Disruption of Epithalamic Left-Right Asymmetry Increases Anxiety in Zebrafish.

Differences between the left and right sides of the brain are found throughout the animal kingdom, but the consequences of altered neural asymmetry are not well understood. In the zebrafish epithalamus, the parapineal is located on the left side of the brain where it influences development of the adjacent dorsal habenular (dHb) nucleus, causing the left and right dHb to differ in their organization, gene expression, and connectivity. Left-right (L-R) reversal of parapineal position and dHb asymmetry occurs spontaneously in a small percentage of the population, whereas the dHb develop symmetrically following experimental ablation of the parapineal. The habenular region was previously implicated in modulating fear in both mice and zebrafish, but the relevance of its L-R asymmetry is unclear. We now demonstrate that disrupting directionality of the zebrafish epithalamus causes reduced exploratory behavior and increased cortisol levels, indicative of enhanced anxiety. Accordingly, exposure to buspirone, an anxiolytic agent, significantly suppresses atypical behavior. Axonal projections from the parapineal to the dHb are more variable when it is located on the right side of the brain, revealing that L-R reversals do not necessarily represent a neuroanatomical mirror image. The results highlight the importance of directional asymmetry of the epithalamus in the regulation of stress responses in zebrafish.

Isolation and genetic characterization of mother-of-snow-white, a maternal effect allele affecting laterality and lateralized behaviors in zebrafish.

In the present work we report evidence compatible with a maternal effect allele affecting left-right development and functional lateralization in vertebrates. Our study demonstrates that the increased frequency of reversed brain asymmetries in a zebrafish line isolated through a behavioral assay is due to selection of mother-of-snow-white (msw), a maternal effect allele involved in early stages of left-right development in zebrafish. msw homozygous females could be identified by screening of their progeny for the position of the parapineal organ because in about 50% of their offspring we found an altered, either bilateral or right-sided, expression of lefty1 and spaw. Deeper investigations at earlier stages of development revealed that msw is involved in the specification and differentiation of precursors of the Kupffer's vesicle, a structure homologous to the mammalian node. To test the hypothesis that msw, by controlling Kupffer's vesicle morphogenesis, controls lateralized behaviors related to diencephalic asymmetries, we analyzed left- and right-parapineal offspring in a "viewing test". As a result, left- and right-parapineal individuals showed opposite and complementary eye preference when scrutinizing a model predator, and a different degree of lateralization when scrutinizing a virtual companion. As maternal effect genes are expected to evolve more rapidly when compared to zygotic ones, our results highlight the driving force of maternal effect alleles in the evolution of vertebrates behaviors.

Lines of Danio rerio selected for opposite behavioural lateralization show differences in anatomical left-right asymmetries.

Previous studies suggest that laterality of the viscera, morphological asymmetries of the brain, and lateralization of cognitive functions have a common genetic origin. To test this hypothesis, we conducted an artificial selection experiment for behavioural lateralization of eye use in two strains (TL and GT) of zebrafish (Danio rerio), maintaining one selected line in each strain for five generations. In addition, we investigated, using molecular markers, whether there was any correlation among directionality in eye preference, diencephalic left-right asymmetries in the brain and positioning of the viscera. After one generation of selection, the right- and left-eye lines of both strains showed a significant difference in the behavioural trait. This difference was maintained for all the five generations even though we observed a progressive decline in the response to artificial selection in subsequent generations for both strains. Overall, anatomical evidence suggests that selection for right-eye use significantly increased the frequency of reversed asymmetry in the epithalamus while selection for left-eye use decreased it. However, the response was irregular since not all samples conformed to this pattern. The association between the direction of behavioural selection and pancreas position was less clear-cut, although the concordance between visceral and brain asymmetries exceeded 90% in both strains.

Determining the function of zebrafish epithalamic asymmetry.

As in many fishes, amphibians and reptiles, the epithalamus of the zebrafish, Danio rerio, develops with pronounced left-right (L-R) asymmetry. For example, in more than 95 per cent of zebrafish larvae, the parapineal, an accessory to the pineal organ, forms on the left side of the brain and the adjacent left habenular nucleus is larger than the right. Disruption of Nodal signalling affects this bias, producing equal numbers of larvae with the parapineal on the left or the right side and corresponding habenular reversals. Pre-selection of live larvae using fluorescent transgenic reporters provides a useful substrate for studying the effects of neuroanatomical asymmetry on behaviour. Previous studies had suggested that epithalamic directionality is correlated with lateralized behaviours such as L-R eye preference. We find that the randomization of epithalamic asymmetry, through perturbation of the nodal-related gene southpaw, does not alter a variety of motor behaviours, including responses to lateralized stimuli. However, we discovered significant deficits in swimming initiation and in the total distance navigated by larvae with parapineal reversals. We discuss these findings with respect to previous studies and recent work linking the habenular region with control of the motivation/reward pathway of the vertebrate brain.

Artificial selection on laterality in the teleost fish Girardinus falcatus.

We performed five generations of artificial selection on laterality of eye preference in Girardinus falcatus using a detour test. Two lines were selected for right turning when encountering a potential predator, two for left turning, one for no turning bias and one unselected line was used as control. We observed a prompt response to directional selection in all lines and the response was approximately symmetrical in left and right turning lines. However, the response to selection ceased after the first or the second generation and unexpectedly in all lines the average laterality score slowly decreased in subsequent generations. After selection was suspended for three generations, no significant variation in mean laterality was observed in most cases, indicating that natural selection was not actively opposing artificial selection during the experiment. After five generations, selected lines maintained substantial additive variance as evidenced by the possibility of rapidly reversing the direction of laterality bias in just one generation of counter-selection.