Mosaic gene expression analysis of semaphorin-plexin interactions in Caenorhabditis elegans using the IR-LEGO single-cell gene induction system.

Genetic mosaic analysis is a powerful means of addressing the sites of gene action in multicellular organisms. In conventional genetic analysis, the generation of desired mosaic patterns is difficult to control due to the randomness of generating the genetic mosaic which often renders the analysis laborious and time consuming. The infrared laser-evoked gene operator (IR-LEGO) microscope system facilitates genetic mosaic analysis by enabling gene induction in targeted single cells in a living organism. However, the level of gene induction is not controllable due to the usage of a heat-shock promoter. Here, we applied IR-LEGO to examine the cell-cell interactions mediated by semaphoring-plexin signaling in Caenorhabditis elegans by inducing wild-type semaphorin/plexin in single cells within the population of mutant cells lacking the relevant proteins. We found that the cell contact-dependent termination of the extension of vulval precursor cells is elicited by the forward signaling mediated by the semaphorin receptor, PLX-1, but not by the reverse signaling via the transmembrane semaphorin, SMP-1. By utilizing Cre/loxP recombination coupled with the IR-LEGO system to induce SMP-1 at a physiological level, we found that SMP-1 interacts with PLX-1 only in trans upon contact between vulval precursor cells. In contrast, when overexpressed, SMP-1 exhibits the ability to cis-interact with PLX-1 on a single cell. These results indicate that mosaic analysis with IR-LEGO, especially when combined with an in vivo recombination system, efficiently complements conventional methods.

Neuronal Circuits That Control Rhythmic Pectoral Fin Movements in Zebrafish.

The most basic form of locomotion in limbed vertebrates consists of alternating activities of the flexor and extensor muscles within each limb coupled with left/right limb alternation. Although larval zebrafish are not limbed, their pectoral fin movements exhibit the following fundamental aspects of this basic movement: abductor/adductor alternation (corresponding to flexor/extensor alternation) and left/right fin alternation. Because of the simplicity of their movements and the compact neural organization of their spinal cords, zebrafish can serve as a good model to identify the neuronal networks of the central pattern generator (CPG) that controls rhythmic appendage movements. Here, we set out to investigate neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish, using transgenic fish that specifically express GFP in abductor or adductor motor neurons (MNs) and candidate CPG neurons. First, we showed that spiking activities of abductor and adductor MNs were essentially alternating. Second, both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively, indicating that the MN spiking activities are controlled in a push-pull manner. Further, we obtained the following evidence that dmrt3a-expressing commissural inhibitory neurons are involved in regulating the activities of abductor MNs: (1) strong inhibitory synaptic connections were found from dmrt3a neurons to abductor MNs; and (2) ablation of dmrt3a neurons shifted the spike timing of abductor MNs. Thus, in this simple system of abductor/adductor alternation, the last-order inhibitory inputs originating from the contralaterally located neurons play an important role in controlling the firing timings of MNs.SIGNIFICANCE STATEMENT Pectoral fin movements in larval zebrafish exhibit fundamental aspects of basic rhythmic appendage movement: alternation of the abductor and adductor (corresponding to flexor-extensor alternation) coupled with left-right alternation. We set out to investigate the neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish. We showed that both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively. This indicates that MN activities are controlled in a push-pull manner. We further obtained evidence that dmrt3a-expressing commissural inhibitory neurons exert an inhibitory effect on abductor MNs. The current study marks the first step toward the identification of central pattern generator organization for rhythmic fin movements.

Phosphorylation of Gephyrin in Zebrafish Mauthner Cells Governs Glycine Receptor Clustering and Behavioral Desensitization to Sound.

The process by which future behavioral responses are shaped by past experiences is one of the central questions in neuroscience. To gain insight into this process at the molecular and cellular levels, we have applied zebrafish larvae to explore behavioral desensitization to sound. A sudden loud noise often evokes a defensive response known as the acoustic startle response (ASR), which is triggered by firing Mauthner cells in teleosts and amphibians. The probability of evoking ASR by suprathreshold sound is reduced after exposure to repetitive auditory stimuli insufficient in amplitude to evoke the ASR (subthreshold). Although it has been suggested that the potentiation of inhibitory glycinergic inputs into Mauthner cell is involved in this desensitization of the ASR, the molecular basis for the potentiation of glycinergic transmission has been unclear. Through the in vivo monitoring of fluorescently-tagged glycine receptors (GlyRs), we here showed that behavioral desensitization to sound in zebrafish is governed by GlyR clustering in Mauthner cells. We further revealed that CaMKII-dependent phosphorylation of the scaffolding protein gephyrin at serine 325 promoted the synaptic accumulation of GlyR on Mauthner neurons through the enhancement of the gephyrin-GlyR binding, which was indispensable for and could induce desensitization of the ASR. Our study demonstrates an essential molecular and cellular basis of sound-induced receptor dynamics and thus of behavioral desensitization to sound.SIGNIFICANCE STATEMENT Behavioral desensitization in the acoustic startle response of fish is known to involve the potentiation of inhibitory glycinergic input to the Mauthner cell, which is a command neuron for the acoustic startle response. However, the molecular and cellular basis for this potentiation has been unknown. Here we show that an increase in glycine receptor (GlyR) clustering at synaptic sites on zebrafish Mauthner cells is indispensable for and could induce desensitization. Furthermore, we demonstrate that CaMKII-mediated phosphorylation of the scaffolding protein gephyrin promotes GlyR clustering by increasing the binding between the ß-loop of GlyRs and gephyrin. Thus, the phosphorylation of gephyrin is a key event which accounts for the potentiation of inhibitory glycinergic inputs observed during sound-evoked behavioral desensitization.

Behavioral Role of the Reciprocal Inhibition between a Pair of Mauthner Cells during Fast Escapes in Zebrafish.

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 STATEMENT Reciprocal 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.

Specialized movement and laterality of fin-biting behaviour in Genyochoromis mento in Lake Malawi.

Several vertebrates, including fish, exhibit behavioural laterality and associated morphological asymmetry. Laterality may increase individual fitness as well as foraging strength, accuracy and speed. However, little is known about which behaviours are affected by laterality or what fish species exhibit obvious laterality. Previous research on the predatory behaviour of the scale-eating Lake Tanganyika cichlid Perissodus microlepis indicates behavioural laterality that reflects asymmetric jaw morphology. The Lake Malawi cichlid Genyochromis mento feeds on the fins of other fish, a behaviour that G. mento developed independently from the Tanganyikan Perissodini scale eaters. We investigated stomach contents and behavioural laterality of predation in aquarium to clarify the functional roles and evolution of laterality in cichlids. We also compared the behavioural laterality and mouth asymmetry of G. mento and P. microlepis The diet of G. mento mostly includes fin fragments, but also scales of several fish species. Most individual G. mento specimens showed significant attack bias favouring the skew mouth direction. However, there was no difference in success rate between attacks from the preferred side and those from the non-preferred side, and no lateralized kinetic elements in predation behaviour. Genyochromismento showed weaker laterality than P. microlepis, partly because of their different feeding habits, the phylogenetic constraints from their shorter evolutionary history and their origin from ancestor Haplochromini omnivorous/herbivorous species. Taken together, this study provides new insights into the functional roles of behavioural laterality: predatory fish aiming for prey that show escape behaviours frequently exhibit lateralized behaviour in predation.

Astroglial Ca2+ signaling is generated by the coordination of IP3R and store-operated Ca2+ channels.

Astrocytes play key roles in the central nervous system and regulate local blood flow and synaptic transmission via intracellular calcium (Ca2+) signaling. Astrocytic Ca2+ signals are generated by multiple pathways: Ca2+ release from the endoplasmic reticulum (ER) via the inositol 1, 4, 5-trisphosphate receptor (IP3R) and Ca2+ influx through various Ca2+ channels on the plasma membrane. However, the Ca2+ channels involved in astrocytic Ca2+ homeostasis or signaling have not been fully characterized. Here, we demonstrate that spontaneous astrocytic Ca2+ transients in cultured hippocampal astrocytes were induced by cooperation between the Ca2+ release from the ER and the Ca2+ influx through store-operated calcium channels (SOCCs) on the plasma membrane. Ca2+ imaging with plasma membrane targeted GCaMP6f revealed that spontaneous astroglial Ca2+ transients were impaired by pharmacological blockade of not only Ca2+ release through IP3Rs, but also Ca2+ influx through SOCCs. Loss of SOCC activity resulted in the depletion of ER Ca2+, suggesting that SOCCs are activated without store depletion in hippocampal astrocytes. Our findings indicate that sustained SOCC activity, together with that of the sarco-endoplasmic reticulum Ca2+-ATPase, contribute to the maintenance of astrocytic Ca2+ store levels, ultimately enabling astrocytic Ca2+ signaling.

Dissection of local Ca(2+) signals inside cytosol by ER-targeted Ca(2+) indicator.

Calcium (Ca(2+)) is a versatile intracellular second messenger that operates in various signaling pathways leading to multiple biological outputs. The diversity of spatiotemporal patterns of Ca(2+) signals, generated by the coordination of Ca(2+) influx from the extracellular space and Ca(2+) release from the intracellular Ca(2+) store the endoplasmic reticulum (ER), is considered to underlie the diversity of biological outputs caused by a single signaling molecule. However, such Ca(2+) signaling diversity has not been well described because of technical limitations. Here, we describe a new method to report Ca(2+) signals at subcellular resolution. We report that OER-GCaMP6f, a genetically encoded Ca(2+) indicator (GECI) targeted to the outer ER membrane, can monitor Ca(2+) release from the ER at higher spatiotemporal resolution than conventional GCaMP6f. OER-GCaMP6f was used for in vivo Ca(2+) imaging of C. elegans. We also found that the spontaneous Ca(2+) elevation in cultured astrocytes reported by OER-GCaMP6f showed a distinct spatiotemporal pattern from that monitored by plasma membrane-targeted GCaMP6f (Lck-GCaMP6f); less frequent Ca(2+) signal was detected by OER-GCaMP6f, in spite of the fact that Ca(2+) release from the ER plays important roles in astrocytes. These findings suggest that targeting of GECIs to the ER outer membrane enables sensitive detection of Ca(2+) release from the ER at subcellular resolution, avoiding the diffusion of GECI and Ca(2+). Our results indicate that Ca(2+) imaging with OER-GCaMP6f in combination with Lck-GCaMP6f can contribute to describing the diversity of Ca(2+) signals, by enabling dissection of Ca(2+) signals at subcellular resolution.

Functional motifs composed of morphologically homologous neurons repeated in the hindbrain segments.

Segmental organization along the neuraxis is a prominent feature of the CNS in vertebrates. In a wide range of fishes, hindbrain segments contain orderly arranged reticulospinal neurons (RSNs). Individual RSNs in goldfish and zebrafish hindbrain are morphologically identified. RSNs sharing similar morphological features are called segmental homologs and repeated in adjacent segments. However, little is known about functional relationships among segmental homologs. Here we investigated the electrophysiological connectivity between the Mauthner cell (M-cell), a pair of giant RSNs in segment 4 (r4) that are known to trigger fast escape behavior, and different series of homologous RSNs in r4-r6. Paired intracellular recordings in adult goldfish revealed unidirectional connections from the M-cell to RSNs. The connectivity was similar in morphological homologs. A single M-cell spike produced IPSPs in dorsally located RSNs (MiD cells) on the ipsilateral side and excitatory postsynaptic depolarization on the contralateral side, except for MiD2cm cells. The inhibitory or excitatory potentials effectively suppressed or enhanced target RSNs spiking, respectively. In contrast to the lateralized effects on MiD cells, single M-cell spiking elicited equally strong depolarizations on bilateral RSNs located ventrally (MiV cells), and the depolarization was high enough for MiV cells to burst. Therefore, the morphological homology of repeated RSNs in r4-r6 and their functional M-cell connectivity were closely correlated, suggesting that each functional connection works as a functional motif during the M-cell-initiated escape.

Coexpression of auxiliary Kvß2 subunits with Kv1.1 channels is required for developmental acquisition of unique firing properties of zebrafish Mauthner cells.

Each neuron possesses a unique firing property, which is largely attributed to heterogeneity in the composition of voltage-gated ion channel complexes. Zebrafish Mauthner (M) cells, which are bilaterally paired giant reticulospinal neurons (RSNs) in the hindbrain and induce rapid escape behavior, generate only a single spike at the onset of depolarization. This single spiking is in contrast with the repetitive firing of the M cell's morphologically homologous RSNs, MiD2cm and MiD3cm, which are also involved in escapes. However, how the unique firing property of M cells is established and the underlying molecular mechanisms remain unclear. In the present study, we first demonstrated that the single-spiking property of M cells was acquired at 4 days postfertilization (dpf), accompanied by an increase in dendrotoxin I (DTX)-sensitive low-threshold K(+) currents, prior to which the M cell repetitively fires as its homologs. Second, in situ hybridization showed that among DTX-sensitive Kv1 channel α-subunits, zKv1.1a was unexpectedly expressed even in the homologs and the bursting M cells at 2 dpf. In contrast, zKvß2b, an auxiliary ß-subunit of Kv1 channels, was expressed only in the single-spiking M cells. Third, zKv1.1a expressed in Xenopus oocytes functioned as a low-threshold K(+) channel, and its currents were enhanced by coexpression of zKvß2b subunits. Finally, knockdown of zKvß2b expression in zebrafish larvae resulted in repetitive firing of M cells at 4 dpf. Taken together, these results suggest that associative expression of Kvß2 subunits with Kv1.1 channels is crucial for developmental acquisition of the unique firing properties of the M cells among homologous neurons.

Glycinergic transmission and postsynaptic activation of CaMKII are required for glycine receptor clustering in vivo.

Synaptic transmission-dependent regulation of neurotransmitter receptor accumulation at postsynaptic sites underlies the formation, maintenance and maturation of synaptic function. Previous in vitro studies showed that glycine receptor (GlyR) clustering requires synaptic inputs. However, in vivo GlyR regulation by synaptic transmission is not fully understood. Here, we established a model system using developing zebrafish, in which GlyRs are expressed in Mauthner cells (M-cells), a pair of giant, reticulospinal, hindbrain neurons, thereby enabling analysis of GlyR clusters over time in identifiable cells. Bath application of a glycinergic blocker, strychnine, to developing zebrafish prevented postsynaptic GlyR cluster formation in the M-cells. After strychnine removal, the GlyR clusters appeared in the M-cells. At a later stage, glycinergic transmission blockade impaired maintenance of GlyR clusters. We also found that pharmacological blockade of either L-type Ca(2+) channels or calcium-/calmodulin-dependent protein kinase II (CaMKII) disturbed GlyR clustering. In addition, the M-cell-specific CaMKII inactivation using the Gal4-UAS system significantly impaired GlyR clustering in the M-cells. Thus, the formation and maintenance of GlyR clusters in the M-cells in the developing animals are regulated in a synaptic transmission-dependent manner, and CaMKII activation at the postsynapse is essential for GlyR clustering. This is the first demonstration of synaptic transmission-dependent modulation of synaptic GlyRs in vivo.

Effective sensory modality activating an escape triggering neuron switches during early development in zebrafish.

Developing nervous systems grow to integrate sensory signals from different modalities and to respond through various behaviors. Here, we examined the development of escape behavior in zebrafish [45-170 h postfertilization (hpf)] to study how developing sensory inputs are integrated into sensorimotor circuits. Mature fish exhibit fast escape upon both auditory/vestibular (AV) and head-tactile stimuli. Newly hatched larvae, however, do not respond to AV stimuli before 75 hpf. Because AV-induced fast escape in mature fish is triggered by a pair of hindbrain neurons known as Mauthner (M) cells, we studied functional development of the M-cell circuit accounting for late acquisition of AV-induced escape. In fast escape elicited by head-directed water jet, minimum onset latency decreased throughout development (5 ms at 45-59 hpf, 3 ms after 75 hpf). After 75 hpf, lesioning the otic vesicle (OV) to eliminate AV input resulted in loss of short-latency (<5 ms) fast escape, whereas ablation of the sensory trigeminal ganglion (gV) to block head-tactile input did not. Before 75 hpf, however, fast escape persisted after OV lesion but disappeared after gV ablation. Laser ablation of the M-cell and Ca²âº imaging of the M-cell during escape demonstrated that M-cell firing is required to initiate short-latency fast escapes at every developmental stage and further suggest that head-tactile input activates the M-cell before 75 hpf, but that after this point AV input activates the M-cell instead. Thus, a switch in the effective sensory input to the M-cells mediates the acquisition of a novel modality for initiating fast escape.

Biogenesis of GPI-anchored proteins is essential for surface expression of sodium channels in zebrafish Rohon-Beard neurons to respond to mechanosensory stimulation.

In zebrafish, Rohon-Beard (RB) neurons are primary sensory neurons present during the embryonic and early larval stages. At 2 days post-fertilization (dpf), wild-type zebrafish embryos respond to mechanosensory stimulation and swim away from the stimuli, whereas mi310 mutants are insensitive to touch. During approximately 2-4 dpf, wild-type RB neurons undergo programmed cell death, which is caused by sodium current-mediated electrical activity, whereas mutant RB cells survive past 4 dpf, suggesting a defect of sodium currents in the mutants. Indeed, electrophysiological recordings demonstrated the generation of action potentials in wild-type RB neurons, whereas mutant RB cells failed to fire owing to the reduction of voltage-gated sodium currents. Labeling of dissociated RB neurons with an antibody against voltage-gated sodium channels revealed that sodium channels are expressed at the cell surface in wild-type, but not mutant, RB neurons. Finally, in mi310 mutants, we identified a mis-sense mutation in pigu, a subunit of GPI (glycosylphosphatidylinositol) transamidase, which is essential for membrane anchoring of GPI-anchored proteins. Taken together, biogenesis of GPI-anchored proteins is necessary for cell surface expression of sodium channels and thus for firings of RB neurons, which enable zebrafish embryos to respond to mechanosensory stimulation.

Origin of inner ear hair cells: morphological and functional differentiation from ciliary cells into hair cells in zebrafish inner ear.

Auditory and vestibular functions in vertebrates depend on the transduction of sound vibration or head acceleration into electrical responses in inner ear hair cells. Mechanoelectrical transduction occurs at the tip of stereocilia, which are polarized to form an orientational arrangement that determines directional sensitivity. It remains to be clarified when and how premature hair cells acquire their specialized structure and function in living animals. The developmental origin of inner ear hair cells has been studied in vivo in zebrafish embryos. Tether cells, a small number of ciliated cells associated with an "ear stone" (or otolith) in the embryonic zebrafish inner ear, are believed to be precocious hair cells. However, whether or not tether cells acquire hair bundles and mechanosensitivity remains unknown. In the present study, we investigated the morphological and functional development of tether cells. Immunohistochemical examination revealed that stereocilia appeared on the tether cell apex in a polarized arrangement at 22 h postfertilization (hpf). Labeling with FM1-43, a marker of functional mechanotransduction channels, and the in vivo electrophysiological recording of mechanotransducer responses in the developing inner ear demonstrated that tether cells acquired direction-selective mechanosensitivity at 23 hpf. These results revealed that tether cells begin to function as hair cells within an hour after the appearance of a polarized array of stereociliary bundles. Thus, the ciliary cells morphologically and functionally differentiate into the first sensory hair cells in the inner ear of the zebrafish.

Experience-dependent learning of behavioral laterality in the scale-eating cichlid Perissodus microlepis occurs during the early developmental stage.

Behavioral laterality-typically represented by human handedness-is widely observed among animals. However, how laterality is acquired during development remains largely unknown. Here, we examined the effect of behavioral experience on the acquisition of lateralized predation at different developmental stages of the scale-eating cichlid fish Perissodus microlepis. Naïve juvenile fish without previous scale-eating experience showed motivated attacks on prey goldfish and an innate attack side preference. Following short-term predation experience, naïve juveniles learned a pronounced lateralized attack using their slightly skewed mouth morphology, and improved the velocity and amplitude of body flexion to succeed in foraging scales during dominant-side attack. Naïve young fish, however, did not improve the dynamics of flexion movement, but progressively developed attack side preference and speed to approach the prey through predation experience. Thus, the cichlid learns different aspects of predation behavior at different developmental stages. In contrast, naïve adults lost the inherent laterality, and they neither developed the lateralized motions nor increased their success rate of predation, indicating that they missed appropriate learning opportunities for scale-eating skills. Therefore, we conclude that behavioral laterality of the cichlid fish requires the integration of genetic basis and behavioral experiences during early developmental stages, immediately after they start scale-eating.

Auditory input to CNS is acquired coincidentally with development of inner ear after formation of functional afferent pathway in zebrafish.

Auditory perception in vertebrates depends on transduction of sound into neural signals in the inner ear hair cells (HCs) and on transmission of these signals to the brain through auditory (VIIIth) nerve afferents. To investigate the developmental acquisition of auditory inputs by the CNS, we have electrophysiologically and morphologically examined the process of acquisition of auditory responsiveness by zebrafish macular HCs and the Mauthner cells (M-cells) in vivo. The M-cells are a paired large reticulospinal neurons in the hindbrain; they receive direct inputs from the VIIIth nerve afferents and initiate an acoustic startle response. Whole-cell recordings from the M-cells showed that sound-evoked postsynaptic currents were first observed around 40 h postfertilization (hpf); during subsequent development, onset latency decreased and amplitude increased. The appearance and development of microphonic potentials in the inner ear coincided with those of the acoustic responses of the M-cell, whereas the functional auditory circuits from the macular HCs to the M-cell were already formed at 27 hpf. These results suggest that the functional maturation of inner ear after formation of the auditory pathway is a critical process in the acquisition of auditory inputs by CNS neurons.

Functional role of a specialized class of spinal commissural inhibitory neurons during fast escapes in zebrafish.

In teleost fish, the Mauthner (M) cell, a large reticulospinal neuron in the brainstem, triggers escape behavior. Spinal commissural inhibitory interneurons that are electrotonically excited by the M-axon have been identified, but the behavioral roles of these neurons have not yet been addressed. Here, we studied these neurons, named CoLo (commissural local), in larval zebrafish using an enhancer-trap line in which the entire population of CoLos was visualized by green fluorescent protein. CoLos were present at one cell per hemi-segment. Electrophysiological recordings showed that an M-spike evoked a spike in CoLos via electrotonic transmission and that CoLos made monosynaptic inhibitory connections onto contralateral primary motoneurons, consistent with the results in adult goldfish. We further showed that CoLos were active only during escapes. We examined the behavioral roles of CoLos by investigating escape behaviors in CoLo-ablated larvae. The results showed that the escape behaviors evoked by sound/vibration stimuli were often impaired with a reduced initial bend of the body, indicating that CoLos play important roles in initiating escapes. We obtained several lines of evidence that strongly suggested that the impaired escapes occurred during bilateral activation of the M-cells: in normal larvae, CoLo-mediated inhibitory circuits enable animals to perform escapes even in these occasions by silencing the output of the slightly delayed firing of the second M-cell. This study illustrates (1) a clear example of the behavioral role of a specialized class of interneurons and (2) the capacity of the spinal circuits to filter descending commands and thereby produce the appropriate behavior.

Initiation of Mauthner- or non-Mauthner-mediated fast escape evoked by different modes of sensory input.

Brainstem reticulospinal neurons (RSNs) serve as the major descending system in vertebrate sensorimotor integration. One of the paired RSNs in zebrafish, the Mauthner (M) cell, is thought to initiate fast escape from sudden noxious stimuli. Two other paired RSNs, morphologically homologous to the M-cell, are also suggested to play key roles in controlling fast escape. However, the relationship among activities of the M-cell and its homologs during fast escape and the sensory inputs that elicit escape via their activation are unclear. We have monitored hindbrain RSN activity simultaneously with tail flip movement during fast escape in zebrafish. Confocal calcium imaging of RSNs was performed on larvae rostrally embedded in agar but with their tails allowed to move freely. Application of a pulsed waterjet to the otic vesicle (OV) to activate acousticovestibular input elicited contralateral fast tail flips with short latency and an apparent Ca(2+) increase, reflecting a single action potential, in the ipsilateral M-cell (M-escape). Application of waterjet to head skin for tactile stimulation elicited fast escapes, but onset was delayed and the M-cell did not fire (non-M-escape). After eliminating either the M-cell or OV, only non-M-escape was initiated. Simultaneous high-speed confocal imaging of the M-cell and one of its homologs, MiD3cm, revealed complementary activation during fast escape: MiD3cm activity was low during M-escape but high during non-M-escape. These results suggest that M-cell firing is necessary for fast escape with short latency elicited by acousticovestibular input and that MiD3cm is more involved in non-M-escape driven by head-tactile input.

[Neural Mechanisms Underlying Bilaterally Asymmetric Behavior].

Lateralized behaviors are key movements of symmetrically organized animals. These behaviors are controlled by asymmetric activity of the bilateral brain. The neural mechanisms underlying these activities were recently revealed.

[Functional organization of escape circuits built in teleost hindbrain segments].

Hindbrain reticulospinal (RS) neurons in goldfish and zebrafish consist of seven clusters spaced periodically along the rostrocaudal axis. Morphologically similar RS neurons, homologs, are arranged in adjacent segments. Electrophysiological examination in goldfish suggests that paired, large M-cells, in the fourth segment (r4) and their homologs, MiD 2 cm in r5 and MiD 3 cm in r6, receive auditory inputs similarly but show different firing pattern in response to depolarization. In addition, there is inhibitory connection from M-cell to its homologs but not in the reverse direction. Calcium imaging of these neurons during escape behavior in larval zebrafish reveals that the M-cell and its homologs fire in a complementary fashion. Thus, the segmentally homologous RS neurons may work as a functional unit to initiate and control escape behavior.

Lateralized expression of left-right axis formation genes is shared by adult brains of lefty and righty scale-eating cichlids.

Variation in the laterality often exists within species and can be maintained by frequency-dependent selection. Although the molecular developmental mechanisms underlying the left-right axis formation have been investigated, the genomic mechanisms underlying variation in laterality remain largely unknown. The scale-eating cichlid Perissodus microlepis in Lake Tanganyika exhibit lateralized predation; lefty individuals with the mouth opening toward the right preferentially attack on the prey's left trunk, while righty individuals with the opposite opening attacks on the right trunk. Here, we performed RNA-sequencing and subsequent confirmation with quantitative-PCR in the telencephalon, optic tectum, and hindbrain of the cichlid and identified five genes (pkd1b, ntn1b, ansn, pde6g, and rbp4l1) that were differentially expressed between the hemispheres regardless of the laterality. Surprisingly, pkd1b and ntn1b are involved in nodal and netrin signalling, respectively, which are important for left-right asymmetry formation during early embryogenesis. This result indicates that nodal- and netrin-related signals may also play important roles in the maintenance of asymmetry in adult brain. By contrast, no genes showed reversal of lateral differences between lefty and righty individuals in any brain regions examined, suggesting that laterality in the scale-eating cichlid does not simply result from inversion of the left-right asymmetry of gene expression.