Dual-polarity voltage imaging of the concurrent dynamics of multiple neuron types

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR. Our indicators enable 0.4- to 1-kilohertz voltage recordings from >50 spiking neurons per field of view in awake mice and ~30-minute continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered brain state–dependent antagonism between neocortical somatostatin-expressing (SST+) and vasoactive intestinal peptide–expressing (VIP+) interneurons and contributions to hippocampal field potentials from cell ensembles with distinct axonal projections. By combining three mutually compatible indicators, we performed simultaneous triple-population imaging. These approaches will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution. Description Real-time voltage imaging of neurons How different subpopulations of neurons coordinate their activity patterns in real time to control network output is still not fully understood. This is in part because of our inability to simultaneously measure the voltage dynamics within and across targeted cell ensembles. Kannan et al. developed a suite of mutually compatible, genetically encoded voltage indicators for high-speed voltage imaging of multiple genetically identified neurons in awake behaving animals. These indicators can be uniquely combined for simultaneous recordings of spiking by ensembles of neurons. This technical advance enables the first comprehensive studies of the real-time, millisecond-scale interactions between neurons of distinct genetic classes. —PRS A new suite of voltage sensors enables simultaneous cellular-resolution neuronal activity imaging in awake mice and flies. INTRODUCTION Signal processing in the brain involves the concerted dynamics of multiple distinct cell subtypes that belong to excitatory and inhibitory neuron populations. To uncover how they synergize their activities in real time, it is important to record the voltage dynamics of multiple identified subtypes simultaneously. Genetically encoded fluorescent voltage indicators (GEVIs) are ideally suited to reveal the millisecond-scale activation dynamics of one or more genetically identified neuron types in awake animals. RATIONALE We previously introduced the green and red fluorescence resonance energy transfer (FRET)–opsin GEVIs Ace-mNeon and VARNAM, which are fusion proteins of voltage- sensitive rhodopsin from Acetabularia with bright fluorescent proteins mNeonGreen and mRuby3, respectively. Ace-mNeon and VARNAM exhibit fluorescence decreases during voltage depolarization events such as action potentials (APs) and together constitute a pair of mutually compatible GEVIs that can be genetically targeted to two distinct neuron types for simultaneous dual-color voltage recordings. Using protein engineering and high-throughput voltage screening, we uncovered the second-generation GEVIs Ace-mNeon2 and VARNAM2, which have improved voltage sensitivities. We also identified the respective reverse response-polarity variants, pAce and pAceR, which exhibit fluorescence increases during APs. We reasoned that the four new indicators make up a suite of mutually compatible tools for multipopulation voltage recordings in behaving animals. When targeted to distinct neuron types, the indicators enable unambiguous cell-type identification from the voltage readout based on their fluorescence spectra, optical spike polarity, or both. RESULTS Our improved indicators exhibit enhanced voltage sensitivities to enable low-power (<25 mW mm−2) recordings of spiking and transmembrane voltage dynamics from >50 individual neurons at a time in running mice and ~30-min continuous imaging in flies. Ace-mNeon2 voltage imaging from ~1200 neurons revealed visual cortical cell type–specific responses to behavioral-state transitions in awake mice. Cortical interneurons expressing somatostatin (SST) exhibited the most profound activation during a quiescence-to-arousal transition. Further, using simultaneous dual-polarity voltage recordings with Ace-mNeon2 and pAce, we examined the functional dynamics of pairs of neuron types. We unveiled a time-varying, state-dependent mutual antagonism in the voltage dynamics of SST- and vasoactive intestinal peptide (VIP)–expressing interneurons. In the hippocampus, dual-polarity voltage imaging uncovered the differential contributions of distinct cell classes to the local field potential (LFP). Spiking in entorhinal cortex (EC)–projecting neurons, but not in SST interneurons, consistently phase-locked to the LFP. Moreover, using dual-color voltage imaging, we found that anterior cingulate cortex (ACC)– and EC-projecting excitatory subclasses differ in their responses to state change; the former uniformly increase their firing rates, but the latter do not. Finally, by combining dual-polarity and dual-color imaging using three mutually compatible sensors from our suite, we extracted the simultaneous, real-time voltage dynamics of three neuron types. These subtypes belong to excitatory and inhibitory interneuronal subpopulations in the visual cortex and hippocampus of running mice. CONCLUSION Our work reveals functional correlations between targeted neuron types in visual cortical circuits during altered behavioral states as well as the contributions of distinct cell subclasses to hippocampal LFPs in running mice. Together, our findings benchmark multipopulation voltage imaging for investigations of the concerted dynamics of multiple cell classes in behaving animals. Dual-polarity imaging of the simultaneous voltage dynamics of two neuron types in running mice. Newly characterized negative- and positive-polarity GEVIs enable unambiguous identification of neuron types based on the optical spike polarity in single-color voltage imaging. Using this technique, we uncover a state-dependent antagonism between visual cortical SST and VIP interneurons as well as relative contributions to the LFP from hippocampal excitatory and inhibitory neurons. N81S, Asn81→Ser; R78K, Arg78→Lys; S81D, Ser81→Asp; D92N, Asp92→Asn; W178F, Trp178→Phe; LED, light-emitting diode; PN, pyramidal neuron.