Left-right Asymmetry in Neural Connectivity of Zebrafish Brain

Fig.1: Asymmetric projections from the habenular nuclei to the IPN. (A) Schematic illustration of a lateral view of an adult zebrafish brain. (B), (C) Transverse sections at the level of the habenulae (B) and the IPN (C). Medial sub-nuclei, green; lateral sub-nuclei, red. (D) Parasagittal section showing axonal projections from the right (green) and left (red) habenula to the IPN. (E), (F) Horizontal sections showing the spiraling of left (E, red) and right (F, green) habenular axons in the IPN at the midline. In both cases, the labeled axons surround the cell bodies (blue) at the center of the nucleus. Abbreviations: Cbll, cerebellum; d, dorsal; Hb, habenula; Hy, hypothalamus; IPN, interpeduncular nucleus; l, left; OB, olfactory bulb; r, right; Tel, telencephalon; TeO, optic tectum; v,ventral.

Fig.2: Reversal of Nodal activation correlates with reversal of habenular laterality and inversion of projections in about face mutant fish. (A, B) Dorsal views of wild-type (A) and heart-reversed about face mutant (B) embryos showing the direction of Nodal signal in the habenular primordium by green fluorescent protein in the left (A) and in the right (B). (C, D) Dorsal views of the habenulae of a wild-type (C) and a heart-reversed about face mutant (D) larvae showing prominent leftover expression (marker for the left-sided lateral sub-nuculeus). (G, H) Dorsal views of habenular projections in the IPN of a wild-type (G) and an about face larvae with reversed Nodal activation (H). Axons from the left habenula have been labeled green and axons from the right habenula labeled red.

Fig.3: Nodal activity determines the orientation of asymmetry in habenular projections. The top panels show dorsal views of embryos (A-D) and the bottom panels show sub-nuclear organization of the habenulae and its asymmetric projections in normal (E) and reversed-laterality (F) adult fish. In wild-type embryos with Nodal activity in the left habenular primordium (A) leads to expansion of the lateral sub-nucleus (red) in the left habenula and the medial sub-nucleus (green) in the right habenula, resulting in projection of the left habenula predominantly to the dorsal IPN and of the right habenula to the ventral IPN (E). This pattern is completely reversed in the mutant with Nodal signaling on the right side (D, F). When Nodal signaling is activated bilaterally (B) or is absent (C), habenular asymmetry is still established, but laterality of the sub-nuclear organization becomes randomized.

Functional asymmetry in cerebral hemispheres of the human brain is a characteristic of a variety of cognitive tasks, including the processing of speech and perception. Recent studies have also found asymmetric activation in response to external stimuli in the sub-cortical structures such as limbic system. Upon seeing a fearful face, for example, one side of the amygdala will show activation. These asymmetries are not unique to humans and may be a universal feature of nervous systems across species. Chickens, fish, toads show preferential use of one visual field for specific tasks, a preference that might extend to all vertebrates. Increasing evidence suggests that lateralization in information processing in the brain is evolutionarily conserved.

Surprisingly, the neuronal connectivity patterns that regulate this functional asymmetry remain poorly understood. How, for instance, is specific information conveyed from the left or right sensory processing structures to the motor regions of the brain. As motor output triggered by lateralized brain activity involves both sides of the body, circuitry that conveys that information from left or right neural structures to nuclei on both sides of the brain should exist. Nevertheless, the location of such circuits structures, if they exist, had yet to be identified.

We uncovered a novel characteristic of neural circuitry that might provide a mechanism that shuttles information from the left and right sides of the brain to specific dorso-ventral (DV) regions of a bilaterally positioned target nucleus by analyzing the output circuitry of the lateralized habenular nuclei in zebrafish. The habenulae, evolutionarily highly conserved parts of a conduction pathway within the limbic system, connect telencephalic nuclei to the interpeduncular nucleus (IPN) of the midbrain. These nuclei probably regulate dopaminergic and serotonergic neuronal activities. Using zebrafish, we demonstrated asymmetric connectivity in the habenulo-interpeduncular projection: a stereotypic, topographic projection from left-sided habenular axons predominantly to the dorsal region of the IPN and from right-sided habenular axons predominantly to the ventral IPN (Fig. 1). A prominent left-right (LR) difference in the size ratio of the medial and lateral sub-nuclei of the habenulae can account for this asymmetry because each sub-nuclei projects to either a specific ventral or dorsal IPN target (Fig. 1).

The Nodal signaling pathway regulates laterality decisions in the viscera. In zebrafish, unilateral activation of this pathway in the left brain specifies lateral asymmetry in habenular size (Fig. 2). Asymmetric Nodal signaling also determines the DV polarity of innervations of the IPN by left and right habenular axons (Fig. 2), however axon terminal segregation can still occur in mutant IPN if Nodal signaling is symmetric or absent (Fig. 3). The latter finding is consistent with Nodal pathway activity for coordinated lateralization in the circuitry between all individuals at the population level.

In this way, we successfully identified a mechanism by which information that is distributed between the left and right sides of the brain is transmitted bilaterally without losing LR coding. We have also started to elucidate the signaling pathways that direct the establishment of lateralized circuitry.