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The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability. • Voltron2 is an improved version of the chemigenetic voltage indicator Voltron • A point mutation enhances the sensitivity of several rhodopsin-based sensors • Voltron2 was tested in vivo in flies, fish, and mice Abdelfattah et al. describe a point mutation that increases the sensitivity of voltage indicators. A new voltage indicator carrying that mutation (Voltron2) is found to have improved sensitivity over its predecessor. Voltron2 is validated in vivo and used to study the subthreshold activity of interneurons in the living mouse brain. [ABSTRACT FROM AUTHOR]

Summary: Calcitonin gene-related peptide (CGRP) is a classic molecular marker of peptidergic primary somatosensory neurons. Despite years of research, it is unknown whether these neurons are required to sense pain or other sensory stimuli. Here, we found that genetic ablation of CGRPα-expressing sensory neurons reduced sensitivity to noxious heat, capsaicin, and itch (histamine and chloroquine) and impaired thermoregulation but did not impair mechanosensation or β-alanine itch—stimuli associated with nonpeptidergic sensory neurons. Unexpectedly, ablation enhanced behavioral responses to cold stimuli and cold mimetics without altering peripheral nerve responses to cooling. Mechanistically, ablation reduced tonic and evoked activity in postsynaptic spinal neurons associated with TRPV1/heat, while profoundly increasing tonic and evoked activity in spinal neurons associated with TRPM8/cold. Our data reveal that CGRPα sensory neurons encode heat and itch and tonically cross-inhibit cold-responsive spinal neurons. Disruption of this crosstalk unmasks cold hypersensitivity, with mechanistic implications for neuropathic pain and temperature perception. [ABSTRACT FROM AUTHOR]