Taizo Kawano

Identification of Genes Involved in Synaptogenesis Using a Fluorescent Active Zone Marker in Caenorhabditis elegans

Active zones are presynaptic regions where synaptic vesicles fuse with plasma membrane to release neurotransmitters. Active zones are highly organized structurally and are functionally conserved among different species. Synapse defective-2 (SYD-2) family proteins regulate active zone morphology in Caenorhabditis elegans and Drosophila. Here, we demonstrate by immunoelectron microscopy that at C. elegans synapses, SYD-2 localizes strictly at active zones and can be used as an active zone marker when fused to green fluorescent protein (GFP). By driving expression of SYD-2::GFP fusion protein in GABAergic neurons, we are able to visualize discrete fluorescent puncta corresponding to active zones in living C. elegans. During development, the number of GABAergic synapses made by specific motoneurons increases only slightly from larvae to adult stages. In contrast, the number of SYD-2::GFP puncta doubles, suggesting that individual synapses accommodate the increasing size of their synaptic targets mainly by incorporating more active zone materials. Furthermore, we used this marker to perform a genetic screen to identify genes involved in the development of active zones. We recovered 16 mutants with altered SYD-2::GFP expression, including alleles of five genes that have been implicated previously in synapse formation or nervous-system development. Mapping of 11 additional mutants suggests that they may represent novel genes involved in active zone formation.

The NCA sodium leak channel is required for persistent motor circuit activity that sustains locomotion

Persistent neural activity, a sustained circuit output that outlasts the stimuli, underlies short-term or working memory, as well as various mental representations. Molecular mechanisms that underlie persistent activity are not well understood. Combining in situ whole-cell patch clamping and quantitative locomotion analyses, we show here that the Caenorhabditis elegans neuromuscular system exhibits persistent rhythmic activity, and such an activity contributes to the sustainability of basal locomotion, and the maintenance of acceleration after stimulation. The NALCN family sodium leak channel regulates the resting membrane potential and excitability of invertebrate and vertebrate neurons. Our molecular genetics and electrophysiology analyses show that the C. elegans NALCN, NCA, activates a premotor interneuron network to potentiate persistent motor circuit activity and to sustain C. elegans locomotion. Collectively, these results reveal a mechanism for, and physiological function of, persistent neural activity using a simple animal model, providing potential mechanistic clues for working memory in other systems.

Caenorhabditis elegans Innexins Regulate Active Zone Differentiation

In a genetic screen for active zone defective mutants in Caenorhabditis elegans, we isolated a loss-of-function allele of unc-7, a gene encoding an innexin/pannexin family gap junction protein. Innexin UNC-7 regulates the size and distribution of active zones at C. elegans neuromuscular junctions. Loss-of-function mutations in another innexin, UNC-9, cause similar active zone defects as unc-7 mutants. In addition to presumptive gap junction localizations, both UNC-7 and UNC-9 are also localized perisynaptically throughout development and required in presynaptic neurons to regulate active zone differentiation. Our mosaic analyses, electron microscopy, as well as expression studies suggest a novel and likely nonjunctional role of specific innexins in active zone differentiation in addition to gap junction formations.

The C2H2 zinc-finger protein SYD-9 is a putative posttranscriptional regulator for synaptic transmission.

Communication between neurons is largely achieved through chemical synapses, where neurotransmitters are released from synaptic vesicles at presynaptic terminals to activate postsynaptic cells. Exo- and endocytosis are coordinated to replenish the synaptic vesicle pool for sustained neuronal activity. We identified syd-9 (syd, synapse defective), a gene that encodes multiple C2H2 zinc-finger domain-containing proteins specifically required for synaptic function in Caenorhabditis elegans. syd-9 loss-of-function mutants exhibit locomotory defects, a diffuse distribution of synaptic proteins, and decreased synaptic transmission with unaffected neurodevelopment. syd-9 mutants share phenotypic and ultrastructural characteristics with mutants that lack synaptic proteins that are required for endocytosis. syd-9 mutants also display genetic interactions with these endocytotic mutants, suggesting that SYD-9 regulates endocytosis. SYD-9 proteins are enriched in the nuclei of both neuron and muscle cells, but their neuronal expression plays a major role in locomotion. SYD-9 isoforms display a speckle-like expression pattern that is typical of RNA-binding proteins that regulate premRNA splicing. Furthermore, syd-9 functions in parallel with unc-75 (unc, uncoordinated), the C. elegans homologue of the CELF/BrunoL family protein that regulates mRNA alternative splicing and processing, and is also required specifically for synaptic transmission. We propose that neuronal SYD-9 proteins are previously uncharacterized and specific posttranscriptional regulators of synaptic vesicle endocytosis.

Hyperactivation of B-Type Motor Neurons Results in Aberrant Synchrony of the Caenorhabditis elegans Motor Circuit

Excitatory acetylcholine motor neurons drive Caenorhabditis elegans locomotion. Coordinating the activation states of the backward-driving A and forward-driving B class motor neurons is critical for generating sinusoidal and directional locomotion. Here, we show by in vivo calcium imaging that expression of a hyperactive, somatodendritic ionotropic acetylcholine receptor ACR-2(gf) in A and B class motor neurons induces aberrant synchronous activity in both ventral- and dorsal-innervating B and A class motor neurons. Expression of ACR-2(gf) in either ventral- or dorsal-innervating B neurons is sufficient for triggering the aberrant synchrony that results in arrhythmic convulsions. Silencing of AVB, the premotor interneurons that innervate B motor neurons suppresses ACR-2(gf)-dependent convulsion; activating AVB by channelrhodopsin induces the onset of convulsion. These results support that the activity state of B motor neurons plays an instructive role for the coordination of motor circuit.

An extrasynaptic GABAergic signal modulates a pattern of forward movement in Caenorhabditis elegans

As a common neurotransmitter in the nervous system, γ-aminobutyric acid (GABA) modulates locomotory patterns in both vertebrates and invertebrates. However, the signaling mechanisms underlying the behavioral effects of GABAergic modulation are not completely understood. Here, we demonstrate that a GABAergic signal in C. elegans modulates the amplitude of undulatory head bending through extrasynaptic neurotransmission and conserved metabotropic receptors. We show that the GABAergic RME head motor neurons generate undulatory activity patterns that correlate with head bending and the activity of RME causally links with head bending amplitude. The undulatory activity of RME is regulated by a pair of cholinergic head motor neurons SMD, which facilitate head bending, and inhibits SMD to limit head bending. The extrasynaptic neurotransmission between SMD and RME provides a gain control system to set head bending amplitude to a value correlated with optimal efficiency of forward movement. DOI: http://dx.doi.org/10.7554/eLife.14197.001

Excitatory motor neurons are local oscillators for backward locomotion

Cell- or network-driven oscillators underlie motor rhythmicity. The identity of C. elegans oscillators remains unknown. Through cell ablation, electrophysiology, and calcium imaging, we show: (1) forward and backward locomotion is driven by different oscillators; (2) the cholinergic and excitatory A-class motor neurons exhibit intrinsic and oscillatory activity that is sufficient to drive backward locomotion in the absence of premotor interneurons; (3) the UNC-2 P/Q/N high-voltage-activated calcium current underlies A motor neuron’s oscillation; (4) descending premotor interneurons AVA, via an evolutionarily conserved, mixed gap junction and chemical synapse configuration, exert state-dependent inhibition and potentiation of A motor neuron’s intrinsic activity to regulate backward locomotion. Thus, motor neurons themselves derive rhythms, which are dually regulated by the descending interneurons to control the reversal motor state. These and previous findings exemplify compression: essential circuit properties are conserved but executed by fewer numbers and layers of neurons in a small locomotor network.

Descending pathway facilitates undulatory wave propagation in Caenorhabditis elegans through gap junctions

Significance A deep understanding of the neural basis of motor behaviors must integrate neuromuscular dynamics, mechanosensory feedback, as well as global command signals, to predict behavioral dynamics. Here, we report on an integrative approach to define the circuit logic underlying locomotion in Caenorhabditis elegans. Our combined experimental and computational analyses revealed that (i) motor neurons in C. elegans function as oscillators; (ii) descending interneuron inputs and proprioceptive coupling between motor neurons work synergistically to facilitate the sequential activation of motor neuron activities, allowing bending waves to propagate efficiently along the body. Our work represents a key step toward an integrative view of animal locomotion. Descending signals from the brain play critical roles in controlling and modulating locomotion kinematics. In the Caenorhabditis elegans nervous system, descending AVB premotor interneurons exclusively form gap junctions with the B-type motor neurons that execute forward locomotion. We combined genetic analysis, optogenetic manipulation, calcium imaging, and computational modeling to elucidate the function of AVB-B gap junctions during forward locomotion. First, we found that some B-type motor neurons generate rhythmic activity, constituting distributed oscillators. Second, AVB premotor interneurons use their electric inputs to drive bifurcation of B-type motor neuron dynamics, triggering their transition from stationary to oscillatory activity. Third, proprioceptive couplings between neighboring B-type motor neurons entrain the frequency of body oscillators, forcing coherent bending wave propagation. Despite substantial anatomical differences between the motor circuits of C. elegans and higher model organisms, converging principles govern coordinated locomotion.

A descending pathway through electrical coupling facilitates undulatory wave propagation in C. elegans

Descending signals from the brain play critical roles in controlling and modulating locomotion kinematics. The anatomical wiring diagram of the C. elegans nervous system suggests that the premotor interneurons AVB, the hub for sensorimotor transformation, make exclusively electrical synapses with the B-type motor neurons that activate body wall muscles and drive forward locomotion. Here, we combined genetic analysis, optogenetic manipulation, and computational modeling to elucidate the functions of AVB-B electrical couplings. First, we found that the B-type motor neurons could intrinsically generate rhythmic activity, constituting distributed center pattern generators. Second, AVB-B electrical couplings provide a descending pathway to drive bifurcation of motor neuron dynamics, triggering their transition from being stationary to generating rhythmic activity. Third, directional proprioceptive couplings between neighboring B-type motor neurons entrain the undulation frequency, forcing coherent bending waves to propagate along the body. Together, we propose that AVB-B electrical couplings work synergistically with proprioceptive couplings to enhance sequential activation of motor activity, and to facilitate the propagation of body undulation from head to tail during C. elegans forward locomotion.Descending signals from the brain play critical roles in controlling and modulating locomotion kinematics. In the Caenorhabditis elegans nervous system, descending AVB premotor interneurons exclusively form gap junctions with B-type motor neurons that drive forward locomotion. We combined genetic analysis, optogenetic manipulation, and computational modeling to elucidate the function of AVB-B gap junctions during forward locomotion. First, we found that some B-type motor neurons generated intrinsic rhythmic activity, constituting distributed central pattern generators. Second, AVB premotor interneurons drove bifurcation of B-type motor neuron dynamics, triggering their transition from stationary to oscillatory activity. Third, proprioceptive couplings between neighboring B-type motor neurons entrained the frequency of body oscillators, forcing coherent propagation of bending waves. Despite substantial anatomical differences between the worm motor circuit and those in higher model organisms, we uncovered converging principles that govern coordinated locomotion. Significance Statement A deep understanding of the neural basis of motor behavior must integrate neuromuscular dynamics, mechanosensory feedback, as well as global command signals, to predict behavioral dynamics. Here, we report on an integrative approach to defining the circuit logic underlying coordinated locomotion in C. elegans. Our combined experimental and computational analysis revealed that (1) motor neurons in C. elegans could function as intrinsic oscillators; (2) Descending inputs and proprioceptive couplings work synergistically to facilitate the sequential activation of motor neuron activities, allowing bending waves to propagate efficiently along the body. Our work thus represents a key step towards an integrative view of animal locomotion.

Excitatory Motor Neurons are Local Central Pattern Generators in an Anatomically Compressed Motor Circuit for Reverse Locomotion

Central pattern generators are cell- or network-driven oscillators that underlie motor rhythmicity. The existence and identity of C. elegans CPGs are unknown. Through cell ablation, electrophysiology, and calcium imaging, we identified oscillators for C. elegans reverse locomotion. We show that the cholinergic and excitatory class A motor neurons exhibit intrinsic and oscillatory activity, and such an activity can drive reverse locomotion without premotor interneurons. Regulation of their oscillatory activity, either through effecting the P/Q/N high voltage-activated calcium channel, an endogenous constituent of their intrinsic oscillation, or, via the dual regulation by descending premotor interneurons, determines the propensity, velocity, and sustention of reverse locomotion. Thus, the reversal motor neuron themselves serve as distributed local oscillators; regulation of their intrinsic activity controls the reversal motor state. These findings exemplify anatomic and functional compression: motor executors integrate the role of rhythm generation in a locomotor network that is constrained by small cell numbers. Highlights The class A motor neurons (A-MNs) intrinsically oscillate. High voltage-activated calcium channel is a constituent of A-MN oscillation. Regulation of A-MN oscillation by interneurons determines the reversal state. A-MNs integrate the role of multiple neuron types in large locomotor networks.