Summary Locomotion requires coordinated motor activity throughout an animal’s body. In both vertebrates and invertebrates, chains of coupled Central Pattern Generators (CPGs) are commonly evoked to explain local rhythmic behaviors. In C. elegans, we report that proprioception within the motor circuit is responsible for propagating and coordinating rhythmic undulatory waves from head to tail during forward movement. Proprioceptive coupling between adjacent body regions transduces rhythmic movement initiated near the head into bending waves driven along the body by a chain of reflexes. Using optogenetics and calcium imaging to manipulate and monitor motor circuit activity of moving C. elegans held in microfluidic devices, we found that the B-type cholinergic motor neurons transduce the proprioceptive signal. In C. elegans, a sensorimotor feedback loop operating within a specific type of motor neuron both drives and organizes body movement.

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.

The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal's position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal's position and orientation. Custom software tracks the 3D position of the animal's head in real time and two feedback loops adjust a motorized stage and objective to keep the animal's head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal's behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.calcium imaging | large-scale recording | behavior | C. elegans | microscopy H ow do patterns of neural activity generate an animal's behavior? To answer this question, it is important to develop new methods for recording from large populations of neurons in animals as they move and behave freely. The collective activity of many individual neurons appears to be critical for generating behaviors including arm reach in primates (1), song production in zebrafinch (2), the choice between swimming or crawling in leech (3), and decision-making in mice during navigation (4). New methods for recording from larger populations of neurons in unrestrained animals are needed to better understand neural coding of these behaviors and neural control of behavior more generally.Calcium imaging has emerged as a promising technique for recording dynamics from populations of neurons. Calcium-sensitive proteins are used to visualize changes in intracellular calcium levels in neurons in vivo which serve as a proxy for neural activity (5). To resolve the often weak fluorescent signal of an individual neuron in a dense forest of other labeled cells requires a high magnification objective with a large numerical aperture that, consequently, can image only a small field of view. Calcium imaging has traditionally been performed on animals that are stationary from anesthetization or immobilization to avoid imaging artifacts induced by animal motion. As a result, calcium imaging studies have historically focused on small brain regions in immobile animals that exhibit little or no behavior (6).No previous neurophysiological study has attained whole-brain imaging with cellular resolution in a...

Box 1. GlossaryBehavioral representation-a quantitative distillation of any aspect of the time-varying behavior exhibited by an animal in an experiment. Such representations can vary in form from classical ethograms to low-dimensional plots capturing the trajectory of an animal in space. Naturalistic-as with ''ecologically relevant'' (see below), there are many definitions for naturalistic, and indeed most experiments in behavioral neuroscience can justifiably be argued to be naturalistic at some level. Here, we take the word ''naturalistic'' to mean behaviors that are representative of actions generated during real-world tasks, like exploring new environments, obtaining food, finding shelter, and identifying mates; naturalistic behaviors as referred to herein are also largely self-motivated and expressed freely without physical restraint. This definition is meant to distinguish such behaviors from those that are imposed by researchers on animals through overtraining or those that are more constrained due to, e.g., head fixation (although, as mentioned above, there are contexts in which those types of behaviors are quite reasonably referred to as ''naturalistic''). Ecologically relevant-as with ''naturalistic'' (see above), ''ecologically relevant'' is an adjective whose meaning is in the eye of the beholder; again, this term can be appropriately applied to many kinds of behavioral experiments, including those in which animals are subject to training and restraint. Here, we take ''ecologicallyrelevant'' to mean a set of behaviors that support tasks animals have to address as part of the existential challenge of living in a particular environmental or ecological niche. Behavioral label-a behavioral label is a descriptor applied to an epoch of behavioral data. Behavioral labels can cover descriptions of behavior at many levels of granularity and run the gamut from ''a twitch of motor unit 72 in the soleus muscle'' to ''hibernating.'' Behavioral motif-a stereotyped and re-used unit of movement. The terms ''motif,'' ''moveme,'' ''module,'' ''primitive,'' and ''syllable'' have all been used interchangeably, and none of these terms is linked to a rigorous definition of the spatiotemporal scale at which a unit of behavior is organized. Similarly, action and behavior here and elsewhere are used to refer to collections of units of behavior, but again, there is no rigorous line that separates these or related terms. Perona and Anderson have argued for a taxonomy in which moveme is the simplest movement associated with a behavior, an action is a sequence of movemes, and an activity is a species-characteristic set of movemes and actions (Anderson and Perona, 2014). Behavioral sequence-an epoch in which more than one behavioral motif is expressed; sequences of motifs can be either deterministic (e.g., motif A always follows motif B) or probabilistic (e.g., motif A follows motif B fifty percent of the time). Artificial neural network-class of machine learning algorithms that operate by simulating a network of simplified neuron...

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here we use DNA-origami technology to construct sub-micrometer nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be unambiguously decoded using epifluorescence or total internal reflection fluorescence (TIRF) microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ~40 nm. One species of the barcodes was used to tag yeast surface receptors, suggesting their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.