Narayan, A., et al. Contrasting responses within a single neuron class enable sex- specific attraction in C. elegans. Proceedings of the National Academy of Sciences USA 113, 10, E1392–E1401 (2016). Publisher's VersionAbstract
Animals find mates and food, and avoid predators by navigating to regions within a favorable range of available sensory cues. How are these ranges set and recognized? Here we show that male C. elegans exhibit strong concentration preferences for sex- specific small molecule cues secreted by hermaphrodites, and that these preferences emerge from the collective dynamics of a single male-specific class of neurons, the CEMs. Within a single worm, CEM responses are dissimilar, not determined by anatomical classification and can be excitatory or inhibitory. Response kinetics vary by concentration, suggesting a mechanism for establishing preferences. CEM responses are enhanced in the absence of synaptic transmission, and worms with only one intact CEM show non-preferential attraction to all concentrations of ascaroside for which CEM is the primary sensor, suggesting that synaptic modulation of CEM responses is necessary for establishing preferences. A heterogeneous concentration-dependent sensory representation thus appears to allow a single neural class to set behavioral preferences and recognize ranges of sensory cues. 
Knecht, Z.A., et al. Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila. eLife 5, e17879 (2016). Publisher's VersionAbstract
Ionotropic Receptors (IRs) are a large subfamily of variant ionotropicglutamate receptors present across Protostomia. While these receptors are mostextensively studied for their roles in chemosensory detection in insects, recent work has implicated two family members, IR21a and IR25a, in thermosensation in Drosophila. Here we characterize one of the most deeply conserved receptors, IR93a, and show that it is co-expressed and functions with IR21a and IR25a to mediate physiological and behavioral responses to cool temperatures. IR93a is also co-expressed with IR25a and a distinct receptor, IR40a, in a discrete population of sensory neurons in the sacculus, a multi-chambered pocket within the antenna. We demonstrate that this combination of receptors is important for neuronal responses to dry air and behavioral discrimination of humidity differences. Our results identify IR93a as a common component of molecularlyand cellularly distinct IR pathways underlying thermosensation and hygrosensation in insects. 
Shen, Y., et al. An extrasynaptic GABAergic signal modulates a pattern of forward movement in Caenorhabditis elegans. eLife 5, e14197 (2016). Publisher's VersionAbstract
As a common neurotransmitter in the nervous system, γ-aminobutyric acid (GABA) modulates locomotory patterns in both vertebrates and invertebrates. However, the signaling mechanisms underlying the behavioral effects of GABAergic modulation are not completely understood. Here, we demonstrate that a GABAergic signal in C. elegans modulates the amplitude of undulatory head bending through extrasynaptic neurotransmission and conserved metabotropic receptors. We show that the GABAergic RME head motor neurons generate undulatory activity patterns that correlate with head bending and the activity of RME causally links with head bending amplitude. The undulatory activity of RME is regulated by a pair of cholinergic head motor neurons SMD, which facilitate head bending, and inhibits SMD to limit head bending. The extrasynaptic neurotransmission between SMD and RME provides a gain control system to set head bending amplitude to a value correlated with optimal efficiency of forward movement.
Ni, L., et al. The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila. eLife 5, e13254 (2016). Publisher's VersionAbstract
Animals rely on highly sensitive thermoreceptors to seek out optimal temperatures, but the molecular mechanisms of thermosensing are not well understood. The Dorsal Organ Cool Cells (DOCCs) of the Drosophila larva are a set of exceptionally thermosensitive neurons critical for larval cool avoidance. Here we show that DOCC cool-sensing is mediated by Ionotropic Receptors (IRs), a family of sensory receptors widely studied in invertebrate chemical sensing. We find that two IRs, IR21a and IR25a, are required to mediate DOCC responses to cooling and are required for cool avoidance behavior. Furthermore, we find that ectopic expression of IR21a can confer cool-responsiveness in an Ir25a-dependent manner, suggesting an instructive role for IR21a in thermosensing. Together, these data show that IR family receptors can function together to mediate thermosensation of exquisite sensitivity.
van Giesen, L., et al. Multimodal stimulus coding by a gustatory sensory neuron in Drosophila larvae. Nature Communications 7, 10687 (2016). Publisher's VersionAbstract
Accurate perception of taste information is crucial for animal survival. In adult Drosophila, gustatory receptor neurons (GRNs) perceive chemical stimuli of one specific gustatory modality associated with a stereotyped behavioural response, such as aversion or attraction. We show that GRNs of Drosophila larvae employ a surprisingly different mode of gustatory information coding. Using a novel method for calcium imaging in the larval gustatory system, we identify a multimodal GRN that responds to chemicals of different taste modalities with opposing valence, such as sweet sucrose and bitter denatonium, reliant on different sensory receptors. This multimodal neuron is essential for bitter compound avoidance, and its artificial activation is sufficient to mediate aversion. However, the neuron is also essential for the integration of taste blends. Our findings support a model for taste coding in larvae, in which distinct receptor proteins mediate different responses within the same, multimodal GRN. 
Lim, M.A., et al. Neuroendocrine Modulation Sustains the C. elegans Forward Motor State. eLIFE 5, e19887 (2016). Publisher's VersionAbstract
Neuromodulators shape neural circuit dynamics. Combining electron microscopy, genetics, transcriptome profiling, calcium imaging, and optogenetics, we discovered a peptidergic neuron that modulates C. elegans motor circuit dynamics. The Six/SO-family homeobox transcription factor UNC-39 governs lineage-specific neurogenesis to give rise to a neuron RID. RID bears the anatomic hallmarks of a specialized endocrine neuron: it harbors near-exclusive dense core vesicles that cluster periodically along the axon, and expresses multiple neuropeptides, including the FMRF-amide-related FLP-14. RID activity increases during forward movement. Ablating RID reduces the sustainability of forward movement, a phenotype partially recapitulated by removing FLP-14. Optogenetic depolarization of RID prolongs forward movement, an effect reduced in the absence of FLP-14. Together, these results establish the role of a neuroendocrine cell RID in sustaining a specific behavioral state in C. elegans.
Venkatachalam, V., et al. Panneuronal Imaging in Roaming C. elegans. Proceedings of the National Academy of Sciences USA 113, E1082-1088 (2016). Publisher's VersionAbstract
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.
Berck, M., et al. The wiring diagram of a glomerular olfactory system. eLife 5, e14859 (2016). Publisher's VersionAbstract
The sense of smell enables animals to detect and react to long-distance cues according to internalized valences. Odors evoke responses from olfactory receptor neurons (ORNs), whose activities are integrated and processed in olfactory glomeruli and then relayed by projection neurons (PNs) to higher brain centers. The wiring diagram with synaptic resolution, which is unknown for any glomerular olfactory system, would enable the formulation of circuit function hypotheses to explain physiological and behavioral observations. Here, we have mapped with electron microscopy the complete wiring diagram of the left and right antennal lobes of Drosophila larva, an olfactory neuropil similar to the vertebrate olfactory bulb. We found two parallel circuits processing ORN inputs. First, a canonical circuit that consists of uniglomerular PNs that relay gain-controlled ORN inputs to the learning and memory center (mushroom body) and the center for innate behaviors (lateral horn). Second, a novel circuit where multiglomerular PNs and hierarchically structured local neurons (LNs) extract complex features from odor space and relay them to multiple brain areas. We found two types of panglomerular inhibitory LNs: one primarily providing presynaptic inhibition (onto ORNs) and another also providing postsynaptic inhibition (onto PNs), indicating that these two functionally different types of inhibition are susceptible to independent modulation. The wiring diagram revealed an LN circuit that putatively implements a bistable gain control mechanism, which either computes odor saliency through panglomerular inhibition, or allows a subset of glomeruli to respond to faint aversive odors in the presence of strong appetitive odor concentrations. This switch between operational modes is regulated by both neuromodulatory neurons and non-olfactory sensory neurons. Descending neurons from higher brain areas further indicate the context-dependent nature of early olfactory processing. The complete wiring diagram of the first olfactory neuropil of a genetically tractable organism will support detailed experimental and theoretical studies of circuit function towards bridging the gap between circuits and behavior.
Kain, J.S., et al. Variability in thermal and phototactic preferences in Drosophila may reflect an adaptive bet-hedging strategy. Evolution 69, 12, 3171-3185 (2015). Publisher's VersionAbstract
Organisms use various strategies to cope with fluctuating environmental conditions. In diversified bet-hedging, a single genotype exhibits phenotypic heterogeneity with the expectation that some individuals will survive transient selective pressures. To date, empirical evidence for bet-hedging is scarce. Here, we observe that individual Drosophila melanogaster flies exhibit striking variation in light- and temperature-preference behaviors. With a modeling approach that combines real world weather and climate data to simulate temperature preference-dependent survival and reproduction, we find that a bet-hedging strategy may underlie the observed interindividual behavioral diversity. Specifically, bet-hedging outcompetes strategies in which individual thermal preferences are heritable. Animals employing bet-hedging refrain from adapting to the coolness of spring with increased warm-seeking that inevitably becomes counterproductive in the hot summer. This strategy is particularly valuable when mean seasonal temperatures are typical, or when there is considerable fluctuation in temperature within the season. The model predicts, and we experimentally verify, that the behaviors of individual flies are not heritable. Finally, we model the effects of historical weather data, climate change, and geographic seasonal variation on the optimal strategies underlying behavioral variation between individuals, characterizing the regimes in which bet-hedging is advantageous.
Fang-Yen, C., Alkema, M.J. & Samuel, A.D.T. Illuminating neural circuits and behaviour in Caenorhabditis elegans with optogenetics. Philosophical Transactions of the Royal Society B: Biological Sciences 370, 1677, 20140212 (2015). Publisher's VersionAbstract
The development of optogenetics, a family of methods for using light to control neural activity via light-sensitive proteins, has provided a powerful new set of tools for neurobiology. These techniques have been particularly fruitful for dissecting neural circuits and behaviour in the compact and transparent roundworm Caenorhabditis elegans. Researchers have used optogenetic reagents to manipulate numerous excitable cell types in the worm, from sensory neurons, to interneurons, to motor neurons and muscles. Here, we show how optogenetics applied to this transparent roundworm has contributed to our understanding of neural circuits.
Zhen, M. & Samuel, A.D.T. C. elegans locomotion: small circuits, complex functions. Current Opinion in Neurobiology 33, 117-126 (2015). Publisher's VersionAbstract
With 302 neurons in the adult Caenorhabditis elegans nervous system, it should be possible to build models of complex behaviors spanning sensory input to motor output. The logic of the motor circuit is an essential component of such models. Advances in physiological, anatomical, and neurogenetic analysis are revealing a surprisingly complex signaling network in the worm’s small motor circuit. We are progressing towards a systems level dissection of the network of premotor interneurons, motor neurons, and muscle cells that move the animal forward and backward in its environment.
Hernandez-Nunez, L., et al. Reverse-correlation analysis of navigation dynamics in Drosophila larva using optogenetics. eLife 4, 9 (2015). Publisher's VersionAbstract
Neural circuits for behavior transform sensory inputs into motor outputs in patterns with strategic value. Determining how neurons along a sensorimotor circuit contribute to this transformation is central to understanding behavior. To do this, a quantitative framework to describe behavioral dynamics is needed. Here, we built a high-throughput optogenetic system for Drosophila larva to quantify the sensorimotor transformations underlying navigational behavior. We express CsChrimson, a red-shifted variant of Channelrhodopsin, in specific chemosensory neurons, and expose large numbers of freely moving animals to random optogenetic activation patterns. We quantify their behavioral responses and use reverse correlation analysis to uncover the linear and static nonlinear components of navigation dynamics as functions of optogenetic activation patterns of specific sensory neurons. We find that linear-nonlinear (LN) models accurately predict navigational decision-making for different optogenetic activation waveforms. We use our method to establish the valence and dynamics of navigation driven by optogenetic activation of different combinations of bitter sensing gustatory neurons. Our method captures the dynamics of optogenetically-induced behavior in compact, quantitative transformations that can be used to characterize circuits for sensorimotor processing and their contribution to navigational decision-making.
Klein, M., et al. Sensory determinants of behavioral dynamics in Drosophila thermotaxis. Proceedings of the National Academy of Sciences USA 112, 2, E220-229 (2015). Publisher's VersionAbstract
Complex animal behaviors are built from dynamical relationships between sensory inputs, neuronal activity, and motor outputs in patterns with strategic value. Connecting these patterns illuminates how nervous systems compute behavior. Here, we study Drosophila larva navigation up temperature gradients towards preferred temperatures (positive thermotaxis). By tracking the movements of animals responding to fixed spatial temperature gradients or random temperature fluctuations, we calculate the sensitivity and dynamics of the conversion of thermosensory inputs into motor responses. We discover three thermosensory neurons in each dorsal organ ganglion (DOG) that are required for positive thermotaxis. Random optogenetic stimulation of the DOG thermosensory neurons evokes behavioral patterns that mimic the response to temperature variations.  In vivo calcium and voltage imaging reveals that the DOG thermosensory neurons exhibit activity patterns with sensitivity and dynamics matched to the behavioral response. Temporal processing of temperature variations carried out by the DOG thermosensory neurons emerges in distinct motor responses during thermotaxis.
Goodman, M.B., et al. Thermotaxis navigation behavior. WormBook : the online review of C. elegans biology 1-10 (2014). Publisher's VersionAbstract
This chapter describes four different protocols used to assay thermotaxis navigation behavior of single, or populations of, C. elegans hermaphrodites on spatial thermal gradients within the physiological temperature range (15-25°C). A method to assay avoidance of noxious temperatures is also described.
Luo, L., et al. Bidirectional thermotaxis in Caenorhabditis elegans is mediated by distinct sensorimotor strategies driven by the AFD thermosensory neurons. Proceedings of the National Academy of Sciences USA 111, 2776-2781 (2014). Publisher's VersionAbstract
The nematode Caenorhabditis elegans navigates toward a pre- ferred temperature setpoint (Ts) determined by long-term temper- ature exposure. During thermotaxis, the worm migrates down temperature gradients at temperatures above Ts (negative ther- motaxis) and performs isothermal tracking near Ts. Under some conditions, the worm migrates up temperature gradients below Ts (positive thermotaxis). Here, we analyze positive and negative thermotaxis toward Ts to study the role of specific neurons that have been proposed to be involved in thermotaxis using genetic ablation, behavioral tracking, and calcium imaging. We find differ- ences in the strategies for positive and negative thermotaxis. Neg- ative thermotaxis is achieved through biasing the frequency of reorientation maneuvers (turns and reversal turns) and biasing the direction of reorientation maneuvers toward colder temper- atures. Positive thermotaxis, in contrast, biases only the direction of reorientation maneuvers toward warmer temperatures. We find that the AFD thermosensory neuron drives both positive and negative thermotaxis. The AIY interneuron, which is postsyn- aptic to AFD, may mediate the switch from negative to positive thermotaxis below Ts. We propose that multiple thermotactic behaviors, each defined by a distinct set of sensorimotor trans- formations, emanate from the AFD thermosensory neurons. AFD learns and stores the memory of preferred temperatures, detects temperature gradients, and drives the appropriate thermotactic behavior in each temperature regime by the flexible use of downstream circuits.
Luo, L., et al. Dynamic encoding of perception, memory and movement in a C. elegans chemotaxis circuit. Neuron 82, 1115-1128 (2014). Publisher's VersionAbstract
Brain circuits endow behavioral flexibility. Here, we study circuits encoding flexible 26 chemotaxis in C. elegans, where the animal navigates up or down NaCl gradients (positive or negative chemotaxis) to reach the salt concentration of previous growth (the setpoint). The ASER sensory neuron mediates positive and negative chemotaxis by regulating the frequency and direction of reorientation movements in response to salt gradients. Both salt gradients and setpoint memory are encoded in ASER temporal activity patterns. Distinct temporal activity patterns in interneurons immediately downstream of ASER encode chemotactic movement decisions. Different interneuron combinations regulate positive vs. negative chemotaxis. We conclude that sensorimotor pathways are segregated immediately after the primary sensory neuron in the chemotaxis circuit, and sensory representation is rapidly transformed to motor representation at the first interneuron layer. Our study reveals compact encoding of perception, memory, and locomotion in an experience dependent navigational behavior in C. elegans.
Smith, H.K., et al. Defining specificity determinants of cyclic GMP-mediated gustatory sensory transduction in Caenorhabditis elegans. Genetics 194, 4, 885-901 (2013). Publisher's VersionAbstract
Cyclic guanosine monophosphate (cGMP) is a key secondary messenger used in signal transduction in various types of sensory neurons. The importance of cGMP in the ASE gustatory receptor neurons of the nematode Caenorhabditis elegans was deduced by the observation that multiple receptor-type guanylyl cyclases (rGCs), encoded by the gcygenes, and two presently known cyclic nucleotide-gated ion channel subunits, encoded by the tax-2 and tax-4 genes, are essential for ASE-mediated gustatory behavior. We describe here specific mechanistic features of cGMP-mediated signal transduction in the ASE neurons. First, we assess the specificity of the sensory functions of individual rGC proteins. We have previously shown that multiple rGC proteins are expressed in a left/right asymmetric manner in the functionally lateralized ASE neurons and are required to sense distinct salt cues. Through domain swap experiments among three different rGC proteins, we show here that the specificity of individual rGC proteins lies in their extracellular domains and not in their intracellular, signal-transducing domains. Furthermore, we find that rGC proteins are also sufficient to confer salt sensory responses to other neurons. Both findings support the hypothesis that rGC proteins are salt receptor proteins. Second, we identify a novel, likely downstream effector of the rGC proteins in gustatory signal transduction, a previously uncharacterized cyclic nucleotide-gated (CNG) ion channel, encoded by the che-6 locus. che-6 mutants show defects in gustatory sensory transduction that are similar to defects observed in animals lacking thetax-2 and tax-4 CNG channels. In contrast, thermosensory signal transduction, which also requires tax-2 and tax-4, does not require che-6, but requires another CNG, cng-3. We propose that CHE-6 may form together with two other CNG subunits, TAX-2 and TAX-4, a gustatory neuron-specific heteromeric CNG channel complex.
Williams, D.C., et al. Rapid and Permanent Neuronal Inactivation In Vivo via Subcellular Generation of Reactive Oxygen with the Use of KillerRed. Cell Reports 5, 2, 553-563 (2013). Publisher's VersionAbstract
Inactivation of selected neurons in vivo can define their contribution to specific developmental outcomes, circuit functions, and behaviors. Here, we show that the optogenetic tool KillerRed selec- tively, rapidly, and permanently inactivates different classes of neurons in C. elegans in response to a single light stimulus, through the generation of reactive oxygen species (ROS). Ablation scales from individual neurons in single animals to multiple neurons in populations and can be applied to freely behaving animals. Using spatially restricted illumination, we demonstrate that localized KillerRed activation in either the cell body or the axon triggers neuronal degeneration and death of the targeted cell. Finally, targeting KillerRed to mitochondria results in organelle fragmentation without killing the cell, in contrast to the cell death observed when KillerRed is targeted to the plasma membrane. We expect this genetic tool to have wide-ranging applications in studies of circuit function and subcellular responses to ROS.
Kane, E.A., et al. Sensorimotor structure of Drosophila larva phototaxis. Proceedings of the National Academy of Sciences 110, 40, E3868-77 (2013). Publisher's VersionAbstract
The avoidance of light by fly larvae is a classic paradigm for sensorimotor behavior. Here, we use behavioral assays and video microscopy to quantify the sensorimotor structure of phototaxis using the Drosophila larva. Larval locomotion is composed of sequences of runs (periods of forward movement) that are interrupted by abrupt turns, during which the larva pauses and sweeps its head back and forth, probing local light information to deter- mine the direction of the successive run. All phototactic responses are mediated by the same set of sensorimotor transformations that require temporal processing of sensory inputs. Through functional imaging and genetic inactivation of specific neurons down- stream of the sensory periphery, we have begun to map these sensorimotor circuits into the larval central brain. We find that specific sensorimotor pathways that govern distinct light-evoked responses begin to segregate at the first relay after the photosensory neurons.
Reina, A., Subramaniam, A.B., Laromaine, A., Samuel, A.D. & Whitesides, G.M. Shifts in the distribution of mass densities is a signature of caloric restriction in Caenorhabditis elegans. PLoS ONE 8, 7, e69651 (2013). Publisher's VersionAbstract
Although the starvation response of the model multicellular organism Caenorhabditis elegans is a subject of much research, there is no convenient phenotypic readout of caloric restriction that can be applicable to large numbers of worms. This paper describes the distribution of mass densities of populations of C. elegans, from larval stages up to day one of adulthood, using isopycnic centrifugation, and finds that density is a convenient, if complex, phenotypic readout in C. elegans. The density of worms in synchronized populations of wildtype N2 C. elegans grown under standard solid-phase culture conditions was normally distributed, with distributions peaked sharply at a mean of 1.091 g/cm3 for L1, L2 and L3 larvae, 1.087 g/cm3 for L4 larvae, 1.081 g/cm3 for newly molted adults, and 1.074 g/cm3 at 24 hours of adulthood. The density of adult worms under starvation stress fell well outside this range, falling to a mean value of 1.054 g/cm3 after eight hours of starvation. This decrease in density correlated with the consumption of stored glycogen in the food-deprived worms. The density of the worms increased when deprived of food for longer durations, corresponding to a shift in the response of the worms: worms sacrifice their bodies by retaining larvae, which consume the adults from within. Density- based screens with the drug Ivermectin on worms cultured on single plates resulted in a clear bimodal (double-peaked) distribution of densities corresponding to drug exposed and non-exposed worms. Thus, measurements of changes in density could be used to conduct screens on the effects of drugs on several populations of worms cultured on single plates.