Brainstem circuits help zebrafish get into the swim of things.

In this issue of Neuron, Berg et al.1 investigate the functional contribution of two molecularly distinct subpopulations of spinal-projecting midbrain neurons in adult zebrafish, shedding light on mechanisms regulating properties of locomotion such as speed and bout duration.

Spinal sensory neurons project onto the hindbrain to stabilize posture and enhance locomotor speed.

In the spinal cord, cerebrospinal fluid-contacting neurons (CSF-cNs) are GABAergic interoceptive sensory neurons that detect spinal curvature via a functional coupling with the Reissner fiber. This mechanosensory system has recently been found to be involved in spine morphogenesis and postural control but the underlying mechanisms are not fully understood. In zebrafish, CSF-cNs project an ascending and ipsilateral axon reaching two to six segments away. Rostralmost CSF-cNs send their axons ipsilaterally into the hindbrain, a brain region containing motor nuclei and reticulospinal neurons (RSNs), which send descending motor commands to spinal circuits. Until now, the synaptic connectivity of CSF-cNs has only been investigated in the spinal cord, where they synapse onto motor neurons and premotor excitatory interneurons. The identity of CSF-cN targets in the hindbrain and the behavioral relevance of these sensory projections from the spinal cord to the hindbrain are unknown. Here, we provide anatomical and molecular evidence that rostralmost CSF-cNs synapse onto the axons of large RSNs including Mauthner cells and V2a neurons. Functional anatomy and optogenetically assisted mapping reveal that rostral CSF-cNs also synapse onto the soma and dendrites of cranial motor neurons innervating hypobranchial muscles. During acousto-vestibular evoked escape responses, ablation of rostralmost CSF-cNs results in a weaker escape response with a decreased C-bend amplitude, lower speed, and deficient postural control. Our study demonstrates that spinal sensory feedback enhances speed and stabilizes posture, and reveals a novel spinal gating mechanism acting on the output of descending commands sent from the hindbrain to the spinal cord.

Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities.

BACKGROUND: Dynactin subunit 1 is the largest subunit of the dynactin complex, an activator of the molecular motor protein complex dynein. Reduced levels of DCTN1 mRNA and protein have been found in sporadic amyotrophic lateral sclerosis (ALS) patients, and mutations have been associated with disease, but the role of this protein in disease pathogenesis is still unknown. METHODS: We characterized a Dynactin1a depletion model in the zebrafish embryo and combined in vivo molecular analysis of primary motor neuron development with live in vivo axonal transport assays in single cells to investigate ALS-related defects. To probe neuromuscular junction (NMJ) function and organization we performed paired motor neuron-muscle electrophysiological recordings and GCaMP calcium imaging in live, intact larvae, and the synapse structure was investigated by electron microscopy. RESULTS: Here we show that Dynactin1a depletion is sufficient to induce defects in the development of spinal cord motor neurons and in the function of the NMJ. We observe synapse instability, impaired growth of primary motor neurons, and higher failure rates of action potentials at the NMJ. In addition, the embryos display locomotion defects consistent with NMJ dysfunction. Rescue of the observed phenotype by overexpression of wild-type human DCTN1-GFP indicates a cell-autonomous mechanism. Synaptic accumulation of DCTN1-GFP, as well as ultrastructural analysis of NMJ synapses exhibiting wider synaptic clefts, support a local role for Dynactin1a in synaptic function. Furthermore, live in vivo analysis of axonal transport and cytoskeleton dynamics in primary motor neurons show that the phenotype reported here is independent of modulation of these processes. CONCLUSIONS: Our study reveals a novel role for Dynactin1 in ALS pathogenesis, where it acts cell-autonomously to promote motor neuron synapse stability independently of dynein-mediated axonal transport.

Mechanosensory neurons control the timing of spinal microcircuit selection during locomotion.

Despite numerous physiological studies about reflexes in the spinal cord, the contribution of mechanosensory feedback to active locomotion and the nature of underlying spinal circuits remains elusive. Here we investigate how mechanosensory feedback shapes active locomotion in a genetic model organism exhibiting simple locomotion-the zebrafish larva. We show that mechanosensory feedback enhances the recruitment of motor pools during active locomotion. Furthermore, we demonstrate that inputs from mechanosensory neurons increase locomotor speed by prolonging fast swimming at the expense of slow swimming during stereotyped acoustic escape responses. This effect could be mediated by distinct mechanosensory neurons. In the spinal cord, we show that connections compatible with monosynaptic inputs from mechanosensory Rohon-Beard neurons onto ipsilateral V2a interneurons selectively recruited at high speed can contribute to the observed enhancement of speed. Altogether, our study reveals the basic principles and a circuit diagram enabling speed modulation by mechanosensory feedback in the vertebrate spinal cord.

Three-dimensional spatiotemporal focusing of holographic patterns.

Two-photon excitation with temporally focused pulses can be combined with phase-modulation approaches, such as computer-generated holography and generalized phase contrast, to efficiently distribute light into two-dimensional, axially confined, user-defined shapes. Adding lens-phase modulations to 2D-phase holograms enables remote axial pattern displacement as well as simultaneous pattern generation in multiple distinct planes. However, the axial confinement linearly degrades with lateral shape area in previous reports where axially shifted holographic shapes were not temporally focused. Here we report an optical system using two spatial light modulators to independently control transverse- and axial-target light distribution. This approach enables simultaneous axial translation of single or multiple spatiotemporally focused patterns across the sample volume while achieving the axial confinement of temporal focusing. We use the system's capability to photoconvert tens of Kaede-expressing neurons with single-cell resolution in live zebrafish larvae.

Optimization of a Neurotoxin to Investigate the Contribution of Excitatory Interneurons to Speed Modulation In Vivo.

Precise control of speed during locomotion is essential for adaptation of behavior in different environmental contexts [1-4]. A central question in locomotion lies in understanding which neural populations set locomotor frequency during slow and fast regimes. Tackling this question in vivo requires additional non-invasive tools to silence large populations of neurons during active locomotion. Here we generated a stable transgenic line encoding a zebrafish-optimized botulinum neurotoxin light chain fused to GFP (BoTxBLC-GFP) to silence synaptic output over large populations of motor neurons or interneurons while monitoring active locomotion. By combining calcium imaging, electrophysiology, optogenetics, and behavior, we show that expression of BoTxBLC-GFP abolished synaptic release while maintaining characterized activity patterns and without triggering off-target effects. As chx10(+) V2a interneurons (V2as) are well characterized as the main population driving the frequency-dependent recruitment of motor neurons during fictive locomotion [5-14], we validated our silencing method by testing the effect of silencing chx10(+) V2as during active and fictive locomotion. Silencing of V2as selectively abolished fast locomotor frequencies during escape responses. In addition, spontaneous slow locomotion occurred less often and at frequencies lower than in controls. Overall, this silencing approach confirms that V2a excitation is critical for the production of fast stimulus-evoked swimming and also reveals a role for V2a excitation in the production of slower spontaneous locomotor behavior. Altogether, these results establish BoTxBLC-GFP as an ideal tool for in vivo silencing for probing the development and function of neural circuits from the synaptic to the behavioral level.

State-Dependent Modulation of Locomotion by GABAergic Spinal Sensory Neurons.

The cerebrospinal fluid (CSF) constitutes an interface through which chemical cues can reach and modulate the activity of neurons located at the epithelial boundary within the entire nervous system. Here, we investigate the role and functional connectivity of a class of GABAergic sensory neurons contacting the CSF in the vertebrate spinal cord and referred to as CSF-cNs. The remote activation of CSF-cNs was shown to trigger delayed slow locomotion in the zebrafish larva, suggesting that these cells modulate components of locomotor central pattern generators (CPGs). Combining anatomy, electrophysiology, and optogenetics in vivo, we show that CSF-cNs form active GABAergic synapses onto V0-v glutamatergic interneurons, an essential component of locomotor CPGs. We confirmed that activating CSF-cNs at rest induced delayed slow locomotion in the fictive preparation. In contrast, the activation of CSF-cNs promptly inhibited ongoing slow locomotion. Moreover, selective activation of rostral CSF-cNs during ongoing activity disrupted rostrocaudal propagation of descending excitation along the spinal cord, indicating that CSF-cNs primarily act at the premotor level. Altogether, our results demonstrate how a spinal GABAergic sensory neuron can tune the excitability of locomotor CPGs in a state-dependent manner by projecting onto essential components of the excitatory premotor pool.

Inhibition and motor control in the developing zebrafish spinal cord.

Vertebrate locomotion relies on oscillatory activity along the spinal cord. Inhibition is involved in controlling the alternation of activity between each side and contributes in modulating propagation and termination of locomotor activity. Spinal inhibitory neurons are thought to regulate these mechanisms but the exact contribution of specific cell types remains difficult to tackle during active locomotion. In the past two decades, use of the transparent zebrafish larva has enabled morphological, functional, and genetic characterization of specific inhibitory spinal neurons. A wide range of new optical tools has been developed to monitor and to manipulate the activity of genetically targeted spinal populations. Combining these techniques with conventional electrophysiology will provide a better understanding of the contribution of inhibitory spinal interneurons in regulating essential features of locomotor patterns.

CDK7 regulates the mitochondrial localization of a tail-anchored proapoptotic protein, Hid.

The mitochondrial outer membrane is a major site of apoptosis regulation across phyla. Human and C. elegans Bcl-2 family proteins and Drosophila Hid require the C-terminal tail-anchored (TA) sequence in order to insert into the mitochondrial membrane, but it remains unclear whether cytosolic proteins actively regulate the mitochondrial localization of these proteins. Here, we report that the cdk7 complex regulates the mitochondrial localization of Hid and its ability to induce apoptosis. We identified cdk7 through an in vivo RNAi screen of genes required for cell death. Although CDK7 is best known for its role in transcription and cell-cycle progression, a hypomorphic cdk7 mutant suppressed apoptosis without impairing these other known functions. In this cdk7 mutant background, Hid failed to localize to the mitochondria and failed to bind to recombinant inhibitors of apoptosis (IAPs). These findings indicate that apoptosis is promoted by a newly identified function of CDK7, which couples the mitochondrial localization and IAP binding of Hid.

Metabolic regulation of cellular plasticity in the pancreas.

Obese individuals exhibit an increase in pancreatic ß cell mass; conversely, scarce nutrition during pregnancy has been linked to ß cell insufficiency in the offspring [reviewed in 1, 2]. These phenomena are thought to be mediated mainly through effects on ß cell proliferation, given that a nutrient-sensitive ß cell progenitor population in the pancreas has not been identified. Here, we employed the fluorescent ubiquitination-based cell-cycle indicator system to investigate ß cell replication in real time and found that high nutrient concentrations induce rapid ß cell proliferation. Importantly, we found that high nutrient concentrations also stimulate ß cell differentiation from progenitors in the intrapancreatic duct (IPD). Furthermore, using a new zebrafish line where ß cells are constitutively ablated, we show that ß cell loss and high nutrient intake synergistically activate these progenitors. At the cellular level, this activation process causes ductal cell reorganization as it stimulates their proliferation and differentiation. Notably, we link the nutrient-dependent activation of these progenitors to a downregulation of Notch signaling specifically within the IPD. Furthermore, we show that the nutrient sensor mechanistic target of rapamycin (mTOR) is required for endocrine differentiation from the IPD under physiological conditions as well as in the diabetic state. Thus, this study reveals critical insights into how cells modulate their plasticity in response to metabolic cues and identifies nutrient-sensitive progenitors in the mature pancreas.