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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 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 the vertebrate olfactory system, odor information is represented as a topographic map in the olfactory bulb (OB). However, it remains unknown how this odor map is transferred from the OB to higher olfactory centers. Using genetic labeling techniques in zebrafish, we found that the OB output neurons, mitral cells (MCs), are heterogeneous with respect to transgene expression profiles and spatial distributions. Tracing MC axons at single-cell resolution revealed that (1) individual MCs send axons to multiple target regions in the forebrain; (2) MCs innervating the same glomerulus do not necessarily display the same axon trajectory; (3) MCs innervating distinct glomerular clusters tend to project axons to different, but partly overlapping, target regions; (4) MCs innervating the medial glomerular cluster directly and asymmetrically send axons to the right habenula. We propose that the topographic odor map in the OB is not maintained intact, but reorganized in higher olfactory centers. Moreover, our finding of asymmetric bulbo-habenular projection renders the olfactory system an attractive model for the studies of brain asymmetry and lateralized behaviors.

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 byalx, a zebrafish homolog of mammalianChx10. We performed morphological and physiological studies using transgenic zebrafish expressing fluorescent reporter constructs in cells that had at any time point expressedalx(alxneurons). Our data reveal that zebrafishalxneurons 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 betweenalxneurons 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 analxneuron of the weak movement class and a motoneuron. Collectively, our data demonstrate thatalxmarks ipsilateral descending neurons that are involved in the regulation of motoneuron activity during forms of locomotion, such as escape and swimming.

In the adult olfactory bulb (OB), particular chemical classes of odorants preferentially activate glomeruli within loosely defined regions, resulting in a coarse and fractured “chemotopic” map. In zebrafish, amino acids and bile acids predominantly stimulate glomeruli in the lateral and medial OB, respectively. We studied the development of these spatial response maps in zebrafish. At 3 d postfertilization (dpf), the OB contained protoglomerular structures that became refined and more numerous during subsequent days. In a transgenic zebrafish line expressing the Ca(2+) indicator protein inverse pericam, mainly in mitral cells, odor responses in the OB were first detected at 2.5-3 dpf. Already at this stage, amino acids and bile acids evoked activity predominantly in the lateral and medial OB, respectively. Two-photon Ca(2+) imaging using a synthetic indicator was used to reconstruct activity patterns at higher resolution in three dimensions. Responses to amino acids and bile acids were detected predominantly in the lateral and medial OB, respectively, with little overlap. Between 2.5 and 6 dpf, the number of odor-responsive units increased, but the overall spatial organization of activity persisted. Hence, a coarse spatial organization of functional activity maps is established very early during OB development when glomeruli are not yet differentiated. This spatial organization is maintained during development and growth of neuronal circuits and may have important functions for odor processing in larvae, for the differentiation of glomeruli, and for the refinement of activity maps at later developmental stages.

Studies in chicks and mice have suggested that transcription factors mark functional subtypes of interneurons in the developing spinal cord. We used genetic, morphological, and physiological studies to test this proposed association in zebrafish. We found that Engrailed-1 expression uniquely marks a class of ascending interneurons, called circumferential ascending (CiA) interneurons, with ipsilateral axonal projections in both motor and sensory regions of spinal cord. These cells express the glycine transporter 2 gene and are the only known ipsilateral interneurons positive for this marker of inhibitory transmission. Patch recordings show that the CiA neurons are rhythmically active during swimming. Pairwise recordings from the CiA interneurons and postsynaptic cells reveal that the Engrailed-1 neurons produce monosynaptic, strychnine-sensitive inhibition of dorsal sensory interneurons and also inhibit more ventral neurons, including motoneurons and descending interneurons. We conclude that Engrailed-1 expression marks a class of inhibitory interneuron that seems to provide all of the ipsilateral glycinergic inhibition in the spinal cord of embryonic and larval fish. Individual Engrailed-1-positive cells are multifunctional, playing roles in both sensory gating and motor pattern generation. This primitive cell type may have given rise to several, more specialized glycinergic inhibitory interneurons in birds and mammals. Our data support the view that the subdivision of spinal cord into different regions by transcription factors defines a primitive functional organization of spinal interneurons that formed a developmental and evolutionary foundation on which more complex systems were built.

Upon touch, twitch once zebrafish respond with one or two swimming strokes instead of typical full-blown escapes. This use-dependent fatigue is shown to be a consequence of a mutation in the tetratricopeptide domain of muscle rapsyn, inhibiting formation of subsynaptic acetylcholine receptor clusters. Physiological analysis indicates that reduced synaptic strength, attributable to loss of receptors, is augmented by a potent postsynaptic depression not seen at normal neuromuscular junctions. The synergism between these two physiological processes is causal to the use-dependent muscle fatigue. These findings offer insights into the physiological basis of human myasthenic syndrome and reveal the first demonstration of a role for rapsyn in regulating synaptic function.

Physiological analysis of two lines of paralytic mutant zebrafish, relaxed and sofa potato , reveals defects in distinct types of receptors in skeletal muscle. In sofa potato the paralysis results from failed synaptic transmission because of the absence of acetylcholine receptors, whereas relaxed mutants lack dihydropyridine receptor-mediated release of internal calcium in response to the muscle action potential. Synaptic structure and function appear normal in relaxed , showing that muscle paralysis per se does not impede proper synapse development. However, sofa potato mutants show incomplete development of the postsynaptic complex. Specifically, in the absence of ACh receptors, clusters of the receptor-aggregating protein rapsyn form in the extrasynaptic membrane but generally fail to localize to the subsynaptic region. Our results indicate that, although rapsyn molecules are capable of self-aggregation, interaction with ACh receptors is required for proper subsynaptic localization.

We generated germ line-transmitting transgenic zebrafish that express green fluorescent protein (GFP) in the cranial motor neurons. This was accomplished by fusing GFP sequences toIslet-1promoter/enhancer sequences that were sufficient for neural-specific expression. The expression of GFP by the motor neurons in the transgenic fish enabled visualization of the cell bodies, main axons, and the peripheral branches within the muscles. GFP-labeled motor neurons could be followed at high resolution for at least up to day four, when most larval neural circuits become functional, and larvae begin to swim and capture prey. Using this line, we analyzed axonal outgrowth by the cranial motor neurons. Furthermore, by selective application of DiI to specific GFP-positive nerve branches, we showed that the two clusters of trigeminal motor neurons in rhombomeres 2 and 3 innervate different peripheral targets. This finding suggests that the trigeminal motor neurons in the two clusters adopt distinct fates. In future experiments, this transgenic line of zebrafish will allow for a genetic analysis of cranial motor neuron development.

Understanding the neuronal networks in the mammal spinal cord is hampered by the diversity of neurons and their connections. The simpler networks in developing lower vertebrates may offer insights into basic organization. To investigate the function of spinal inhibitory interneurons inXenopustadpoles, paired whole-cell recordings were used. We show directly that one class of interneuron, with distinctive anatomy, produces glycinergic, negative feedback inhibition that can limit firing in motoneurons and interneurons of the central pattern generator during swimming. These same neurons also produce inhibitory gating of sensory pathways during swimming. This discovery raises the possibility that some classes of interneuron, with distinct functions later in development, may differentiate from an earlier class in which these functions are shared. Preliminary evidence suggests that these inhibitory interneurons express the transcription factor engrailed, supporting a probable homology with interneurons in developing zebrafish that also express engrailed and have very similar anatomy and functions.