Octopamine biosynthesis requires tyrosine decarboxylase to convert tyrosine into tyramine and tyramine beta-hydroxylase to convert tyramine into octopamine. We identified and characterized a Caenorhabditis elegans tyrosine decarboxylase gene, tdc-1, and a tyramine beta-hydroxylase gene, tbh-1. The TBH-1 protein is expressed in a subset of TDC-1-expressing cells, indicating that C. elegans has tyraminergic cells that are distinct from its octopaminergic cells. tdc-1 mutants have behavioral defects not shared by tbh-1 mutants. We show that tyramine plays a specific role in the inhibition of egg laying, the modulation of reversal behavior, and the suppression of head oscillations in response to anterior touch. We propose a model for the neural circuit that coordinates locomotion and head oscillations in response to anterior touch. Our findings establish tyramine as a neurotransmitter in C. elegans, and we suggest that tyramine is a genuine neurotransmitter in other invertebrates and possibly in vertebrates as well.

We present an optogenetic illumination system capable of real-time light delivery with high spatial resolution to specified targets in freely moving Caenorhabditis elegans. A tracking microscope records the motion of an unrestrained worm expressing Channelrhodopsin-2 or Halorhodopsin/NpHR in specific cell types. Image processing software analyzes the worm’s position within each video frame, rapidly estimates the locations of targeted cells, and instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Since each cell in an unrestrained worm is a rapidly moving target, our system operates at high speed (~50 frames per second) to provide high spatial resolution (~30 µm). To demonstrate the accuracy, flexibility, and utility of our system, we present optogenetic analyses of the worm motor circuit, egg-laying circuit, and mechanosensory circuits that were not possible with previous methods.

SUMMARY Differences in expression, protein interactions and DNA binding of paralogous transcription factors (“TF parameters”) are thought to be important determinants of regulatory and biological specificity. However, both the extent of TF divergence and the relative contribution of individual TF parameters remain undetermined. We comprehensively identify dimerization partners, spatiotemporal expression patterns and DNA binding specificities for the C. elegans bHLH family of TFs, and model these data into an integrated network. This network displays both specificity and promiscuity, as some bHLH proteins, DNA sequences, and tissues are highly connected, whereas others are not. By comparing all bHLH TFs, we find extensive divergence, and that all three parameters contribute equally to bHLH divergence. Our approach provides a framework for examining divergence for other protein families in C. elegans and in other complex multicellular organisms, including humans. Cross-species comparisons of integrated networks may provide further insights into molecular features underlying protein family evolution.

We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal's posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.C. elegans | Drosophila | calcium imaging | thermotaxis U nderstanding how brain dynamics creates behaviors requires quantifying the flow and transformation of sensory information to motor output in behaving animals. Optical imaging using genetically encoded calcium or voltage fluorescent probes offers a minimally invasive method to record neural activity in intact animals. The nematode Caenorhabditis elegans is particularly ideal for optical neurophysiology owing to its small size, optical transparency, compact nervous system, and ease of genetic manipulation. Imaging systems for tracking the activity of small numbers of neurons have been effective in determining their role during nematode locomotion and navigational behaviors like chemotaxis, thermotaxis, and the escape response (1-6).Recordings from large numbers of interconnected neurons are required to understand how neuronal ensembles carry out the systematic transformations of sensory input into motor patterns that build behavioral decisions.Several methods for fast 3D imaging of neural activity in a fixed imaging volume have been developed for different model organisms (7)(8)(9)(10)(11)(12)(13)(14). High-speed light sheet microscopy, light field microscopy, multifocus microscopy, and two-photon structured illumination microscopy have proved effective for rapidly recording large numbers of neurons in immobilized, intact, transparent animals like larval zebrafish and nematodes (15)(16)(17)(18)(19). However, these methods are problematic when attempting to track many neurons within the bending and moving body of a behaving animal. Panneuronal recording in moving animals poses higher demands on spatial and temporal resolution. Furthermore, extracting neuronal signals from recordings in a behaving animal requires an effective analysis pipel...