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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. Their expression must also 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 addresses 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. In every neuron we express a nonconventional optical actuator, the gustatory receptor homolog GUR-3 + PRDX-2, under the control of a drug-inducible system QF + hGR, and a calcium indicator GCAMP6s, in a background with additional fluorophores from 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. 

Abstract Establishing how neural function emerges from network properties is a fundamental problem in neuroscience¹. Here, to better understand the relationship between the structure and the function of a nervous system, we systematically measure signal propagation in 23,433 pairs of neurons across the head of the nematodeCaenorhabditis elegansby direct optogenetic activation and simultaneous whole-brain calcium imaging. We measure the sign (excitatory or inhibitory), strength, temporal properties and causal direction of signal propagation between these neurons to create a functional atlas. We find that signal propagation differs from model predictions that are based on anatomy. Using mutants, we show that extrasynaptic signalling not visible from anatomy contributes to this difference. We identify many instances of dense-core-vesicle-dependent signalling, including on timescales of less than a second, that evoke acute calcium transients—often where no direct wired connection exists but where relevant neuropeptides and receptors are expressed. We propose that, in such cases, extrasynaptically released neuropeptides serve a similar function to that of classical neurotransmitters. Finally, our measured signal propagation atlas better predicts the neural dynamics of spontaneous activity than do models based on anatomy. We conclude that both synaptic and extrasynaptic signalling drive neural dynamics on short timescales, and that measurements of evoked signal propagation are crucial for interpreting neural function.