The zebrafish dorsal habenula (dHb) shows conspicuous asymmetry in its connection with the interpeduncular nucleus (IPN) and is equivalent to the mammalian medial habenula. Genetic inactivation of the lateral subnucleus of dHb (dHbL) biased fish towards freezing rather than the normal flight response to a conditioned fear stimulus, suggesting that the dHbL-IPN pathway is important for controlling experience-dependent modification of fear responses.

Anticipation of danger at first elicits panic in animals, but later it helps them to avoid the real threat adaptively. In zebrafish, as fish experience more and more danger, neurons in the ventral habenula (vHb) showed tonic increase in the activity to the presented cue and activated serotonergic neurons in the median raphe (MR). This neuronal activity could represent the expectation of a dangerous outcome and be used for comparison with a real outcome when the fish is learning how to escape from a dangerous to a safer environment. Indeed, inhibiting synaptic transmission from vHb to MR impaired adaptive avoidance learning, while panic behavior induced by classical fear conditioning remained intact. Furthermore, artificially triggering this negative outcome expectation signal by optogenetic stimulation of vHb neurons evoked place avoidance behavior. Thus, vHb-MR circuit is essential for representing the level of expected danger and behavioral programming to adaptively avoid potential hazard.

The mammalian habenula consists of the medial and lateral habenulae. Recent behavioral and electrophysiological studies suggested that the lateral habenula plays a pivotal role in controlling motor and cognitive behaviors by influencing the activity of dopaminergic and serotonergic neurons. Despite the functional significance, manipulating neural activity in this pathway remains difficult because of the absence of a genetically accessible animal model such as zebrafish. To address the level of lateral habenula conservation in zebrafish, we applied the tract-tracing technique to GFP (green fluorescent protein)-expressing transgenic zebrafish to identify habenular neurons that project to the raphe nuclei, a major target of the mammalian lateral habenula. Axonal tracing in live and fixed fish showed projection of zebrafish ventral habenula axons to the ventral part of the median raphe, but not to the interpeduncular nucleus where the dorsal habenula projected. The ventral habenula expressed protocadherin 10a, a specific marker of the rat lateral habenula, whereas the dorsal habenula showed no such expression. Gene expression analyses revealed that the ventromedially positioned ventral habenula in the adult originated from the region of primordium lateral to the dorsal habenula during development. This suggested that zebrafish habenulae emerge during development with mediolateral orientation similar to that of the mammalian medial and lateral habenulae. These findings indicated that the lateral habenular pathways are evolutionarily conserved pathways and might control adaptive behaviors in vertebrates through the regulation of monoaminergic activities.

When animals encounter conflict they initiate and escalate aggression to establish and maintain a social hierarchy. The neural mechanisms by which animals resolve fighting behaviors to determine such social hierarchies remain unknown. We identified two subregions of the dorsal habenula (dHb) in zebrafish that antagonistically regulate the outcome of conflict. The losing experience reduced neural transmission in the lateral subregion of dHb (dHbL)-dorsal/intermediate interpeduncular nucleus (d/iIPN) circuit. Silencing of the dHbL or medial subregion of dHb (dHbM) caused a stronger predisposition to lose or win a fight, respectively. These results demonstrate that the dHbL and dHbM comprise a dual control system for conflict resolution of social aggression.

A simple and efficient procedure for labeling neurons is a prerequisite for investigating the development of neural networks in zebrafish. To label neurons we used Kaede, a fluorescent protein with a photoconversion property allowing conversion from green to red fluorescence following irradiation with UV or violet light. We established a zebrafish stable transgenic line, Tg(HuC:Kaede), expressing Kaede in neurons under the control of the HuC promoter. This transgenic line was used to label a small number of neurons in the trigeminal ganglion. Also, using embryos injected with the transgene, we labeled peripheral axon arbors of a Rohon-Beard neuron at 4 days postfertilization and observed the dendrite development of a tectal neuron for 3 days. These data indicate that Kaede is a useful tool to selectively label neural networks in zebrafish.

The encoding of long-term associative memories for learned behaviors is a fundamental brain function. Yet, how behavior is stably consolidated and retrieved in the vertebrate cortex is poorly understood. We trained zebrafish in aversive reinforcement learning and measured calcium signals across their entire brain during retrieval of the learned response. A discrete area of dorsal telencephalon that was inactive immediately after training became active the next day. Analysis of the identified area indicated that it was specific and essential for long-term memory retrieval and contained electrophysiological responses entrained to the learning stimulus. When the behavioral rule changed, a rapid spatial shift in the functional map across the telencephalon was observed. These results demonstrate that the retrieval of long-term memories for learned behaviors can be studied at the whole-brain scale in behaving zebrafish in vivo. Moreover, the findings indicate that consolidated memory traces can be rapidly modified during reinforcement learning.

The Hedgehog (Hh) signal plays a pivotal role in induction of ventral neuronal and muscle cell types around the midline during vertebrate development [1]. We report that the gene disrupted in zebrafish you mutants, in which Hh signaling is impaired, encodes the secreted matrix protein Scube2. Consistently, epistasis analyses suggested that Scube2 functions upstream of Hh ligands or through a parallel pathway. In addition, overexpression analyses suggested that Scube2 is an essential, but a permissive, mediator of Hh signaling in zebrafish embryos. Surprisingly, the you gene is expressed in the dorsal neural tube, raising the possibility that Scube2 could indirectly act via a long-range regulator of Hh signaling. The dorsal Bmps have a long-range and opposing influence on Hh signaling [2-5]. We show that neural plate patterning is affected in you mutants in a way that is consistent with the aberrant long-range action of a Bmp-dependent signal. We further show that Bmp activity can be attenuated by the coexpression of Scube2. Our data support the idea that Scube2 can modulate the long-range action of Bmp-dependent signaling in the neural tube and somites.

In the Supplemental Data of this article, the strain described as smu b571 was actually b641. The authors regret the error.