Shawn Lockery (2003) International Worm Meeting "Theoretical analysis of locomotory switching in C. elegans"

Shawn Lockery

[International Worm Meeting, 2003]

Locomotory state in C. elegans is thought to be regulated by a network of command interneurons in two functional pools: the forward command neurons AVB and PVC, and the reverse command neurons AVA and AVD. It has been proposed that the circuit functions as a bistable switch in which only one pool is on at a time. This model is consistent with the observation that presumptive depolarization of both pools (by activated glutamate-receptor knock-in) decreases dwell time in the forward state, whereas presumptive hyperpolarization (by reducing tonic glutamate transmission) increases dwell time in the forward state. However, the bistable switch model does not easily account for the paradoxical observation that dwell time in the forward state is decreased whether one kills forward command neurons (AVB) or reverse command neurons (AVA).Here I propose an alternative model based on four assumptions. (1) Neurons switch stochastically between on and off states with a probability that increases with net synaptic input. (2) Forward command neurons act as a single unit, as do reverse command neurons. (3) Units in the model can adopt four activation states: (i) both off, (ii) forward on, reverse off, (iii) reverse on, forward off, (iv) both off. (4) The four activation states correspond, respectively, to the four main locomotory states: stop, forward, reverse, and omega turn. Unit activity is governed by three synaptic parameters: h, net input from sensory systems; w, the strength of the connections from the unit to its partner; and z, the strength of connections among neurons in the same pool. A key feature of the model is that the rate constants for transitions between its states can be expressed terms of the underlying neuronal parameters h, w, and z. This feature, together with the correspondence between activation states in the model and locomotory states in the animal, allows one to predict synaptic strengths in the command network from the rate constants for locomotory state transitions in the animal.Using a curve fitting routine, I adjusted h, w, and z to fit the rate constants for switching between locomotory states measured in real animals. I tested the model by increasing or decreasing h to mimic, respectively, depolarization and hyperpolarization of command neurons. I found that dwell time in the forward state was modulated as in real worms. In a second test, I decreased the values of h, w, and z in the forward unit by 50% to mimic ablation of the forward neuron AVB and found, as expected, that forward dwell time was decreased. Remarkably, mimicking ablation of the reverse neuron AVA also decreased forward dwell time. The new model therefore provides a successful alternative to the bistable switch hypothesis. NIH MH51383.

Shawn Lockery et al. (2007) International Worm Meeting "Sensory regulation of command neuron state transitions: Theory and experiment."

Thad Lindsay, Shawn Lockery, Tod Thiele, Nathan Dunn, Serge Faumont

[International Worm Meeting, 2007]

Locomotory state in C. elegans (forward vs. reverse) is thought to be controlled by a network of forward and reverse command neurons in two reciprocally connected pools, but how this network is regulated by sensory input is poorly understood. According to one hypothesis, known as the Stochastic Switch Model, each pool acts as a probabilistic binary unit (OFF = 0, ON = 1). The model assumes that the rate constant for transitions to the ON state (a₀₁) at any point in time t is a sigmoidal function S(x) of net synaptic input I such that a₀₁(t) = a_max S(I(t)) where a_max is a scale factor. According to the Stochastic Switch Model, sensory input to each unit acts to bias the probability of the ON state. In one configuration of the model, modulation of bias is reciprocal. For example, a sensory stimulus that promotes forward locomotion would excite the forward unit and inhibit the reverse unit; a stimulus that promotes reverse locomotion would produce the opposite pattern of synaptic effects. Alternatively, the modulation may be non-reciprocal such that sensory input affects the bias of one unit but not the other. To distinguish between these two configurations in the context of chemotaxis, we presented worms with stepwise changes in the concentration of the chemoattractant NaCl and measured the rate constants for forward-to-reverse and reverse-to-forward transitions. Rate constants were analyzed in terms of a reduced version of the Stochastic Switch Model that enabled us to compute I(t) for each unit using the above equation. We found that in response to upward steps in NaCl, a forward-promoting stimulus, the forward unit was excited whereas the reverse unit was inhibited. This result indicates that upsteps promote forward locomotion by a reciprocal control mechanism. In contrast, we found that in response to downward steps in NaCl, a reverse-promoting stimulus, the forward unit was unaffected whereas the reverse unit was excited. This result indicates that downsteps promote reverse locomotion by a non-reciprocal mechanism. Taken together, these results suggest the hypothesis that locomotory bias in response to chemosensory inputs is established by different control mechanisms depending on stimulus polarity. We plan to test this hypothesis by recording synaptic potentials from command neurons in response to direct activation of chemosensory neurons. Support: NIH MH051383 and NSF IOB-0543643.

Shawn Lockery et al. (2001) Worm Breeder's Gazette "Tight-seal whole-cell patch clamping of C. elegans neurons: erratum"

Miriam Goodman, Shawn Lockery

[Worm Breeder's Gazette, 2001]

We regret to inform the C. elegans community that the published recipe for internal saline for whole-cell recordings[1,2] from neurons was incorrect. The published recipe was (in mM): KGluconate 125, KCl 18, NaCl 0, CaCl2 0.7, MgCl2 1, HEPES 10, EGTA 10. The recipe actually used was (in mM): KGluconate 125, KCl 18, NaCl 4, CaCl2 0.6, MgCl2 1, HEPES 10, EGTA 10. The main effect of this error resides in the difference in NaCl concentration. The correct saline will produce a predicted Na reversal potential of 90 mV with the published external saline, while the erroneous published saline has an undefined ENa. Because C. elegans lacks voltage-gated Na channels, this difference in salines may have little or no effect on recordings of voltage-gated currents. It may, however, affect measurements of currents carried by ligand-gated currents and currents carried by DEG/ENaC channels. We apologize for any inconvenience this error may have caused. 1. Goodman, M.B., Hall, D.H., Avery, L., and Lockery, S.R. (1998) Active Currents Regulate Sensitivity and Dynamic Range in C. elegans Neurons. Neuron 20:763-772. 2. Lockery, S.R. and Goodman, M.B. (1998) Tight-seal whole-cell patch clamping of C. elegans neurons. Methods in Enzymology 295:201-217.

Shawn Lockery et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "A Predictive Model of Locomotory State Transitions in C. elegans"

Steven Augustine, Rebecca Anderson, Shawn Lockery, Kristy Lawton

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Locomotory state in C. elegans is regulated by a network of command neurons in two reciprocally connected pools-the forward neurons AVB and PVC and the reverse neurons AVA and AVD-but how this network functions is poorly understood. We propose a model in which the network functions as a stochastic, bi-stable switch. The model makes three simple assumptions: (A) forward command neurons act as a single unit, as do reverse command neurons; (B) unit activation switches stochastically between two states: 0 (off) and 1 (on); (C) the stochastic processes underlying the state changes of the forward and reverse units are uncorrelated. Accordingly, the network can exist in four states: both units off (00); forward on (10), reverse on (01), and both on (11). Previous neuronal ablations suggest that states 10, 01, and 00 correspond, respectively, to forward locomotion, reverse locomotion, and a pause state; we propose that state 11 is also a pause state because co-activation of forward and reverse motor systems could cause the body musculature to lock up. A key consequence of (C) is that only one unit changes state at a time. This means that transitions between the forward and reverse states (01 to 10, or 10 to 01) must pass through the intermediate state 00 or 11. Thus the model predicts pauses during transitions between forward and reverse locomotion. The model also predicts pauses during apparently continuous bouts of forward or reverse locomotion. To test these predictions, we constructed a novel tracking system capable of recording the worm's speed with a precision of 19 um/sec at a rate of 30 samples/sec. We found clear evidence for pauses, which were typified by a precipitous drop in speed and, after a variable delay, a precipitous rise in speed. Mean dwell time in the pause state was 0.15 sec (SEM = 0.01). Pauses were observed during all transitions between forward and reverse, and also during bouts of forward and reverse locomotion that appeared continuous to the naked eye. These results support the stochastic switch model and suggest revised definitions of fundamental locomotory states in C. elegans.

Shawn R Lockery et al. (2005) International Worm Meeting "Kinetic analysis of locomotory switching in C. elegans"

Shawn R Lockery, Steven B Augustine, Kristy J Lawton

[International Worm Meeting, 2005]

Locomotory state in C. elegans is regulated by a network of command neurons in two functional pools: the forward neurons AVB and PVC, and the reverse neurons AVA and AVD. These two pools are linked by reciprocal synaptic connections, but how this network functions to regulate locomotion is not well understood.In one model, the network functions as a stochastic, bi-stable switch. This model embodies two main assumptions: (1) Forward command neurons act as a single unit, as do reverse command neurons, (2) Units switch stochastically between the on (1) and off (0) states. According to these assumptions, the network can exist in four different states: both units off (00); forward unit on, reverse unit off (10); forward unit off, reverse unit on (01); both units on (11). Previous neuronal ablation studies suggest that state 10 is forward locomotion, state 01 is reverse locomotion, and state 00 is a pause state. Here it is further assumed that state 11 is also a pause state, because co-activation of forward and reverse motor systems might cause the body musculature to lock-up.A key consequence of assumption (2) is that only one unit changes state at a time, because the probability of simultaneous random events is zero. This means that transitions in either direction between the forward and reverse state (10<sym17>01 and 01<sym17>10, respectively) must involve the intermediate states 00 or 11. Thus the model predicts that a worm pauses briefly during transitions between forward and reverse. The model also predicts that brief pauses occur during apparently continuous bouts of forward or reverse locomotion.To test these predictions we videotaped wild type worms moving on foodless agar plates. Frame-by-frame analysis (30 frames/sec) revealed the presence of brief but measurable pauses (mean SD

Izquierdo, Eduardo et al. (2009) International Worm Meeting "A minimal neural network model of klinotaxis behavior in C. elegans."

Lockery, Shawn, Izquierdo, Eduardo

[International Worm Meeting, 2009]

Previous experiments have shown that C. elegans chemotaxis is based in part on a biased random walk. Recently, however, Iino and colleagues have described a complementary strategy, called klinotaxis, in which the direction of locomotion is continuously aligned to the direction of the chemical gradient. Here we combined neural network modeling and mathematical analysis to identify simple circuit motifs for klinotaxis and to understand how they function. The model involves an idealized representation of C. elegans sensory neurons, motor neurons, and neck musculature. The direction of locomotion is determined by the angle of the head with respect to the body, which is set by the difference between the activation levels of dorsal and ventral neck muscles. The circuit has two sensory neurons, representing the chemosensory neurons ASEL and ASER, and two motor neurons collectively representing the dorsal and ventral neck muscle motor neurons. In keeping with functional differences between ASE neurons in the biological network, the sensory neurons in the model respond to the derivative of the attractant concentration such that the model''s ASEL neuron acts like an ON cell, whereas the model''s ASER neuron acts like an OFF cell. The motor neurons are modeled as simple nonlinear dynamical neurons. In addition to input from the sensory neurons, the motor neurons also receive out-of-phase sinusoidal inputs from a central pattern generator, which is not modeled explicitly; however, connections between motor neurons were not included. We used an evolutionary algorithm to optimize synaptic strengths and intrinsic neuronal properties to generate klinotaxis behavior in simulated radial gradients. Connections were constrained to be symmetric across the dorsal-ventral midline. We found that it was possible to generate realistic klinotaxis behavior within the constraints described above. Importantly, the effects of simulated ablations of sensory neurons in the model were consistent with the same ablations in real animals. In particular, ablation of ASER produced a strong chemotaxis deficit whereas ablation of ASEL produced little or no deficit, unless combined with ablation of ASER. Further analysis of the simulated ablations showed that ASEL contributes mainly to the time it takes the worm to reach the gradient peak (efficiency), whereas ASER contributes mainly to the likelihood of reaching the peak (reliability). Finally, a dynamical systems analysis of the motor neurons suggested a phase-dependent sensitivity to input. It is this mechanism that enables the state-dependence necessary for klinotaxis. These findings provide novel hypotheses that can now be tested on real worms.

Katzen, Abraham W. et al. (2013) International Worm Meeting "Electrical recordings of naturalistic feeding behavior in a microfluidic environment."

Katzen, Abraham W., Lockery, Shawn R.

[International Worm Meeting, 2013]

Food choice - the decision of what to eat - is critical to survival and reproduction. To advance the study of food choice in C. elegans, we have devised a microfluidic device for measuring feeding behavior in single worms automatically and accurately in response to a variety of food types under naturalistic conditions. Feeding behavior in C. elegans is quantified by observing the pumping rate of the pharynx. There are currently two main methods for measuring pumping rate: manual observation of slow-motion videos of worms crawling in food, and electrical recordings, known as electropharyngeograms (EPGs). Neither method is practical for experiments requiring naturalistic feeding behavior with precise presentation of food stimuli. Manual observation is labor intensive, limited to short observation periods (~20 sec), and provides little spatiotemporal control of food presentation. EPG recordings, in which a submerged worm is sucked into a hollow recording electrode, necessarily isolate the pharynx from the environment, interfering with delivery of food to the worm. To overcome these limitations we have integrated EPG recording electrodes into an existing microfluidic device [1]. The worm is restrained with the longitudinal axis of its body aligned between two laminar fluid streams, while the head is free to move and explore either stream. The device allows the user to control the delivery, concentration, and type of food that is available to the worm in either fluid stream. Worms exhibit naturalistic feeding behavior in the microfluidic device, and stable recordings lasting 15 minutes are routine. Using this method, we are exploring the feeding response of C. elegans to familiar or novel foods of differing qualities. Additionally, we are determining the effect of food concentration on feeding latency and rate. We anticipate that this method will provide novel insights into the internal and external cues which contribute to adaptive feeding decisions. References: [1] McCormick et al. (2011) PLoS ONE 6(1):e25710.

Katzen, Abraham et al. (2015) International Worm Meeting "Economic decision-making and neural correlates of value in C. elegans."

Harbaugh, William, Katzen, Abraham, Lockery, Shawn

[International Worm Meeting, 2015]

Investigation of the neuronal basis of economic decisions would be accelerated by establishing decision making paradigms in simple, genetically tractable organisms, such as the nematode Caenorhabditis elegans. For an organism to be a valid model of economic decision making its choice behavior must be sensitive to: (i) the difference between high and low quality goods, and (ii) the relative cost of those options.Previous work has shown that the nematode worm C. elegans quickly learns to feed on those foods (species of bacteria) that promote higher rates of growth and reproduction. Worms spend more time foraging in patches of Good bacteria (high worm growth rate) versus Mediocre bacteria (moderate growth rate) when equally abundant. Until now, however, it has not been possible to simultaneously present two food choices of different quality and cost. To that end, we have developed an electro-microfluidic device in which a semi-restrained worm forages between contiguous yet discrete fluid streams containing good and mediocre quality food. This arrangement constitutes a two-alternative forced-choice task, analogous to those used in behavioral economics. Electrodes inserted into the device monitor muscular impulses associated with individual swallowing events. Relative consumption of Good and Mediocre food is measured by counting the number of swallowing events in the respective fluid streams. The fraction of total swallowing events in Good vs Mediocre food serves as an index of food preference. Importantly, we can alter the effective prices of the two foods by adjusting the concentration of the bacteria, with price being inversely related to concentration.Here we present behavioral data delineating preference for Good vs Mediocre food across a range of relative prices. We find that worms exposed to the two species of bacteria at equal prices prefer Good bacteria, indicating that feeding preferences are normal in the device. Worms respond to price adjustments as predicted by economic theory in that increasing the relative price of a food leads to a decline in its consumption. In addition, we present calcium-imaging data from sensory neurons showing that they respond to transitions between Good and Mediocre foods, and the amplitude of calcium signal scales with relative food preference. These results show that C. elegans forages in an economic manner, and that relative value is represented at the level of the sensory neurons.

Levichev, Anastasia et al. (2021) International Worm Meeting "Mechanism for the munchies: endocannabinoid modulation of food preferences in C. elegans."

Lockery, Shawn, Levichev, Anastasia, Faumont, Serge

[International Worm Meeting, 2021]

The mammalian endocannabinoid system, comprised of the endocannabinoids AEA (N-arachidonoyl-ethanolamine) and 2-AG (2-Arachidonoylglycerol), their receptors, CB1 and CB2, and their metabolic enzymes, is thought to integrate internal energy state and sensory food cues to modulate feeding. For example, cannabinoids, acting on CB1, can increase preference for more palatable, calorically dense food: a response called hedonic amplification, colloquially known as "the munchies." In mammals, cannabinoids can increase sensitivity to odors and sweet tastes, which may underlie hedonic amplification. We are developing C. elegans as a model in which to investigate the neurogenetics of hedonic amplification. We have found that exposure to AEA, an endocannabinoid common to mammals and C. elegans, increases the worm's preference for strongly preferred (more palatable) bacteria over weakly preferred (less palatable) bacteria, mimicking hedonic amplification in mammals. Furthermore, AEA acts bidirectionally, increasing consumption of strongly preferred bacteria while decreasing consumption of weakly preferred bacteria. We also found that deletion of the CB1 ortholog, NPR-19, eliminates hedonic amplification, which can be rescued by expression of the human CB1 receptor, establishing a humanized worm for cannabinoid signaling studies. Deletion of the olfactory neuron AWC, which directs chemotaxis to food, abolishes hedonic amplification. Consistent with this finding, calcium imaging revealed that AEA bidirectionally modulates AWC's activity, increasing its responses to strongly preferred food and decreasing its response for weakly preferred food. Furthermore, AEA's effect on AWC requires NPR-19. However, GFP expression analysis revealed that NPR-19 is expressed ~21 neuron classes but, surprisingly, not in AWC. Although AEA's effect could be mediated by NPR-19-expressing neurons presynaptic to AWC, nearly complete elimination of fast synaptic transmission, via a mutation in unc-13, has no effect on modulation. Instead, AEA's effect on AWC is mediated via unc-31-dependent dense-core vesicle release. We are now working to identify the NPR-19-expressing neurons that pass the cannabinoid signal down to AWC, thereby modulating chemosensation and leading to hedonic amplification.

Lanier, Vincent et al. (2021) International Worm Meeting "High-throughput EPG recordings reveal the food exploitation-exploration trade-off"

Lanier, Vincent, Lockery, Shawn, Millet, Jonathan, Weeks, Janis, Roberts, William

[International Worm Meeting, 2021]

Whole-animal screens complement traditional screens based on cultured cells and single-celled organisms. In C. elegans, two of the most common whole-animals screens involve locomotion and pharyngeal pumping. Whereas high-throughput locomotion screens are in wide use, high-throughput pharyngeal screens are lacking. Electrophysiological measures of pharyngeal activity - electropharyngeograms (EPGs) - offer higher temporal resolution and signal to noise ratio than manual or optical methods. Recently, the ease and efficiency of EPGs was improved by placing individual worms in tight-fitting microchannels. Electrical resistance formed where the worm contacts the channel walls generates voltage differences sufficient to resolve individual pharyngeal action potentials. However, this approach is currently limited to only eight worms per recording. To address this deficiency, we explored the utility of bulk EPG recordings made by increasing the length and width of the recording channel to accommodate hundreds or even thousands of worms. In bulk recordings, where worms are loosely arrayed in the channel and unrestrained, the voltage signal produced by each worm is insufficient to resolve individual action potentials. However, the composite voltage signal, which can be conceptualized as the sum of many individual, asynchronous EPGs, can be analyzed by computing its power spectrum. We found that EPG power spectra generally have just two peaks: a low frequency peak at 0-2 Hz, and a high frequency peak at 4-6 Hz. Using serotonin dose-response curves, optogenetic inhibition of pharyngeal muscles, and pharyngeal pumping mutants, we found that the high frequency peak reflects neurogenic pumping whereas the low frequency peak reflects myogenic pumping together with locomotion. As serotonin concentration is increased, the high and low frequency peaks are enlarged and diminished, respectively. We propose that the high frequency peak reflects the proportion of worms that are nearly stationary but feeding vigorously (exploitation), whereas the low frequency peak reflects the proportion of worms that are moving rapidly but feeding intermittently (exploration). Thus, the new method quantifies not only neurogenic pharyngeal pumping, but also the distribution of animals across these mutually exclusive behavioral states.