TWISP: A Transgenic Worm for Interrogating Signal Propagation in C. elegans

Abstract

Genetically encoded optical indicators and actuators of neural activity allow for all-optical investigations of signaling in the nervous system. But commonly used indicators, actuators and expression strategies are poorly suited for systematic measurements of signal propagation at brain scale and cellular resolution. Large scale measurements of the brain require indicators and actuators with compatible excitation spectra to avoid optical crosstalk. They must be highly expressed in every neuron but at the same time avoid lethality and permit the animal to reach adulthood. And finally, their expression must be compatible with additional fluorescent labels to locate and identify neurons, such as those in the NeuroPAL cell identification system. We present TWISP, a Transgenic Worm for Interrogating Signal Propagation, that address these needs and enables optical measurements of evoked calcium activity at brain scale and cellular resolution in the nervous system of the nematode Caenorhabditis elegans. We express in every neuron a non-conventional optical actuator, the gustatory receptor homolog GUR-3+PRDX-2 under the control of a drug-inducible system QF+hGR, and calcium indicator GCAMP6s, in a background with additional fluorophores of the NeuroPAL cell ID system. We show that this combination, but not others tested, avoids optical-crosstalk, creates strong expression in the adult, and generates stable transgenic lines for systematic measurements of signal propagation in the worm brain.

Keywords: C. elegans; Dexamethasone; GUR-3; PRDX-2; calcium imaging; drug-inducible gene expression; functional connectivity; neurons; optogenetics.

Figure 1.. Strategy for all-neuron random-access stimulation and calcium imaging without optical cross talk.

a.) Schematic of a transgenic C. elegans for measuring signal propagation via optogenetic stimulation and calcium imaging. b.) Previously reported action spectra for several neural actuators, compared with the absorbance spectra of GCaMP6s also shown for comparison. Adapted from: (Chen et al. 2013; Husson et al. 2013; Klapoetke et al. 2014; Bhatla and Horvitz 2015; Dana et al. 2016). 505 nm light is close to GCaMP6s absorbance peak at 498 nm. c.) Optogenetic proteins were expressed in every neuron under a rab-3 promotor. Behavior response to 1.5 mW/mm² illumination of either 505 nm or 475 nm is shown. For animals expressing rhodopsin-based optogenetic proteins, behavior is measured with and without the necessary co-factor all-trans retinal (ATR). Actuators in blue show good compatibility with GCaMP imaging wavelengths.

Figure 2.. High concentration injections of actuator-containing plasmids are not viable for transgenesis, but higher expression is desirable.

a.) Injection concentration for plasmids containing either GUR-3+PRDX-2 or TsChR and the viability of transgene expressing progeny is shown. b.) Light-evoked behavior response of worms expressing TsChR (50 ng/μl injection) in all neurons upon 1.5 mW/mm² 475 nm blue light illumination, with and without the cofactor ATR (All-trans-retinal). Most worms had very low expression, based on fluorescent reporter marker. Rare worms with high expression showed stronger responses.

Figure 3.. Drug inducible expression enables robust light response while avoiding lethality

a.) Injections of plasmids containing actuators under the control of the QF+hGR drug inducible expression system are viable at higher injection concentrations (black filled shapes) than injections of plasmids for direct expression of the actuators (gray open shapes, same as Fig 2). b.) Exposure to the drug dexamethasone (Dex) evokes actuator expression and confers robust light-response to 475 nm illumination.

Figure 4.. Drug induced actuator expression modulates health and growth.

Animals are observed as they are propagated on plates across multiple generations either on or off the drug dexamethasone. Observations of the animal’s health and developmental stage are made at time points indicated by the circles. For each observation, the developmental stage of the most developmentally advance animal found on the plate is reported. Health and generation times improve for the progeny of animals that had previously been exposed to the drug.

Figure 5.. Rate of growth and progeny production decrease with transgenic load and actuator expression.

a) Rate of progeny and b) percentage of animals that reach adulthood in 70 hrs or c) in 94 hrs is reported for several strains carrying various components of the TWISP system. Box shows median (line) and 25% percentile and 75% percentile (bottom and top) values, whiskers show min and max values. Exact values and number of plates are reported in Supplementary Tables S3, S4 and S5. Statistical significance is with respect to WT, using Kruskal-Wallis followed by Dunn’s multiple comparisons, *<0.03, **<0.002, ***<0.0002 & ****<0.0001.

Figure 6.. Locomotion decreases with transgenic load and actuator expression.

a) Speed b) reversal rate c) body length and d) fraction of time paused is reported for animals from strains containing various components of the TWISP system. The TWISP strain is measured with and without dexamethasone treatment. The combination of NeuroPAL and GCaMP expression decreases locomotion. Box and whisker plots report the distribution of behavior across animal tracks (not plates). The number of tracks recorded per condition, from left to right, are N= [1706, 304, 1283, 1940, 393, 1374, 723, 647]. Box indicates median and interquartile range. Whiskers indicate range excluding outliers. Mean+/−SD values and number of plates are reported in Supplementary Table S6. We performed a Kolmogorov-Smirnov statistical test for all conditions compared to WT. All tests were significant (p<10⁻⁵) after accounting for multiple hypothesis testing via Bonferroni correction.

Figure 7.. Population calcium activity in response to neural activation measured with TWISP.

Calcium activity of simultaneously recorded identified neurons are shown during targeted optogenetic stimulation of individual neurons. Neuron identities are listed on the left. Gray vertical line and red thunderbolt indicate the timing and targeting of two-photon optogenetic stimulation. The name of the neuron stimulated is listed above. A thunder bolt is shown on top for those instances in which a neuron was stimulated but that neuron’s identity was not determined unambiguously. Unidentified neurons are excluded from the plot.