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Homeobox genes act at the top of genetic hierarchies to regulate cell specification and differentiation during embryonic development. We identified the short stature homeobox domain 2 (shox2) transcription factor that is required for vestibular neuron development. shox2 transcripts are initially localized to the otic placode of the developing inner ear where neurosensory progenitors reside. To study shox2 function, we generated CRISPR-mediated mutant shox2 fish. Mutant embryos display behaviors associated with vestibular deficits and showed reduced number of anterior statoacoustic ganglion neurons that innervate the utricle, the vestibular organ in zebrafish. Moreover, a shox2-reporter fish showed labeling of developing statoacoustic ganglion neurons in the anterior macula of the otic vesicle. Single cell RNA-sequencing of cells from the developing otic vesicle of shox2 mutants revealed altered otic progenitor profiles, while single molecule in situ assays showed deregulated levels of transcripts in developing neurons. This study implicates a role for shox2 in development of vestibular but not auditory statoacoustic ganglion neurons.

Spatio-temporal information about head orientation and movement is fundamental to the sense of balance and motion. Hair cells (HCs) in otolith organs of the vestibular system transduce linear acceleration, including head tilt and vibration. Here, we build a tiltable objective microscope in which an objective lens and specimen tilt together. With in vivo Ca2+imaging of all utricular HCs and ganglion neurons during 360° static tilt and vibration in pitch and roll axes, we reveal the direction- and static/dynamic stimulus-selective topographic responses in larval zebrafish. We find that head vibration is preferentially received by striolar HCs, whereas static tilt is preferentially transduced by extrastriolar HCs. Spatially ordered direction preference in HCs is consistent with hair-bundle polarity and is preserved in ganglion neurons through topographic innervation. Together, these results demonstrate topographically organized selectivity for direction and dynamics of head orientation/movement in the vestibular periphery.

Developmental maturation occurs in slow swimming behavior in larval zebrafish; older larvae acquire the ability to perform slow swimming while keeping their head stable in the yaw dimension. A class of long-distance descending commissural excitatory V0v neurons, called MCoD neurons, are known to develop in a later phase of neurogenesis, and participate in slow swimming in older larvae. We hypothesized that these MCoD neurons play a role in coordinating the activities of trunk muscles in the diagonal dimension (e.g., the rostral left and the caudal right) to produce the S-shaped swimming form that contributes to the stability of the head. Here, we show that MCoD neurons do indeed play this role. In larvae in which MCoD neurons were laser-ablated, the swimming body form often adopted a one-sided (C-shaped) bend with reduced appearance of the normal S-shaped bend. With this change in swimming form, the MCoD-ablated larvae exhibited a greater degree of head yaw displacement during slow swimming. In mice, the long-distance descending commissural V0v neurons have been implicated in diagonal interlimb coordination during walking. Together with this, our study suggests that the long-distance descending commissural V0v neurons form an evolutionarily conserved pathway in the spinal locomotor circuits that coordinates the movements of the diagonal body/limb muscles.

The zebrafish is an important model in systems neuroscience but a key limitation is the lack of viral tools to dissect the structure and function of neuronal circuitry. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 can be combined with the Gal4/UAS system to target cell types with high spatial, temporal and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches.

Summary As sensory information moves through the brain, higher-order areas exhibit more complex tuning than lower areas. Though models predict that complexity arises via convergent inputs from neurons with diverse response properties, in most vertebrate systems, convergence has only been inferred rather than tested directly. Here, we measure sensory computations in zebrafish vestibular neurons across multiple axes in vivo. We establish that whole-cell physiological recordings reveal tuning of individual vestibular afferent inputs and their postsynaptic targets. Strong, sparse synaptic inputs can be distinguished by their amplitudes, permitting analysis of afferent convergence in vivo. An independent approach, serial-section electron microscopy, supports the inferred connectivity. We find that afferents with similar or differing preferred directions converge on central vestibular neurons, conferring more simple or complex tuning, respectively. Together, these results provide a direct, quantifiable demonstration of feedforward input convergence in vivo.

Objective It is well-known that there is a correlation between the neuropathological grade of hippocampal sclerosis (HS) and neuroradiological atrophy of the hippocampus in mesial temporal lobe epilepsy (mTLE) patients. However, there is no strict definition or criterion regarding neuron loss and atrophy of the amygdala neighboring the hippocampus. We examined the relationship between HS and neuronal loss in the amygdala. Materials and methods Nineteen mTLE patients with neuropathological proof of HS were assigned to Group A, while seven mTLE patients without HS were assigned to Group B. We used FreeSurfer software to measure amygdala volume automatically based on pre-operation magnetic resonance images. Neurons observed using Kluver-Barrera (KB) staining in resected amygdala tissue were counted. and the extent of immunostaining with stress marker antibodies was semiquantitatively evaluated. Results There was no significant difference in amygdala volume between the two groups (Group A: 1.41 ± 0.24; Group B: 1.41 ± 0.29 cm 3 ; p = 0.98), nor in the neuron cellularity of resected amygdala specimens (Group A: 3.98 ± 0.97; Group B: 3.67 ± 0.67 10 × −4 number of neurons/μm 2 ; p = 0.40). However, the HSP70 level, representing acute stress against epilepsy, in Group A patients was significantly larger than that in Group B. There was no significant difference in the level of Bcl-2, which is known as a protein that inhibits cell death, between the two groups. Conclusions Neuronal loss and volume loss in the amygdala may not necessarily follow hippocampal sclerosis. From the analysis of stress proteins, epileptic attacks are as likely to damage the amygdala as the hippocampus but do not lead to neuronal death in the amygdala.

Summary: Commissural inhibitory neurons in the spinal cord of aquatic vertebrates coordinate left-right body alternation during swimming. Their developmental origin, however, has been elusive. We investigate this by comparing the anatomy and function of two commissural inhibitory neuron types, dI6dmrt3a and V0d, derived from the pd6 and p0 progenitor domains, respectively. We find that both of these commissural neuron types have monosynaptic, inhibitory connections to neuronal populations active during fictive swimming, supporting their role in providing inhibition to the contralateral side. V0d neurons tend to fire during faster and stronger movements, while dI6dmrt3a neurons tend to fire more consistently during normal fictive swimming. Ablation of dI6dmrt3a neurons leads to an impairment of left-right alternating activity through abnormal co-activation of ventral root neurons on both sides of the spinal cord. Our results suggest that dI6dmrt3a and V0d commissural inhibitory neurons synergistically provide inhibition to the opposite side across different swimming behaviors. : Satou et al. explore the functional properties dI6dmrt3a and V0d neurons in the spinal cord of larval zebrafish. Both populations provide inhibition to the swimming CPG and motor neurons on the contralateral side when the ipsilateral side is active. Ablation of dI6dmrt3a neurons confirms their contribution to left-right alternation. Keywords: zebrafish, mid cycle inhibition, commissural inhibitory neurons, spinal cord circuit, dI6 dmrt3a neurons, V0d neurons, dbx1, glycinergic interneuons

During many behaviors in vertebrates, the CNS generates asymmetric activities between the left and right sides to produce asymmetric body movements. For asymmetrical activations of the CNS, reciprocal inhibition between the left and right sides is believed to play a key role. However, the complexity of the CNS makes it difficult to identify the reciprocal inhibition circuits at the level of individual cells and the contribution of each neuron to the asymmetric activity. Using larval zebrafish, we examined this issue by investigating reciprocal inhibition circuits between a pair of Mauthner (M) cells, giant reticulospinal neurons that trigger fast escapes. Previous studies have shown that a class of excitatory neurons, called cranial relay neurons, is involved in the reciprocal inhibition pathway between the M cells. Using transgenic fish, in which two of the cranial relay neurons (Ta1 and Ta2) expressed GFP, we showed that Ta1 and Ta2 constitute major parts of the pathway. In larvae in which Ta1/Ta2 were laser-ablated, the amplitude of the reciprocal IPSPs dropped to less than one-third. Calcium imaging and electrophysiological recording showed that the occurrence probability of bilateral M-cell activation upon sound/vibration stimuli was greatly increased in the Ta1/Ta2-ablated larvae. Behavioral experiments revealed that the Ta1/Ta2 ablation resulted in shallower body bends during sound/vibration-evoked escapes, which is consistent with the observation that increased occurrence of bilateral M-cell activation impaired escape performance. Our study revealed major components of the reciprocal inhibition circuits in the M cell system and the behavioral importance of the circuits.SIGNIFICANCE STATEMENTReciprocal inhibition between the left and right side of the CNS is considered imperative for producing asymmetric movements in animals. It has been difficult, however, to identify the circuits at the individual cell level and their role in behavior. Here, we address this problem by examining the reciprocal inhibition circuits of the hindbrain Mauthner (M) cell system in larval zebrafish. We determined that two paired interneurons play a critical role in the reciprocal inhibition between the paired M cells and that the reciprocal inhibition prevents bilateral firing of the M cells and is thus necessary for the full body bend during M cell-initiated escape. Further, we discussed the cooperation of multiple reciprocal inhibitions working in the hindbrain and spinal cord to ensure high-performance escapes.

The developing nervous system consists of a variety of cell types. Transgenic animals expressing reporter genes in specific classes of neuronal cells are powerful tools for the study of neuronal network formation. We generated a wide variety of transgenic zebrafish that expressed reporter genes in specific classes of neurons or neuronal progenitors. These include lines in which neurons of specific neurotransmitter phenotypes expressed fluorescent proteins or Gal4, and lines in which specific subsets of the dorsal progenitor domain in the spinal cord expressed fluorescent proteins. Using these, we examined domain organization in the developing dorsal spinal cord, and found that there are six progenitor domains in zebrafish, which is similar to the domain organization in mice. We also systematically characterized neurotransmitter properties of the neurons that are produced from each domain. Given that reporter gene expressions occurs in a wide area of the nervous system in the lines generated, these transgenic fish should serve as powerful tools for the investigation of not only the neurons in the dorsal spinal cord but also neuronal structures and functions in many other regions of the nervous system.

Summary Background During locomotion in vertebrates, reticulospinal neurons in the hindbrain play critical roles in providing descending excitation to the spinal cord locomotor systems. However, despite the fact that many genes that are used to classify the neuronal identities of neurons in the hindbrain have been identified, the molecular identity of the reticulospinal neurons that are critically involved in locomotor drive is not well understood. Chx10-expressing neurons (V2a neurons) are ipsilaterally projecting glutamatergic neurons in the spinal cord and the hindbrain. Many of the V2a neurons in the hindbrain are known to project to the spinal cord in zebrafish, making hindbrain V2a neurons a prime candidate in descending locomotor drive. Results We investigated the roles of hindbrain V2a neurons using optogenetic and electrophysiological approaches. The forced activation of hindbrain V2a neurons using channelrhodopsin efficiently evoked swimming, whereas the forced inactivation of them using Archearhodopsin3 or Halorhodpsin reliably stopped ongoing swimming. Electrophysiological recordings of two populations of hindbrain reticulospinal V2a neurons showed that they were active during swimming. One population of neurons, small V2a neurons in the caudal hindbrain, fired with low rhythmicity, whereas the other population of neurons, large reticulospinal V2a neurons, called MiV1 neurons, fired more rhythmically. Conclusions These results indicated that hindbrain reticulospinal V2a neurons play critical roles in providing excitation to the spinal locomotor circuits during swimming by providing both tonic and phasic inputs to the circuits.

The developing spinal cord is subdivided into distinct progenitor domains, each of which gives rise to different types of neurons. However, the developmental mechanisms responsible for generating neuronal diversity within a domain are not well understood. Here, we have studied zebrafish V0 neurons, those that derive from the p0 progenitor domain, to address this question. We find that all V0 neurons have commissural axons, but they can be divided into excitatory and inhibitory classes. V0 excitatory neurons (V0-e) can be further categorized into three groups based on their axonal trajectories; V0-eA (ascending), V0-eB (bifurcating), and V0-eD (descending) neurons. By using time-lapse imaging of p0 progenitors and their progeny, we show that inhibitory and excitatory neurons are produced from different progenitors. We also demonstrate that V0-eA neurons are produced from distinct progenitors, while V0-eB and V0-eD neurons are produced from common progenitors. We then use birth-date analysis to reveal that V0-eA, V0-eB, and V0-eD neurons arise in this order. By perturbing Notch signaling and accelerating neuronal differentiation, we predictably alter the generation of early born V0-e neurons at the expense of later born ones. These results suggest that multiple types of V0 neurons are produced by two distinct mechanisms; from heterogeneous p0 progenitors and from the same p0 progenitor, but in a time-dependent manner.

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.

The p2 progenitor domain in the ventral spinal cord gives rise to two interneuron subtypes: V2a and V2b. Delta-Notch-mediated cell-cell interactions between postmitotic immature neurons have been implicated in the segregation of neuron subtypes. However, lineage relationships between V2a and V2b neurons have not been reported. We address this issue using Tg[vsx1:GFP]zebrafish, a model system in which high GFP expression is initiated near the final stage of p2 progenitors. Cell fates were followed in progeny using time-lapse microscopy. Results indicate that the vast majority, if not all, of GFP-labeled p2 progenitors divide once to produce V2a/V2b neuron pairs,indicating that V2a and V2b neurons are generated by the asymmetric division of pair-producing progenitor cells. Together with evidence that Notch signaling is involved in the cell fate specification process, our results strongly suggest that Delta-Notch interactions between sister cells play a crucial role in the final outcome of these asymmetric divisions. This mechanism for determining cell fate is similar to asymmetric divisions that occur during Drosophila neurogenesis, where ganglion mother cells divide once to produce distinct neurons. However, unlike in Drosophila, the divisional axes of p2 progenitors in zebrafish were not fixed. We report that the terminal division of pair-producing progenitor cells in vertebrate neurogenesis can reproducibly produce two distinct neurons through a mechanism that may not depend on the orientation of the division axis.

Extracellular matrix (ECM) proteins play an important role in early cortical development, specifically in the formation of neural connections and in controlling the cyto-architecture of the central nervous system. F-spondin and Mindin are a family of matrix-attached adhesion molecules that share structural similarities and overlapping domains of expression. Genes for both proteins contain a thrombospondin type I repeat(s) at the C terminus and an FS1-FS2 (spondin) domain. Both the vertebrate F-spondin and the zebrafish mindins are expressed on the embryonic floor plate. In the current study we have cloned the rat homologue of mindin and studied its expression and activity together with F-spondin in the developing rodent brain. The two genes are abundantly expressed in the developing hippocampus. In vitro studies indicate that both F-spondin and Mindin promote adhesion and outgrowth of hippocampal embryonic neurons. We have also demonstrated that the two proteins bind to a putative receptor(s) expressed on both hippocampal and sensory neurons.

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.

Zebrafish is a powerful model system for investigations of vertebrate neural development. The animal has also become an important model for studies of neuronal function. Both in developmental and functional studies, transgenic zebrafish expressing fluorescent proteins in central nervous system neurons have been playing important roles. We review here the methods for producing transgenic zebrafish. Recent advances in transposon- or bacterial artificial chromosome-based transgenesis greatly facilitate the creation of useful lines. We also present our study on alx-positive neurons to reveal how transgenic zebrafish expressing fluorescent proteins in a specific class of neurons can be used to investigate their development and function.

Zebrafish are an excellent model for studies of the functional organization of neuronal circuits, but little is known regarding the transmitter phenotypes of the neurons in their nervous system. We examined the distribution in spinal cord and hindbrain of neurons expressing markers of transmitter phenotype, including the vesicular glutamate transporter (VGLUT) genes for glutamatergic neurons, the neuronal glycine transporter (GLYT2) for glycinergic neurons, and glutamic acid decarboxylase (GAD65/67) for GABAergic neurons. All three markers were expressed in a large domain in the dorsal two-thirds of spinal cord, with additional, more ventral expression domains for VGLUT2 and GAD/GABA. In the large dorsal domain, dual in situ staining showed that GLYT2-positive cells were intermingled with VGLUT2 cells, with no dual-stained neurons. Many of the neurons in the dorsal expression domain that were positive for GABA markers at embryonic stages were also positive for GLYT2, suggesting that the cells might use both GABA and glycine, at least early in their development. The intermingling of neurons expressing inhibitory and excitatory markers in spinal cord contrasted markedly with the organization in hindbrain, where neurons expressing a particular marker were clustered together to form stripes that were visible running from rostral to caudal in horizontal sections and from dorsomedial to ventrolateral in cross sections. Dual labeling showed that the stripes of neurons labeled with one transmitter marker alternated with stripes of cells labeled for the other transmitter phenotypes. The differences in the distribution of excitatory and inhibitory neurons in spinal cord versus hindbrain may be tied to differences in their patterns of development and functional organization. J. Comp. Neurol. 480:1–18, 2004. © 2004 Wiley-Liss, Inc.

Genetically encoded calcium indicators, such as cameleon, have offered the promise of noninvasively monitoring activity of neurons, but no one has demonstrated whether these indicators can report calcium transients in neurons of behaving vertebrates. We show that cameleon can be expressed at high levels in sensory and spinal cord neurons in zebrafish by using neural-specific promoters in both transient expression experiments and in a stable transgenic line. Using standard confocal microscopy, calcium transients in identified motoneurons and spinal interneurons could be detected during escape behaviors produced by a touch on the head of the fish. Small movements of the restrained fish during the behavior did not represent a major problem for analyzing the calcium responses because of the ratiometric nature of cameleon. We conclude that cameleon can be used to noninvasively study the activity of neurons in an intact, behaving vertebrate. The ability to introduce an indicator genetically allows for studies of the functional roles of local interneurons that cannot easily be monitored with other approaches. Transgenic lines such as the one we generated can also be crossed into mutant lines of fish to study both structural and functional consequences of the mutations.

SummaryThe 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.Video Abstract

We isolated and characterized the eagle gene, encoding a member of the steroid receptor superfamily in Drosophila. In the central nervous system, eagle RNA was expressed in a limited number of cells. During stages 10 and 11, eagle RNA expression was observed in four neuroblasts, NB2-4, NB3–3, NB6–4 and NB7–3. Except for NB6–4, eagle RNA expression reached a maximum at the very beginning of expression or in the period of neuroblast delamination. Weak eagle RNA expression was also observed in a few putative progeny of NB7–3 during stages, late 11 and 12. All eagle RNA in abdominal segments disappeared at stage 13. Using an eagle-kinesin-lacZ fusion gene as a reporter, the division, migration, and axonogenesis in eagle-positive cells and their derivatives were examined. At stage 14, several types of neural or glial cells were detected which include EG and EW interneurons joining to the anterior and posterior commissures, respectively. Lack of eagle expression caused altered axonogenesis in an appreciable fraction of eagle-Kinesin-LacZ-positive neurons. Some EG cells failed to acquire the neural fate or underwent an extremely delayed differentiation, while EW neurons produced neurites in abnormal directions, suggesting that eagle may play a critical role in development of the progeny of eagle-positive neuroblasts.