Andrew M. Leifer

Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans

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 in specific cell types. Image processing software analyzes the worms position in 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. Because 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 test the accuracy, flexibility and utility of our system, we performed optogenetic analyses of the worm motor circuit, egg-laying circuit and mechanosensory circuits that have not been possible with previous methods.

Monoaminergic Orchestration of Motor Programs in a Complex C. elegans Behavior

A single monoamine can orchestrate different phases of a compound motor sequence in C. elegans through the synaptic and extra-synaptic activation of distinct classes of receptors.

Optogenetic manipulation of neural activity in C. elegans: from synapse to circuits and behaviour.

The emerging field of optogenetics allows for optical activation or inhibition of excitable cells. In 2005, optogenetic proteins were expressed in the nematode Caenorhabditis elegans for the first time. Since then, C. elegans has served as a powerful platform upon which to conduct optogenetic investigations of synaptic function, circuit dynamics and the neuronal basis of behaviour. The C. elegans nervous system, consisting of 302 neurons, whose connectivity and morphology has been mapped completely, drives a rich repertoire of behaviours that are quantifiable by video microscopy. This model organisms compact nervous system, quantifiable behaviour, genetic tractability and optical accessibility make it especially amenable to optogenetic interrogation. Channelrhodopsin‐2 (ChR2), halorhodopsin (NpHR/Halo) and other common optogenetic proteins have all been expressed in C. elegans. Moreover, recent advances leveraging molecular genetics and patterned light illumination have now made it possible to target photoactivation and inhibition to single cells and to do so in worms as they behave freely. Here, we describe techniques and methods for optogenetic manipulation in C. elegans. We review recent work using optogenetics and C. elegans for neuroscience investigations at the level of synapses, circuits and behaviour.

Simultaneous optogenetic manipulation and calcium imaging in freely moving C. elegans

Understanding how an organisms nervous system transforms sensory input into behavioral outputs requires recording and manipulating its neural activity during unrestrained behavior. Here we present an instrument to simultaneously monitor and manipulate neural activity while observing behavior in a freely moving animal, the nematode Caenorhabditis elegans. Neural activity is recorded optically from cells expressing a calcium indicator, GCaMP3. Neural activity is manipulated optically by illuminating targeted neurons expressing the optogenetic protein Channelrhodopsin. Real-time computer vision software tracks the animals behavior and identifies the location of targeted neurons in the nematode as it crawls. Patterned illumination from a DMD is used to selectively illuminate subsets of neurons for either calcium imaging or optogenetic stimulation. Real-time computer vision software constantly updates the illumination pattern in response to the worms movement and thereby allows for independent optical recording or activation of different neurons in the worm as it moves freely. We use the instrument to directly observe the relationship between sensory neuron activation, interneuron dynamics and locomotion in the worms mechanosensory circuit. We record and compare calcium transients in the backward locomotion command interneurons AVA, in response to optical activation of the anterior mechanosensory neurons ALM, AVM or both.

Automatically tracking neurons in a moving and deforming brain

Advances in optical neuroimaging techniques now allow neural activity to be recorded with cellular resolution in awake and behaving animals. Brain motion in these recordings pose a unique challenge. The location of individual neurons must be tracked in 3D over time to accurately extract single neuron activity traces. Recordings from small invertebrates like C. elegans are especially challenging because they undergo very large brain motion and deformation during animal movement. Here we present an automated computer vision pipeline to reliably track populations of neurons with single neuron resolution in the brain of a freely moving C. elegans undergoing large motion and deformation. 3D volumetric fluorescent images of the animal’s brain are straightened, aligned and registered, and the locations of neurons in the images are found via segmentation. Each neuron is then assigned an identity using a new time-independent machine-learning approach we call Neuron Registration Vector Encoding. In this approach, non-rigid point-set registration is used to match each segmented neuron in each volume with a set of reference volumes taken from throughout the recording. The way each neuron matches with the references defines a feature vector which is clustered to assign an identity to each neuron in each volume. Finally, thin-plate spline interpolation is used to correct errors in segmentation and check consistency of assigned identities. The Neuron Registration Vector Encoding approach proposed here is uniquely well suited for tracking neurons in brains undergoing large deformations. When applied to whole-brain calcium imaging recordings in freely moving C. elegans, this analysis pipeline located 156 neurons for the duration of an 8 minute recording and consistently found more neurons more quickly than manual or semi-automated approaches.

Temporal processing and context dependency in Caenorhabditis elegans response to mechanosensation

A quantitative understanding of how sensory signals are transformed into motor outputs places useful constraints on brain function and helps to reveal the brain’s underlying computations. We investigate how the nematode Caenorhabditis elegans responds to time-varying mechanosensory signals using a high-throughput optogenetic assay and automated behavior quantification. We find that the behavioral response is tuned to temporal properties of mechanosensory signals, such as their integral and derivative, that extend over many seconds. Mechanosensory signals, even in the same neurons, can be tailored to elicit different behavioral responses. Moreover, we find that the animal’s response also depends on its behavioral context. Most dramatically, the animal ignores all tested mechanosensory stimuli during turns. Finally, we present a linear-nonlinear model that predicts the animal’s behavioral response to stimulus.

Whole-brain calcium imaging with cellular resolution in freely behaving C. elegans

Significance Large-scale neural recordings in freely moving animals are important for understanding how patterns of activity across a population of neurons generates animal behavior. Previously, recordings have been limited to either small brain regions or to immobilized or anesthetized animals exhibiting limited behavior. This work records from neurons with cellular resolution throughout the entire brain of the nematode Caenorhabditis elegans during free locomotion. Neurons are found whose activity correlates with behaviors including forward and backward locomotion and turning. A growing body of evidence suggests that animal behavior is sometimes generated by the collective activity of many neurons. It is hoped that methods like this will provide quantitative datasets that yield insights into how brain-wide neural dynamics encode animal action and perception. 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.

Predicting natural behavior from whole-brain neural dynamics

The activity of an animal’s brain contains information about that animal’s actions and movements. We investigated the neural representation of locomotion in the nematode C. elegans by recording population calcium activity during unrestrained movement. We report that a neural population more accurately decodes locomotion than any single neuron. Relevant signals are distributed across neurons with diverse tunings to locomotion. Two distinct subpopulations are informative for decoding velocity and body curvature, and different neurons’ activities contribute features relevant for different instances of behavioral motifs. We labeled neurons AVAL and AVAR and found their activity was highly correlated with one another. They exhibited expected transients during backward locomotion, although they were not always the most informative neurons for decoding velocity. Finally, we compared population neural activity during movement and immobilization. Immobilization alters the correlation structure of neural activity and its dynamics. Some neurons previously correlated with AVA become anti-correlated and vice versa. The activity of an animal’s brain contains information about that animal’s actions and movements. We investigated the neural representation of locomotion in the nematode C. elegans by recording brain-wide neural dynamics in freely moving animals. We report that a population of neurons more accurately decodes the animal’s locomotion than any single neuron. Neural signals are distributed across neurons in the population with a diversity of tuning to locomotion. Two distinct subpopulations are most informative for decoding velocity and body curvature, and different neurons’ activities contribute features relevant for different instances of behavioral motifs within these subpopulations. We additionally labeled the AVA neurons within our population recordings. AVAL and AVAR exhibit activity that is highly correlated with one another, and they exhibit the expected responses to locomotion, although we find that AVA is not always the most informative neuron for decoding velocity. Finally, we compared brain-wide neural activity during movement and immobilization and observe that immobilization alters the correlation structure of neural activity and its dynamics. Some neurons that were previously correlated with AVA become anti-correlated and vice versa during immobilization. We conclude that neural population codes are important for understanding neural dynamics of behavior in moving animals.Abstract We record calcium activity from the majority of head neurons in freely moving C. elegans to reveal where and how natural behavior is encoded in a compact brain. We find that a sparse subset of neurons distributed throughout the head encode locomotion. A linear combination of these neurons’ activity predicts the animal’s velocity and body curvature and is sufficient to infer its posture. This sparse linear model outperforms single neuron or PCA models at predicting behavior. Among neurons important for the prediction are well-known locomotory neurons, such as AVA, as well as neurons not traditionally associated with locomotion. We compare neural activity of the same animal during unrestrained movement and during immobilization and find large differences between brain-wide neural dynamics during real and fictive locomotion. One Sentence Summary C. elegans behavior is predicted from neural activity.

From the Cover: PNAS Plus: Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

Significance Large-scale neural recordings in freely moving animals are important for understanding how patterns of activity across a population of neurons generates animal behavior. Previously, recordings have been limited to either small brain regions or to immobilized or anesthetized animals exhibiting limited behavior. This work records from neurons with cellular resolution throughout the entire brain of the nematode Caenorhabditis elegans during free locomotion. Neurons are found whose activity correlates with behaviors including forward and backward locomotion and turning. A growing body of evidence suggests that animal behavior is sometimes generated by the collective activity of many neurons. It is hoped that methods like this will provide quantitative datasets that yield insights into how brain-wide neural dynamics encode animal action and perception. 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.