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.

The habenulae are part of an evolutionarily highly conserved limbic-system conduction pathway that connects telencephalic nuclei to the interpeduncular nucleus (IPN) of the midbrain . In zebrafish, unilateral activation of the Nodal signaling pathway in the left brain specifies the laterality of the asymmetry of habenular size . We show "laterotopy" in the habenulo-interpeduncular projection in zebrafish, i.e., the stereotypic, topographic projection of left-sided habenular axons to the dorsal region of the IPN and of right-sided habenular axons to the ventral IPN. This asymmetric projection is accounted for by a prominent left-right (LR) difference in the size ratio of the medial and lateral habenular sub-nuclei, each of which specifically projects either to ventral or dorsal IPN targets. Asymmetric Nodal signaling directs the orientation of laterotopy but is dispensable for the establishment of laterotopy itself. Our results reveal a mechanism by which information distributed between left and right sides of the brain can be transmitted bilaterally without loss of LR coding, which may play a crucial role in functional lateralization of the vertebrate brain .

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.

Motor cortex neurons are activated at different times during self-initiated voluntary movement. However, the manner in which excitatory and inhibitory neurons in distinct cortical layers help to organize voluntary movement is poorly understood. We carried out juxtacellular and multiunit recordings from actively behaving rats and found temporally and functionally distinct activations of excitatory pyramidal cells and inhibitory fast-spiking interneurons. Across cortical layers, pyramidal cells were activated diversely for sequential motor phases (for example, preparation, initiation and execution). In contrast, fast-spiking interneurons, including parvalbumin-positive basket cells, were recruited predominantly for motor execution, with pyramidal cells producing a command-like activity. Thus, fast-spiking interneurons may underlie command shaping by balanced inhibition or recurrent inhibition, rather than command gating by temporally alternating excitation and inhibition. Furthermore, initiation-associated pyramidal cells excited similar and different functional classes of neurons through putative monosynaptic connections. This suggests that these cells may temporally integrate information to initiate and coordinate voluntary movement.

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.

The mammalian habenula is involved in regulating the activities of serotonergic and dopaminergic neurons. It consists of the medial and lateral habenulae, with each subregion having distinct neural connectivity. Despite the functional significance, manipulating neural activity in a subset of habenular pathways remains difficult because of the poor availability of molecular markers that delineate the subnuclear structures. Thus, we examined the molecular nature of neurons in the habenular subnuclei by analyzing the gene expressions of neurotransmitter markers. The results showed that different subregions of the medial habenula (MHb) use different combinations of neurotransmitter systems and could be categorized as either exclusively glutamatergic (superior part of MHb), both substance P-ergic and glutamatergic (dorsal region of central part of MHb), or both cholinergic and glutamatergic (inferior part, ventral region of central part, and lateral part of MHb). The superior part of the MHb strongly expressed interleukin-18 and was innervated by noradrenergic fibers. In contrast, the inferior part, ventral region of the central part, and lateral part of the MHb were peculiar in that acetylcholine and glutamate were cotransmitted from the axonal terminals. In contrast, neurons in the lateral habenula (LHb) were almost uniformly glutamatergic. Finally, the expressions of Htr2c and Drd2 seemed complementary in the medial LHb division, whereas they coincided in the lateral division, suggesting that the medial and lateral divisions of LHb show strong heterogeneity with respect to monoamine receptor expression. These analyses clarify molecular differences between subnuclei in the mammalian habenula that support their respective functional implications.

The habenular neurons on both sides of the zebrafish diencephalon show an asymmetric (laterotopic) axonal projection pattern into the interpeduncular nucleus. We previously revealed that the habenula could be subdivided into medial and lateral subnuclei, and a prominent left-right difference in the size ratio of these subnuclei accounts for the asymmetry in its neural connectivity. In the present study, birth date analysis showed that neural precursors for the lateral subnuclei were born at earlier stages than those for the medial subnuclei. More neurons for the early-born lateral subnuclei were generated on the left side, while more neurons for the late-born medial subnuclei were generated on the right side. Genetic hyperactivation and repression of Notch signaling revealed that differential timing determines both specificity and asymmetry in the neurogenesis of neural precursors for the habenular subnuclei.

It is widely accepted that dorsal striatum neurons participate in either the direct pathway (expressing dopamine D1 receptors) or the indirect pathway (expressing D2 receptors), controlling voluntary movements in an antagonistically balancing manner. The D1-and D2-expressing neurons are activated and inactivated, respectively, by dopamine released from substantia nigra neurons encoding reward expectation. However, little is known about the functional representation of motor information and its reward modulation in individual striatal neurons constituting the two pathways. In this study, we juxtacellularly recorded the spike activity of single neurons in the dorsolateral striatum of rats performing voluntary forelimb movement in a reward-predictable condition. Some of these neurons were identified morphologically by a combination of juxtacellular visualization and in situ hybridization for D1 mRNA. We found that the striatal neurons exhibited distinct functional activations before and during the forelimb movement, regardless of the expression of D1 mRNA. They were often positively, but rarely negatively, modulated by expecting a reward for the correct motor response. The positive reward modulation was independent of behavioral differences in motor performance. In contrast, regular-spiking and fast-spiking neurons in any layers of the motor cortex displayed only minor and unbiased reward modulation of their functional activation in relation to the execution of forelimb movement. Our results suggest that the direct and indirect pathway neurons cooperatively rather than antagonistically contribute to spatiotemporal control of voluntary movements, and that motor information is subcortically integrated with reward information through dopaminergic and other signals in the skeletomotor loop of the basal ganglia.