RubyACRs Enable Red-Shifted Optogenetic Inhibition in Freely Behaving Drosophila

Abstract Optogenetic activators with red-shifted excitation spectra, such as Chrimson, have significantly advanced Drosophila neuroscience. However, until recently, available optogenetic inhibitors required shorter activation wavelengths, which don’t penetrate tissue as effectively and are stronger visual stimuli to the animal, potentially confounding behavioral results. Here, we assess the efficacy of two newly identified anion-conducting channelrhodopsins with spectral sensitivities similar to Chrimson: A1ACR and HfACR (RubyACRs). Electrophysiology and functional imaging confirmed that RubyACRs effectively hyperpolarize neurons, with stronger and faster effects than the widely used inhibitor GtACR1. Activation of RubyACRs led to circuit-specific behavioral changes in three different neuronal groups. In glutamatergic motor neurons, activating RubyACRs suppressed adult locomotor activity. In PPL1-γ1pedc dopaminergic neurons, pairing odors with RubyACR activation during learning produced odor responses consistent with synaptic silencing. Finally, activation of RubyACRs in the pIP10 neuron suppressed pulse song during courtship. Together, these results demonstrate that RubyACRs are effective and reliable tools for neuronal inhibition in Drosophila, expanding the optogenetic toolkit for circuit dissection in freely behaving animals.