The retinas of adult teleost fish can regenerate neurons following injury. The current study provides the first documentation of functional whole retina regeneration in the zebrafish, Danio rerio, following intraocular injection of the cytotoxin, ouabain. Loss and replacement of laminated retinal tissue was monitored by analysis of cell death and cell proliferation, and by analysis of retina-specific gene expression patterns. The spatiotemporal process of retinal ganglion cell (RGC) regeneration was followed through the use of selective markers, and was found to largely recapitulate the spatiotemporal process of embryonic ganglion cell neurogenesis, over a more protracted time frame. However, the re-expression of some ganglion cell markers was not observed. The growth and pathfinding of ganglion cell axons was evaluated by measurement of the optic nerve head (ONH), and the restoration of normal ONH size was found to correspond to the time of recovery of two visually-mediated behaviors. However, some abnormalities were noted, including overproduction of RGCs, and progressive and excessive growth of the ONH at longer recovery times. This model system for whole-retina regeneration has provided an informative view of the regenerative process.

SUMMARY Centrifugal innervation of the neural retina has been documented in many species. In zebrafish Danio rerio, the only so-far described centrifugal pathway originates from terminal nerve (TN) cell bodies that are located in the olfactory bulb. Most of the TN axons terminate in the forebrain and midbrain, but some project via the optic nerve to the neural retina, where they synapse onto dopaminergic interplexiform cells (DA-IPCs). While the anatomical pathway between the olfactory and visual organs has been described, it is unknown if and how olfactory signals influence visual system functions. We demonstrate here that olfactory input is involved in the modulation of visual sensitivity in zebrafish. As determined by a behavioral assay and by electroretinographic (ERG) recording, zebrafish visual sensitivity was increased upon presentation of amino acids as olfactory stimuli. This effect, however, was observed only in the early morning hours when zebrafish are least sensitive to light. The effect of olfactory input on vision was eliminated after lesion of the olfactory bulbs or after the destruction of DA-IPCs. Intraocular injections of a dopamine D2 but not a D1 receptor antagonist blocked the effect of olfactory input on visual sensitivity. Although we cannot exclude the involvement of other anatomical pathways, our data suggest that the TN and DA-IPCs are the prime candidates for olfactory modulation of visual sensitivity.

The vertebrate retina receives centrifugal input from the brain. In zebrafish, the major centrifugal input originates in the terminal nerve (TN). TN cell bodies are located in the olfactory bulb and ventral telencephalon. The TN projects axons to the retina where they branch in the inner plexiform layer (IPL) and synapse onto several inner retinal cell types, including dopaminergic interplexiform cells (DA-IPCs). This olfactoretinal centrifugal input plays a role in modulating retinal ganglion cell (RGC) activity, probably via dopamine-mediated Ca 2+ signalling pathways. Normally, dopamine inhibits RGC firing by decreasing the inward Ca 2+ current. Olfactory stimulation with amino acids decreases dopamine release in the retina, thereby reducing dopaminergic inhibition of RGCs. This model of olfacto-visual integration was directly tested by recording single-unit RGC activity in response to olfactory stimulation in the presence or absence of dopamine receptor blockers. Stimulation of the olfactory neurones increased RGC activity. However, this effect diminished when the dopamine D1 receptors were pharmacologically blocked. In isolated RGCs, the application of dopamine or a dopamine D1 receptor agonist decreased voltage-activated Ca 2+ current and lowered Ca 2+ influx. Together, the data suggest that olfactory input has a modulatory effect on RGC firing, and that this effect is mediated by dopamine D1 receptor-coupled Ca 2+ signalling pathways.

The optomotor response (OMR) is a simple experimental paradigm that is widely used in the study of visual system functions. In the current paper we investigated how spatial and temporal properties of repetitive stimuli determine the OMR in zebrafish. The experiments showed that the OMR has the temporal characteristic of a low-pass filter when the spatial frequencies are low and of a band-pass filter when the spatial frequencies are high. These findings are discussed on the basis of inherent sampling constraints of any motion detector. We found some indications that the strength and direction of the OMR vary with the spatio-temporal frequency of the stimulus pattern as has previously been described for other species.

Circadian rhythms in cardiac function are apparent in e.g., blood pressure, heart rate, and acute adverse cardiac events. A circadian clock in heart tissue has been identified, but entrainment pathways of this clock are still unclear. We cultured tissues of mice carrying bioluminescence reporters of the core clock genes, period 1 or 2 (per1luc or PER2LUC) and compared in vitro responses of atrium to treatment with medium and a synthetic glucocorticoid (dexamethasone [DEX]) to that of the suprachiasmatic nucleus (SCN) and liver. We observed that PER2LUC, but not per1luc is rhythmic in atrial tissue, while both per1luc and PER2LUC exhibit rhythmicity in other cultured tissues. In contrast to the SCN and liver, both per1luc and PER2LUC bioluminescence amplitudes were increased in response to DEX treatment, and the PER2LUC amplitude response was dependent on the time of treatment. Large phase-shift responses to both medium and DEX treatments were observed in the atrium, and phase responses to medium treatment were not attributed to serum content but the treatment procedure itself. The phase-response curves of atrium to both DEX and medium treatments were found to be different to the liver. Moreover, the time of day of the culturing procedure itself influenced the phase of the circadian clock in each of the cultured tissues, but the magnitude of this response was uniquely large in atrial tissue. The current data describe novel entrainment signals for the atrial circadian clock and specifically highlight entrainment by mechanical treatment, an intriguing observation considering the mechanical nature of cardiac tissue.

Zebrafish are a promising model for behavioral and genetic studies of vertebrate visual system development and retinal degeneration. In the past few years, numerous studies on zebrafish vision have been published. While most of the studies focus on the molecular and cellular characterization of mutations that disrupt zebrafish visual system structure in early development, others examine the mechanisms that underlie inherited visual system disorders in adults. Behavioral assays, along with morphologic and electrophysiological methods, are powerful tools for functional analyses of zebrafish visual development and performance.

We report a study of the characterization of voltage-activated potassium (K+) currents in retinal ON bipolar cells in zebrafish. At single-channels levels, the open probability of the K+ channels increased when the membrane potential was increased. The maximal open proportion was 0.76+/-0.05 under our testing conditions. In whole-cell recordings, the K+ current displayed two exponential components with the activation time constants of 11-22 msec (tau1) and 0.8-4 msec (tau2). Dopamine modulated the K+ current. Dopamine reduced the time constant tau2 when the membrane potential was depolarized to high voltages. A decrease in K+ current was seen when dopamine D1 receptors were selectively activated by SKF38393 or when the D1 receptor-coupled G-proteins were activated by GTP-gamma-S. The activation of adenylate cyclase by forskolin or the increase of intracellular cAMP concentrations by 8-Br-cAMP or Sp-cAMPS also resulted in a decrease in K+ current. Together, the data suggest that dopamine modulates the K+ current via D1 receptor-coupled G-protein pathways.

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