Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior

Search strategy is regulated by somatostatin signaling and deep brain photoreceptors in zebrafish

Abstract Background Animals use sensory cues to efficiently locate resources, but when sensory information is insufficient, they may rely on internally coded search strategies. Despite the importance of search behavior, there is limited understanding of the underlying neural mechanisms in vertebrates. Results Here, we report that loss of illumination initiates sophisticated light-search behavior in larval zebrafish. Using three-dimensional tracking, we show that at the onset of darkness larvae swim in a helical trajectory that is spatially restricted in the horizontal plane, before gradually transitioning to an outward movement profile. Local and outward swim patterns display characteristic features of area-restricted and roaming search strategies, differentially enhancing phototaxis to nearby and remote sources of light. Retinal signaling is only required to initiate area-restricted search, implying that photoreceptors within the brain drive the transition to the roaming search state. Supporting this, orthopediaA mutant larvae manifest impaired transition to roaming search, a phenotype which is recapitulated by loss of the non-visual opsin opn4a and somatostatin signaling. Conclusion These findings define distinct neuronal pathways for area-restricted and roaming search behaviors and clarify how internal drives promote goal-directed activity

Additional file 2: Figure S1. of Search strategy is regulated by somatostatin signaling and deep brain photoreceptors in zebrafish

Fractal dimension measurement of path complexity. Illustrative examples of fractal dimension values for 30 s duration movement paths of increasing complexity. Start position of each path is indicated (black circle). (TIF 503 kb

Additional file 10: Figure S8. of Search strategy is regulated by somatostatin signaling and deep brain photoreceptors in zebrafish

Response of enucleated otpa mutant larvae to loss of illumination. Displacement (a), path complexity (fractal dimension, b), rate of re-orientation (meander, c), and trajectory bias (d) for enucleated otpa homozygous mutant larvae, recorded for 10 min under full-field illumination (yellow) or following loss of illumination (black); N = 22. Repeated measures ANOVA showed no significant main effect of enucleation on movement parameters (fractal dimension: F1,38 = 0.292, P = 0.59; displacement: F1,40 = 0.66, P = 0.42; meander: F1,38 = 2.28, P = 0.14; traj. bias: F1,38 = 0.087, P = 0.77). (TIF 1296 kb

Additional file 6: Movies 2a, b. of Search strategy is regulated by somatostatin signaling and deep brain photoreceptors in zebrafish

Representative response of a larva in the local search assay after (a) a 3 s delay following loss of illumination, and (b) after a 3 minute delay following loss of illumination. (ZIP 6033 kb

Additional file 7: Figure S5. of Search strategy is regulated by somatostatin signaling and deep brain photoreceptors in zebrafish

Wall-avoidance behavior after loss of illumination. Illustrative path trajectories for larvae over 10 min, inside a transparent interior barrier (diameter 85 mm) within the recording chamber (a) or outside the barrier (b) under baseline conditions. (c) Time spent within concentric rings of equal area (1000 mm2) progressively further from the interior barrier during 10 min recording as in (b). Differences between time spent in the four rings are not significant (repeated measures ANOVA F3,39 = 0.19, P = 0.90), indicating that larvae do not show preferential swimming in proximity to a convex wall; N = 14 larvae. (d) Representative 2 min trajectories for larva inside the interior barrier during baseline (orange), or 8 min after loss of illumination (black). (e) Quantification of (d) – proportion of time spent by larvae within 3 mm of the wall of the chamber during baseline and at the indicated time-points after loss of illumination; N = 15. * P < 0.05. (TIF 1499 kb

Additional file 3: Figure S2. of Search strategy is regulated by somatostatin signaling and deep brain photoreceptors in zebrafish

Schematic of lock index measure. Left: random turning produces a Lock index of zero. Right: continuous turning in one direction gives a Lock index of 100. (TIF 352 kb

Clock-controlled rhythmic gene expression is disrupted in ΔCLK-expressing pineal glands.

<p>(A) Experimental procedure for transcriptome analysis. Adult fish were kept under DD and pineal glands were sampled at 12 time points (indicated by arrows) throughout two daily cycles. Black and gray bars correspond to subjective night and day, respectively. (B) The mRNA-seq analysis resulted in the identification of 29 circadian genes in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish compared with 290 circadian genes in the pineal gland of Tg(<i>aanat2</i>:EGFP) control fish.</p

Diverse effects of ΔCLK on expression profiles of clock-controlled genes in the pineal gland.

<p>Representative examples of expression profiles of CCGs in the pineal gland of control Tg(<i>aanat2</i>:EGFP) fish (control; blue trendline) compared with the expression profiles of these genes in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish (ΔCLK; red trendline). Black and gray bars denote subjective night and day, respectively. CT, circadian time. While the majority of CCGs became arrhythmic (A–I), a few maintained their circadian profile in the Tg(<i>aanat2</i>:EGFP-ΔCLK) pineal gland (J–L). For some of the CCGs that became arrhythmic in the Tg(<i>aanat2</i>:EGFP-ΔCLK) pineal gland the overall basal expression levels remained relatively intermediate or high (A–D), whereas for others the expression was down-regulated (E–H) or abolished (I).</p