National Institute for Basic Biology

Neurobiology

DIVISION OF BEHAVIORAL NEUROBIOLOGY

Professor

HIGASHIJIMA, Shin-ichi

Assistant Professor:

KIMURA, Yukiko
TANIMOTO, Masashi
Technical Staff:
TAKEUCHI, Yasushi
SOKENDAI Graduate Student: SUGIOKA, Takumi
KAWANO, Kohei
SHIMIZU, Sayaka
Visiting Undergraduate: KATAYAMA, Taisei
Technical Assistant:
WATAKABE, Ikuko
ITO, Hiroko
TAKEUCHI, Yoshiko
SETO, Kumiko

The vertebrate central nervous system (CNS) contains manydifferent types of neurons that form at distinct characteristic positions, and develop specific axonal connections andfunctions. This complexity has made it difficult to perform
detailed functional analysis of neuronal circuits. In particular,
it has been very difficult to reproducibly identify cell types.
However, molecular genetic studies conducted over the past
15 years have strongly suggested that the expression of transcription factors in the developing CNS helps determine themorphological and functional properties of neurons. Thishas opened up the possibility that researchers can use thesetranscription factors as markers to identify cell types in theCNS. Transgenic animals that express fluorescent protein inspecific subsets of neurons are particularly powerful tools instudying the functions of the corresponding neurons in theneuronal circuits.
To fully exploit the methodology described above, we
use larval zebrafish as experimental animals. The biggest
advantage of doing so is that larval zebrafish are almost
completely transparent. This allows us to utilize many opticaltechniques, including morphological/functional imaging andoptogenetics. We can also perform targeted in vivo electrophysiological recording with relative ease using this transparent model. An additional advantage of zebrafish is that their
CNS is much simpler than that of mammals. This enables us
to perform detailed functional analysis of neuronal circuits ata single cell resolution. Our hope is to reveal the operational
principles of vertebrate CNS by using this simple system.
We have been focusing on studying neuronal circuits that
control locomotion. Much of the control of locomotor movements is accomplished by neuronal circuitry located in thespinal cord. Therefore, the focus of our studies has beenspinal neuronal circuits in larval zebrafish.

In addition to zebrafish, we have also started to use medaka
as experimental animals. Medaka have many advantages thatare similar to those of zebrafish Because NIBB is the main
hub of the Medaka National Bioresource Project, we areideally located in regards to experiments using medaka. To
begin with, we explored whether knock-in fish could be efficiently generated using the CRISPR/Cas9 technique.

I. Generation of Transgenic zebrafish

We have been generating transgenic zebrafish that express
fluorescent proteins (GFP or RFP), Gal4, or Cre in specificclasses of neurons in the CNS by using gene promoters/enhancers of genes and are known to be expressed in subsetsof neurons. Most of the genes we used are transcription
factors expressed in subsets of neurons in the developing
CNS. We also used genes whose expressions are tightly
related to neurotransmitter properties of neurons (i.e., vesicular glutamate transporter).

Figure 1. Examples of transgenic fish expressing GFP in specific classesof neurons.

In our early studies, we used a BAC-based transgenic
technique for the generation of transgenic fish. By 2014, we
succeeded in establishing a reliable knock-in method by utilizing the CRISPR-Cas9 system. The method we have developed is highly efficient, so much so that nearly one-third of
the animals we raise become transgenic founders. Thus far,
we have established more than 20 knock-in transgenic fish.
Thus, this method greatly facilitates our functional analysis
on neuronal circuits.

II. Long descending commissural V0v neurons
ensure coordinated swimming movements
along the body axis in larval zebrafish
In the early developmental stage, most animals can onlyexhibit immature forms of behaviors. As development progresses, they acquire the ability to produce more mature orrefined forms of behaviors. Concurrent with such changes,
many new connections are formed in the nervous system,
which suggests that the formation of new connections islinked to the developmental maturation of behaviors. In
animals in which new neurons are generated during development (e.g., fish and amphibians), the incorporation of
new neurons into the pre-existing circuits together with theforming of new connections likely contributes to the maturation of movements.

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National Institute for Basic Biology

Neurobiology

One example of this developmental maturation of behaviors is seen in the swimming behavior of larval zebrafish.
Older larvae (i.e., 4–5 dpf) exhibit more refined forms ofswimming than younger larvae (i.e., 2 dpf), During thisperiod (from 2 dpf to 4–5 dpf), new neurons are generated inboth the brain and spinal cord, with the latter being mainly
responsible for generating swimming outputs. One classof premotor spinal neurons that are added later to the earlyspinal neuronal circuits are MCoD neurons, a subclass of
V0v neurons (V0v neurons represent an excitatory class ofneurons derived from the p0 developmental domain of thespinal cord). MCoD neurons are absent in the early stages
and develop in a later phase of neurogenesis in the spinalcord. MCoD neurons are active during slow swimming. As
for their function, one study showed that they contribute to
the general excitability of spinal swimming circuits, and theirablation decreased the occurrence frequency of spontaneous
swimming. Their function in the more specific aspect of slow
swimming remains elusive, however.
One of the most characteristic features of slow swimmingin older larvae is the stability of the head in the yaw dimension. In slow swimming, the muscle contractions are mostly
confined to the trunk that is caudal to the swim bladder.
Given that the center of the mass is located near the swim
bladder in larval zebrafish (Figure 2A), it is thought thatthe head yaw displacement is produced by the recoil of theyawing moment force generated in the trunk. Considering
this, the stability of the head yaw indicates that the netyawing moment force in the trunk that acts to the center ofthe mass is very small during slow swimming. Swimming
consists of a descending wave of muscle contraction along
the trunk. With this movement, the bending of the bodytransmits force to the surrounding water, and this region of
the body, in turn, receives reaction force. To make the net
yawing moment force minimal, the movements of the rostraland caudal parts of the trunk need to be highly coordinatedwith the diagonal dimension; when the rostral part receives
leftward force, for example, the caudal part needs to receive
rightward force. For this to occur, the swimming body formcannot be C-shaped (unilateral body bend); rather, the shapeneeds to be sinusoidal (S-shaped). MCoD neurons are a goodcandidate for implementing this coordinated movement of
the trunk in the diagonal dimension, because they are active
during slow swimming, and because they are long-distancedescending commissural excitatory neurons that make directconnections onto MNs in the caudal region of the contralateral spinal cord (Figure 2B).
In this study, we tested whether MCoD neurons play a rolein the coordinated movements of the trunk in the diagonal
dimension, thereby ensuring minimal head yaw displacement during slow swimming. Our laser ablation experiments
revealed that MCoD neurons do indeed play the expected role.
In the MCoD-ablated larvae, the normal S-shaped body form
during swimming was often lost with increased appearance of
unilateral C-shaped bends. Concurrently, the head yaw stability was greatly impaired (Figure 3). In addition to swimming,
the present study also sheds light on the evolutionarily conserved role of V0v neurons. In mice, long-distance descendingcommissural V0v neurons have been implicated in interlimb

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coordination during walking in the diagonal dimension18. We
suggest that the long-distance descending commissural V0v
neurons for the coordinated movements of the body/limbs
in the diagonal dimension are the evolutionarily conserved
pathway in spinal locomotor circuits.

Figure 2. (A) One of the swim forms of a zebrafish larva at 4 to 5 dpf.
The cyan circle shows the center of mass, which is located near the swim
bladder. Muscle contractions are presumed to occur in the two locations
marked in red. (B) Projection pattern of an MCoD neuron in the spinalcord. The axon of the MCoD neuron crosses the midline (broken line),
descends on the contralateral spinal cord, and makes mono-synaptic
excitatory connections onto caudally located MNs.

Figure 3. (A) Confocal stacked images of Tg[evx2-hs:GFP] fish before(left) and after (right) laser ablation. Images of two hemi-segments areshown. Magenta arrows show MCoD neurons that were chosen for laserablation. MCoD neurons can be identified by their very ventral location inthe spinal cord. Brown lines show boundaries of muscle segments. Scalebar, 20 μm. (B) Successive images captured at 1000 frames per second oflarval zebrafish swimming. Images of every three frames (3 ms interval)
are shown. Magenta bars depict the head directions in each frame. Top,images of an intact fish. Bottom, images of an MCoD-ablated fish. Scalebar, 500 μm. (C) Graphs of head yaw angle (y axis) versus time (x-axis)
during swimming. Left, intact fish. Right, MCoD-ablated fish.

III. In vivo functional imaging analysis of thevestibular sensory organ

Maintaining head and body orientation relative to theEarth’s vertical gravity axis is vital for survival. Vestibularorgans in the inner ear play a crucial role for this task.
Sensory hair cells in the otolith organs receive linear acceleration, e.g., head tilt, translation and vibration. Direction of
the acceleration is detected by the polarized arrangement of
hair bundles in the hair cells (Figure 4). Each otolith organ
contains hair cells with different but topographically organized hair-bundle polarity that reverses at a line of polarity
reversal (LPR). Although morphofunctional specialization of
the vestibular hair cells has been widely studied, the direction- and modality-selective responses to the head motion
have not been systematically studied in vivo, therefore how
the head motion signals are processed in the vestibular
system remains unclear.

pitch tilts, hair cells medial to the LPR in the rostral utricle
and those lateral to the LPR in the caudal utricle are activated, whereas the rest of the hair cells are activated by thetail-down tilts. Interestingly, hair cells in the medial utricle
exhibited larger responses to the head tilt compared to thelateral hair cells. In contrast to the responses to the head tilt,
the vibratory stimulus in the pitch or roll axis activated thehair cells only in the rostral and lateral utricle near the LPR.

Figure 6. Hair cell responses to head tilt or vibration in the utricle

Figure 4. Hair bundle polarity (arrowheads) in the utricle.

To visualize hair cell responses to the head motion, we built
a microscope in which an objective lens can tilt with a small
sample 360 degree by a motorized stage during Ca2+ imaging(Figure 5). A spinning-disk confocal scanner and an image
splitting optics formed green and red fluorescent images ona digital camera. This ratiometric imaging setup reduced
artifacts derived from non-uniformity of the excitation lightand optical distortion during the optics motion. With thistiltable objective microscope, we imaged neural activity inall the utricular hair cell at the single-cell level during pitch
or roll tilt/vibration in 5-day-old transgenic zebrafish larvae
expressing Ca2+ indicator, jGCaMP7f, and red fluorescentprotein, tdTomato, in the hair cells.

Together, the tiltable objective microscope visualized, forthe first time to our knowledge, the topographically organized response selectivity for the stimulus direction andmodality in the vestibular periphery. The imaging strategy
we have established here is applicable to the central nervous
system, and thus it will provide deeper understanding of thevestibular processing in the brain.

Publication List:〔Original Papers〕

U.L., Kimura, Y., Kawashima, T., Ahrens, M.B., Higashijima,• Bohm,S., Engert, F., and Cohen, A.E. (2022). Voltage imaging identifies spinal••circuits that modulate locomotor adaptation in zebrafish. Neuron 110,1211+. DOI: 10.1016/j.neuron.2022.01.001
Kawano, K., Kato, K., Sugioka, T., Kimura, Y., Tanimoto, M., andHigashijima, S. (2022). Long descending commissural V0v neurons
ensure coordinated swimming movements along the body axis in larvalzebrafish. Sci. Rep. 12, 4348. DOI: 10.1038/s41598-022-08283-0
Suzuki, D.G., Wada, H., and Higashijima, S. (2021). Generation ofknock-in lampreys by CRISPR-Cas9-mediated genome engineering.
Sci. Rep. 11, 19836. DOI: 10.1038/s41598-021-99338-1

Figure 5. Tiltable objective microscope.

Consistent with the morphological hair-bundle polarity, haircells medial to the LPR are activated by the lateral-down rolltilts, whereas those lateral to the LPR are activated by themedial-down tilts (Figure 6). In response to the nose-down

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