The cerebellum is important for the integration of sensory perception and motor control, but its structure has mostly been studied in mammals. Here, we describe the cell types and neural tracts of the adult zebrafish cerebellum using molecular markers and transgenic lines. Cerebellar neurons are categorized to two major groups: GABAergic and glutamatergic neurons. The Purkinje cells, which are GABAergic neurons, express parvalbumin7, carbonic anhydrase 8, and aldolase C like (zebrin II). The glutamatergic neurons are vglut1(+) granule cells and vglut2(high) cells, which receive Purkinje cell inputs; some vglut2(high) cells are eurydendroid cells, which are equivalent to the mammalian deep cerebellar nuclei. We found olig2(+) neurons in the adult cerebellum and ascertained that at least some of them are eurydendroid cells. We identified markers for climbing and mossy afferent fibers, efferent fibers, and parallel fibers from granule cells. Furthermore, we found that the cerebellum-like structures in the optic tectum and antero-dorsal hindbrain show similar Parvalbumin7 and Vglut1 expression profiles as the cerebellum. The differentiation of GABAergic and glutamatergic neurons begins 3 days post-fertilization (dpf), and layers are first detectable 5 dpf. Using anti-Parvalbumin7 and Vglut1 antibodies to label Purkinje cells and granule cell axons, respectively, we screened for mutations affecting cerebellar neuronal development and the formation of neural tracts. Our data provide a platform for future studies of zebrafish cerebellar development.

Animals move over a range of speeds by using rhythmic networks of neurons located in the spinal cord. Here we use electrophysiology and in vivo imaging in larval zebrafish (Danio rerio) to reveal a systematic relationship between the location of a spinal neuron and the minimal swimming frequency at which the neuron is active. Ventral motor neurons and excitatory interneurons are rhythmically active at the lowest swimming frequencies, with increasingly more dorsal excitatory neurons engaged as swimming frequency rises. Inhibitory interneurons follow the opposite pattern. These inverted patterns of recruitment are independent of cell soma size among interneurons, but may be partly explained by concomitant dorso-ventral gradients in input resistance. Laser ablations of ventral, but not dorsal, excitatory interneurons perturb slow movements, supporting a behavioural role for the topography. Our results reveal an unexpected pattern of organization within zebrafish spinal cord that underlies the production of movements of varying speeds.

Recent molecular genetic studies suggest that the expression of transcription factors in the developing spinal cord helps determine the morphological and physiological properties of neurons. Using the zebrafish preparation, we have examined the properties of neurons marked by alx, a zebrafish homolog of mammalian Chx10. We performed morphological and physiological studies using transgenic zebrafish expressing fluorescent reporter constructs in cells that had at any time point expressed alx (alx neurons). Our data reveal that zebrafish alx neurons are all ipsilateral descending neurons that are positive for vesicular glutamate transporter 2, suggesting that they are glutamatergic excitatory interneurons. Patch recordings show that earlier-born neurons are active during stronger movements such as escapes and fast swimming (strong movement class), whereas later-born ones are involved in sustained weak swimming (weak movement class). Paired recordings between alx neurons and motoneurons show that neurons of the strong movement class make frequent monosynaptic excitatory connections onto motoneurons. Thus, neurons of this class are likely premotor interneurons that regulate motoneuron activity during escapes and fast swimming. We also show the existence of a monosynaptic connection between an alx neuron of the weak movement class and a motoneuron. Collectively, our data demonstrate that alx marks ipsilateral descending neurons that are involved in the regulation of motoneuron activity during forms of locomotion, such as escape and swimming.

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.

In mammals, cerebellar neurons are categorized as glutamatergic or GABAergic, and are derived from progenitors that express the proneural genes atoh1 or ptf1a, respectively. In zebrafish, three atoh1 genes, atoh1a, atoh1b, and atoh1c, are expressed in overlapping but distinct expression domains in the upper rhombic lip (URL): ptf1a is expressed exclusively in the ventricular zone (VZ). Using transgenic lines expressing fluorescent proteins under the control of the regulatory elements of atoh1a and ptf1a, we traced the lineages of the cerebellar neurons. The atoh1(+) progenitors gave rise not only to granule cells but also to neurons of the anteroventral rhombencephalon. The ptf1a(+) progenitors generated Purkinje cells. The olig2(+) eurydendroid cells, which are glutamatergic, were derived mostly from ptf1a(+) progenitors in the VZ but some originated from the atoh1(+) progenitors in the URL. In the adult cerebellum, atoh1a, atoh1b, and atoh1c are expressed in the molecular layer of the valvula cerebelli and of the medial corpus cerebelli, and ptf1a was detected in the VZ. The proneural gene expression patterns coincided with the sites of proliferating neuronal progenitors in the adult cerebellum. Our data indicate that proneural gene-linked neurogenesis is evolutionarily conserved in the cerebellum among vertebrates, and that the continuously generated neurons help remodel neural circuits in the adult zebrafish cerebellum.

Despite a number of reports on transgenic zebrafish, there have been no reports on transgenic zebrafish in which the gene is under the control of a promoter of zebrafish origin. Neither have there been reports on transgenic zebrafish in which the gene is under the control of a tissue-specific promoter/enhancer. To investigate whether it is possible to generate transgenic zebrafish which reliably express a reporter gene in specific tissues, we have isolated a zebrafish muscle-specific actin (alpha-actin) promoter and generated transgenic zebrafish in which the green fluorescent protein (GFP) reporter gene was driven by this promoter. In total, 41 GFP-expressing transgenic lines were generated with a frequency of as high as 21% (41 of 194), and GFP was specifically expressed throughout muscle cells in virtually all of the lines (40 of 41). Nonexpressing transgenic lines were rare. This demonstrates that a tissue-specific promoter can reliably drive reporter gene expression in transgenic zebrafish in a manner identical to the control of the endogeneous expression of the gene. Levels of GFP expression varied greatly from line to line; i.e., fluorescence was very weak in some lines, while it was extremely high in others. We also isolated a zebrafish cytoskeletal beta-actin promoter and generated transgenic zebrafish using a beta-actin-GFP construct. In all of the four lines generated, GFP was expressed throughout the body like the beta-actin gene, demonstrating that consistent expression could also be achieved in this case. In the present study, we also examined the effects of factors which potentially affect the transgenic frequency or expression levels. The following results were obtained: (i) expression levels of GFP in the injected embryo were not strongly correlated to transgenic frequency; (ii) the effect of the NLS peptide (SV40 T antigen nuclear localization sequence), which has been suggested to facilitate the transfer of a transgene into embryonic nuclei, remained to be elusive; (iii) a plasmid vector sequence placed upstream of the construct might reduce the expression levels of the reporter gene.

The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.

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.