RubyACRs enable red-shifted optogenetic inhibition in freely behaving Drosophila

Optogenetic neuronal 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 do not 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.