° vertical deviation following nerve crush to 4.5° by week 16. Initial predator-prey interactions were exclusively mediated by the control eye. As regeneration progressed, there was a gradual expansion of the visual field. The reaction distance and attack angles within the visual field of the experimental eye were initially less than controls, however, these differences disappeared by week 10. Improvements in visual function were closely correlated with an increase of regenerating ganglion cell axons into the optic tectum indicating sufficient synaptogenesis to mediate both simple and complex visual behavior.

Spatial vision was studied in the bluegill sunfish, Lepomis macrochirus (9.5–14 cm standard length) to assess the limitations imposed by the optics of the eye, the retinal receptor spacing and the retinotectal projection during regeneration. Examination of images formed by the dioptric elements of the eye showed that spatial frequencies up to 29 c/° could be imaged on the retina. Cone spacing was measured in the retina of fresh, intact eyes. The spacing of rows of double cones predicted 3.4 c/° as the cutoff spatial frequency; the spacing between rows of single and double cones predicted 6.7 c/°. Contrast sensitivity functions were obtained psychophysically in normals and fish with one regenerating optic nerve. Fish were trained to orient to gratings (mean luminance = 25 cd/m2) presented to either eye. In normals, contrast sensitivity functions were similar in shape and bandwidth to those of other species, peaking at 0.4 c/° with a minimum contrast threshold of 0.03 and a cutoff at about 5 c/°, which was within the range predicted by cone spacing. Given that the optical cutoff frequency exceeds that predicted by cone spacing, it is possible that gratings could be detected by aliasing with the bluegill's regular cone mosaic. However, tests with high contrast gratings up to 15 c/° found no evidence of such detection. After crushing one optic nerve in three trained sunfish, recovery of visual avoidance, dorsal light reflex and orienting to gratings, were monitored over 315 days. At 64–69 days postcrush, responses to gratings reappeared, and within 2–5 days contrast sensitivity at low (0.15 c/°) and medium (1.0 c/°) spatial frequencies had returned to normal. At a high spatial frequency (2.93 c/°) recovery was much slower, and complete only in one fish.

Horizontal cells are second order neurons that receive direct synaptic input from photoreceptors. In teleosts horizontal cells can be divided into two categories, cone-connected and rod-connected. Although the anatomy and physiology of fish cone horizontal cells have been extensively investigated, less is known about rod horizontal cells. This study was undertaken to determine whether light and/or the circadian clock regulate gap junctional coupling between goldfish rod horizontal cells. We used fine-tipped, microelectrode intracellular recording to monitor rod horizontal cells under various visual stimulation conditions, and tracer (biocytin) iontophoresis to visualize their morphology and evaluate the extent of coupling. Under dark-adapted conditions, rod horizontal cells were extensively coupled to cells of like-type (homologous coupling) with an average of ∼120 cells coupled. Under these conditions, no differences were observed between day, night, the subjective day, and subjective night. In addition, under dark-adapted conditions, application of the dopamine D2-like agonist quinpirole (1 μM), the D2-like antagonist spiperone (10 μM), or the D1-like antagonist SCH23390 (10 μM) had no effect on rod horizontal cell tracer coupling. In contrast, the extent of tracer coupling was reduced by ∼90% following repetitive light (photopic range) stimulation of the retina or application of the D1-agonist SKF38393 (10 μM) during the subjective day and night. We conclude that similarly to cone horizontal cells, rod horizontal cells are extensively coupled to one another in darkness and that the extent of coupling is dramatically reduced by bright light stimulation or dopamine D1-receptor activation. However, in contrast to cone horizontal cells whose light responses are under the control of the retinal clock, the light responses of rod horizontal cells under dark-adapted conditions were similar during the day, night, subjective day, and subjective night thus demonstrating that they are not under the influence of the circadian clock.

The Syngnathidae are specialized diurnal feeders that are known to possess a retinal fovea and use independent eye movements to locate, track, and strike individual planktonic prey items. In this study, we have investigated the spectral sensitivities of three syngnathid species: a pipefish and two seahorses. We used spectrophotometry to measure the spectral transmission properties of ocular lenses and microspectrophotometry to measure the spectral absorption characteristics of visual pigments in the retinal photoreceptors. The pipefish, Stigmatopora argus, together with the seahorse Hippocampus subelongatus, is found in “green-water” temperate coastal seagrass habitats, whereas the second seahorse, H. barbouri, originates from a “blue-water” tropical coral reef habitat. All species were found to possess short wavelength absorbing pigment(s) in their lenses, with the 50% cut-off point of S. argus and H. subelongatus at 429 and 425 nm respectively, whereas that of H. barbouri was located at 409 nm. Microspectrophotometry of the photoreceptors revealed that the rods of all three species contained visual pigment with the wavelength of maximum absorption (λmax) at approximately 500 nm. The visual pigment complement of the cones varied between the species: all possessed single cones with a λmax close to 460 nm but H. barbouri also possessed an additional class of single cone with λmax at 430 nm. Three classes of visual pigment were found in the double cones, the λmax being approximately 520, 537, and 560 nm in the two seahorses and 520, 537, and 580 nm in the pipefish. The spectral sensitivities of the syngnathids investigated here do not appear to conform to generally accepted trends for fishes inhabiting different spectral environments. The influence of the specialized feeding regime of the syngnathids is discussed in relation to our findings that ultra-violet sensitivity is apparently not necessary for zooplanktivory in certain habitats.

To elucidate the specific properties of retinae with grouped photoreceptors the neural pathways in the outer and inner plexiform layer were studied. Photoreceptor bundles in this species consist of more than 100 rods and up to 50 cones, and are usually regarded as functional units. Golgi impregnation in thick and thin sections and light microscopy were used to identify bipolar cell types linking photoreceptors to amacrine and/or ganglion cells. Nine different types were distinguished based on their dendritic morphology and the position of the axon terminal in the inner plexiform layer. Small cells have dendritic fields smaller than the diameter of a photoreceptor bundle and are contacted mostly by cones. The dendritic field size of bushy cells matches that of a photoreceptor bundle; they are contacted mainly by rods. Flat cells receive about equal input from rods and cones; their dendritic field size exceeds the bundle diameter. Within the three major classes there are subtypes addressing three sublaminae of the inner plexiform layer, the proximal On-centre region (sl b), the distal Off-centre region (sl a) and a central sublayer (sl c) probably with transient activity. These observations suggest that cone vision has a spatial acuity better than the “bundle grain”. In rod dominated vision the resolution matches that of the bundles; for this pathway, the hypothesis of the bundle as a functional unit is confirmed. The mesopic flat cell pathway has a resolution inferior to the “bundle grain”; it may therefore be dedicated to movement detection.

Connexin 35/36 is the most widespread neuronal gap junction protein in the retina and central nervous system. Electrical and/or tracer coupling in a number of neuronal circuits that express this connexin are regulated by light adaptation. In many cases, the regulation of coupling depends on signaling pathways that activate protein kinases such as PKA, and Cx35 has been shown to be regulated by PKA phosphorylation in cell culture systems. To examine whether phosphorylation might regulate Cx35/36 in the retina we developed phospho-specific polyclonal antibodies against the two regulatory phosphorylation sites of Cx35 and examined the phosphorylation state of this connexin in the retina. Western blot analysis with hybrid bass retinal membrane preparations showed Cx35 to be phosphorylated at both the Ser110 and Ser276 sites, and this labeling was eliminated by alkaline phosphatase digestion. The homologous sites of mouse and rabbit Cx36 were also phosphorylated in retinal membrane preparations. Quantitative confocal immunofluorescence analysis showed gap junctions identified with a monoclonal anti-Cx35 antibody to have variable levels of phosphorylation at both the Ser110 and Ser276 sites. Unusual gap junctions that could be identified by their large size (up to 32 μm2) and location in the IPL showed a prominent shift in phosphorylation state from heavily phosphorylated in nighttime, dark-adapted retina to weakly phosphorylated in daytime, light-adapted retina. Both Ser110 and Ser276 sites showed significant changes in this manner. Under both lighting conditions, other gap junctions varied from non-phosphorylated to heavily phosphorylated. We predict that changes in the phosphorylation states of these sites correlate with changes in the degree of coupling through Cx35/36 gap junctions. This leads to the conclusion that connexin phosphorylation mediates changes in coupling in some retinal networks. However, these changes are not global and likely occur in a cell type-specific or possibly a gap junction-specific manner.

Lungfish (order Dipnoi) evolved during the Devonian period and are believed to be the closest living relatives to the land vertebrates. Here we describe the previously unknown morphology of the lungfish eye in order to examine ocular adaptations present in early sarcopterygian fish. Unlike many teleosts, the Australian lungfish Neoceratodus forsteri possesses a mobile pupil with a slow pupillary response similar to amphibians. The structure of the eye changes from juvenile to adult, with both eye and lens becoming more elliptical in shape with growth. This change in structure results in a decrease in focal ratio (the distance from lens center to the retina divided by the lens radius) and increased retinal illumination in adult fish. Despite a degree of lenticular correction for spherical aberration, there is considerable variation across the lens. A re-calculation of spatial resolving power using measured focal ratios from cryosectioning reveals a low ability to discriminate fine detail. The dipnoan eye shares more features with amphibian eyes than with most teleost eyes, which may echo the visual needs of this living fossil.

Absorbance spectra of rods and some cones were measured by microspectrophotometry in 22 fish species from the brackish-water of the Baltic Sea, and when applicable, in the same species from the Atlantic Ocean (3 spp.), the Mediterranean Sea (1 sp.), or Finnish fresh-water lakes (9 spp.). The main purpose was to study whether there were differences suggesting spectral adaptation of rod vision to different photic environments during the short history (<104 years) of postglacial isolation of the Baltic Sea and the Finnish lakes. Rod absorbance spectra of the Baltic subspecies/populations of herring (Clupea harengus membras), flounder (Platichthys flesus), and sand goby (Pomatoschistus minutus) were all long-wavelength-shifted (9.8, 1.9, and 5.3 nm, respectively, at the wavelength of maximum absorbance, λmax) compared with their truly marine counterparts, consistent with adaptation for improved quantum catch, and improved signal-to-noise ratio of vision in the Baltic light environment. Judged by the shape of the spectra, the chromophore was pure A1 in all these cases; hence the differences indicate evolutionary tuning of the opsin. In no species of fresh-water origin did we find significant opsin-based spectral shifts specific to the Baltic populations, only spectral differences due to varying A1/A2 chromophore ratio in some. For most species, rod λmax fell within a wavelength range consistent with high signal-to-noise ratio of vision in the spectral conditions prevailing at depths where light becomes scarce in the respective waters. Exceptions were sandeels in the Baltic Sea, which are active only in bright light, and all species in a “brown” lake, where rod λmax lay far below the theoretically optimal range.

Large field motion detection in goldfish, measured in the optomotor response, is based on the L-cone type, and is therefore color-blind (Schaerer & Neumeyer, 1996). In experiments using a two-choice training procedure, we investigated now whether the same holds for the detection of a small moving object (size: 8 mm diameter; velocity: 7 cm/s). In initial experiments, we found that goldfish did not discriminate between a moving and a stationary stimulus, obviously not taking attention to the cue “moving.” Therefore, random dot patterns were used in which the stimulus was visible only when moving. Using black and white random dot patterns with variable contrast between 0.2 and 1, we found that the fish could see motion only with high (0.8) contrast. In the decisive experiment, a red-green random dot pattern was used. By keeping the intensity of the red dots constant and reducing the intensity of the green dots, a narrow intensity range was found in which goldfish could no longer discriminate between the moving random dot stimulus in random dot surround and the stationary random dot pattern. The same was the case when a red moving disk was presented in green surround. This is the evidence that object motion is red-green color blind, i.e., color information cannot be used to detect the moving object. Calculations of the cone excitation values revealed that the M-cone type is decisive, as this cone type (and not the L-cone type) is not modulated by that particular red-green pattern in which the moving stimulus was invisible.