Post-embryonic remodeling of the C. elegans motor circuit.

During development, animals can maintain behavioral output even as underlying circuitry structurally remodels. After hatching, C. elegans undergoes substantial motor neuron expansion and synapse rewiring while the animal continuously moves with an undulatory pattern. To understand how the circuit transitions from its juvenile to mature configuration without interrupting functional output, we reconstructed the C. elegans motor circuit by electron microscopy across larval development. We observed the following: First, embryonic motor neurons transiently interact with the developing post-embryonic motor neurons prior to remodeling of their juvenile wiring. Second, post-embryonic neurons initiate synapse development with their future partners as their neurites navigate through the juvenile nerve cords. Third, embryonic and post-embryonic neurons sequentially build structural machinery needed for the adult circuit before the embryonic neurons relinquish their roles to post-embryonic neurons. Fourth, this transition is repeated region by region along the body in an anterior-to-posterior sequence, following the birth order of neurons. Through this orchestrated and programmed rewiring, the motor circuit gradually transforms from asymmetric to symmetric wiring. These maturation strategies support the continuous maintenance of motor patterns as the juvenile circuit develops into the adult configuration.

Extrasynaptic signaling enables an asymmetric juvenile motor circuit to produce symmetric undulation.

In many animals, there is a direct correspondence between the motor patterns that drive locomotion and the motor neuron innervation. For example, the adult C. elegans moves with symmetric and alternating dorsal-ventral bending waves arising from symmetric motor neuron input onto the dorsal and ventral muscles. In contrast to the adult, the C. elegans motor circuit at the juvenile larval stage has asymmetric wiring between motor neurons and muscles but still generates adult-like bending waves with dorsal-ventral symmetry. We show that in the juvenile circuit, wiring between excitatory and inhibitory motor neurons coordinates the contraction of dorsal muscles with relaxation of ventral muscles, producing dorsal bends. However, ventral bending is not driven by analogous wiring. Instead, ventral muscles are excited uniformly by premotor interneurons through extrasynaptic signaling. Ventral bends occur in anti-phasic entrainment to activity of the same motor neurons that drive dorsal bends. During maturation, the juvenile motor circuit is replaced by two motor subcircuits that separately drive dorsal and ventral bending. Modeling reveals that the juvenile's immature motor circuit is an adequate solution to generate adult-like dorsal-ventral bending before the animal matures. Developmental rewiring between functionally degenerate circuit solutions, which both generate symmetric bending patterns, minimizes behavioral disruption across maturation.

Imaging whole-brain activity to understand behavior.

The brain evolved to produce behaviors that help an animal inhabit the natural world. During natural behaviors, the brain is engaged in many levels of activity from the detection of sensory inputs to decision-making to motor planning and execution. To date, most brain studies have focused on small numbers of neurons that interact in limited circuits. This allows analyzing individual computations or steps of neural processing. During behavior, however, brain activity must integrate multiple circuits in different brain regions. The activities of different brain regions are not isolated, but may be contingent on one another. Coordinated and concurrent activity within and across brain areas is organized by (1) sensory information from the environment, (2) the animal's internal behavioral state, and (3) recurrent networks of synaptic and non-synaptic connectivity. Whole-brain recording with cellular resolution provides a new opportunity to dissect the neural basis of behavior, but whole-brain activity is also mutually contingent on behavior itself. This is especially true for natural behaviors like navigation, mating, or hunting, which require dynamic interaction between the animal, its environment, and other animals. In such behaviors, the sensory experience of an unrestrained animal is actively shaped by its movements and decisions. Many of the signaling and feedback pathways that an animal uses to guide behavior only occur in freely moving animals. Recent technological advances have enabled whole-brain recording in small behaving animals including nematodes, flies, and zebrafish. These whole-brain experiments capture neural activity with cellular resolution spanning sensory, decision-making, and motor circuits, and thereby demand new theoretical approaches that integrate brain dynamics with behavioral dynamics. Here, we review the experimental and theoretical methods that are being employed to understand animal behavior and whole-brain activity, and the opportunities for physics to contribute to this emerging field of systems neuroscience.

Structural Analysis of the Caenorhabditis elegans Dauer Larval Anterior Sensilla by Focused Ion Beam-Scanning Electron Microscopy.

At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments.

Natural sensory context drives diverse brain-wide activity during C. elegans mating.

Natural goal-directed behaviors often involve complex sequences of many stimulus-triggered components. Understanding how brain circuits organize such behaviors requires mapping the interactions between an animal, its environment, and its nervous system. Here, we use brain-wide neuronal imaging to study the full performance of mating by the C. elegans male. We show that as mating unfolds in a sequence of component behaviors, the brain operates similarly between instances of each component but distinctly between different components. When the full sensory and behavioral context is taken into account, unique roles emerge for each neuron. Functional correlations between neurons are not fixed but change with behavioral dynamics. From individual neurons to circuits, our study shows how diverse brain-wide dynamics emerge from the integration of sensory perception and motor actions in their natural context.

Connectomes across development reveal principles of brain maturation.

An animal's nervous system changes as its body grows from birth to adulthood and its behaviours mature1-8. The form and extent of circuit remodelling across the connectome is unknown3,9-15. Here we used serial-section electron microscopy to reconstruct the full brain of eight isogenic Caenorhabditis elegans individuals across postnatal stages to investigate how it changes with age. The overall geometry of the brain is preserved from birth to adulthood, but substantial changes in chemical synaptic connectivity emerge on this consistent scaffold. Comparing connectomes between individuals reveals substantial differences in connectivity that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. During development, the central decision-making circuitry is maintained, whereas sensory and motor pathways substantially remodel. With age, the brain becomes progressively more feedforward and discernibly modular. Thus developmental connectomics reveals principles that underlie brain maturation.

Temperature regulates synaptic subcellular specificity mediated by inhibitory glutamate signaling.

Environmental factors such as temperature affect neuronal activity and development. However, it remains unknown whether and how they affect synaptic subcellular specificity. Here, using the nematode Caenorhabditis elegans AIY interneurons as a model, we found that high cultivation temperature robustly induces defects in synaptic subcellular specificity through glutamatergic neurotransmission. Furthermore, we determined that the functional glutamate is mainly released by the ASH sensory neurons and sensed by two conserved inhibitory glutamate-gated chloride channels GLC-3 and GLC-4 in AIY. Our work not only presents a novel neurotransmission-dependent mechanism underlying the synaptic subcellular specificity, but also provides a potential mechanistic insight into high-temperature-induced neurological defects.

Corrigendum: A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System.

[This corrects the article DOI: 10.3389/fncir.2018.00094.].

A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System.

The "connectome," a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using Caenorhabditis elegans. Recent advances in vEM allow mapping new C. elegans connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the C. elegans nervous system and beyond.

Interrogating the ccm-3 Gene Network.

Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by mutations in one of three genes (CCM1-3). Loss of CCM3 causes the poorest prognosis, and little is known about how it regulates vascular integrity. The C. elegans ccm-3 gene regulates the development of biological tubes that resemble mammalian vasculature, and in a genome-wide reverse genetic screen, we identified more than 500 possible CCM-3 pathway genes. With a phenolog-like approach, we generated a human CCM signaling network and identified 29 genes in common, of which 14 are required for excretory canal extension and membrane integrity, similar to ccm-3. Notably, depletion of the MO25 ortholog mop-25.2 causes severe defects in tube integrity by preventing CCM-3 localization to apical membranes. Furthermore, loss of MO25 phenocopies CCM3 ablation by causing stress fiber formation in endothelial cells. This work deepens our understanding of how CCM3 regulates vascular integrity and may help identify therapeutic targets for treating CCM3 patients.

Myrf ER-Bound Transcription Factors Drive C. elegans Synaptic Plasticity via Cleavage-Dependent Nuclear Translocation.

Synaptic refinement is a critical step in nervous system maturation, requiring a carefully timed reorganization and refinement of neuronal connections. We have identified myrf-1 and myrf-2, two C. elegans homologs of Myrf family transcription factors, as key regulators of synaptic rewiring. MYRF-1 and its paralog MYRF-2 are functionally redundant specifically in synaptic rewiring. They co-exist in the same protein complex and act cooperatively to regulate synaptic rewiring. We find that the MYRF proteins localize to the ER membrane and that they are cleaved into active N-terminal fragments, which then translocate into the nucleus to drive synaptic rewiring. Overexpression of active forms of MYRF is sufficient to accelerate synaptic rewiring. MYRF-1 and MYRF-2 are the first genes identified to be indispensable for promoting synaptic rewiring in C. elegans. These findings reveal a molecular mechanism underlying synaptic rewiring and developmental circuit plasticity.

Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome.

Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.

ELASPIC web-server: proteome-wide structure-based prediction of mutation effects on protein stability and binding affinity.

UNLABELLED: ELASPIC is a novel ensemble machine-learning approach that predicts the effects of mutations on protein folding and protein-protein interactions. Here, we present the ELASPIC webserver, which makes the ELASPIC pipeline available through a fast and intuitive interface. The webserver can be used to evaluate the effect of mutations on any protein in the Uniprot database, and allows all predicted results, including modeled wild-type and mutated structures, to be managed and viewed online and downloaded if needed. It is backed by a database which contains improved structural domain definitions, and a list of curated domain-domain interactions for all known proteins, as well as homology models of domains and domain-domain interactions for the human proteome. Homology models for proteins of other organisms are calculated on the fly, and mutations are evaluated within minutes once the homology model is available. AVAILABILITY AND IMPLEMENTATION: The ELASPIC webserver is available online at http://elaspic.kimlab.org CONTACT: pm.kim@utoronto.ca or pi@kimlab.orgSupplementary data: Supplementary data are available at Bioinformatics online.