Population imaging of neural activity in awake behaving mice (original) (raw)

A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here we describe a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and is compatible with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, and number of neurons observable several-fold over previously published fully genetically encoded reagents 1-8. Under conventional one-photon microscopy, SomArchon enables the routine population analysis of around 13 neurons at once, in multiple brain regions (cortex, hippocampus, and striatum) of head-fixed, awake, behaving mice. Using SomArchon, we detected both positive and negative responses of striatal neurons during movement, as previously reported by electrophysiology but not easily detected using modern calcium imaging techniques 9-11 , highlighting the power of voltage imaging to reveal bidirectional modulation. We also examined how spikes relate to the subthreshold theta oscillations of individual hippocampal neurons, with SomArchon showing that the spikes of individual neurons are more phase-locked to their own subthreshold theta oscillations than to local field potential theta oscillations. Thus, SomArchon reports both spikes and subthreshold voltage dynamics in awake, behaving mice. Near-infrared genetically encoded voltage indicators (GEVIs) derived from rhodopsins offer high temporal fidelity, and are compatible with optogenetics 1,12,13 , whereas green fluorescent GEVIs derived from the voltage-sensing domains of phosphatases or opsins are often slower and brighter 2,3,14-17. Translating these voltage sensors into the living mammalian brain has been challenging, because of poor membrane localization, low photostability, and low signal-to-noise ratio (SNR). So far, amongst fully genetically encoded reagents, only Ace2N and paQuasAr3-s have been used to optically report voltage dynamics in a living mouse brain, reporting the activities of up to four cells in one field of view (FOV) in awake mice 4,17. Recently, we developed a robotic directed-evolution approach and created the improved GEVI Archon1 13. To further improve SNR in the dense, living mammalian brain, we conducted a screen for peptides to localize Archon1 to the soma 18-21 , so that neuropil contamination could be reduced (Extended Data Fig. 1; see Supplementary Table 1 for the sequences of the motifs). The molecule Archon1-KGC-EGFP-K V 2.1-motif-ER2, which we call SomArchon (Fig. 1a), exhibited the highest relative change in fluorescence (ΔF/F) during 100-mV voltage steps (Fig. 1g) and was welllocalized to the soma (Extended Data Fig. 1h-k). SomArchon fluorescently reported action potentials in mouse brain slices after in utero electroporation (IUE) into the cortex and hippocampus, and after adeno-associated virus (AAV)-mediated expression in the cortex, striatum, and thalamus (Extended Data Fig. 2). SomArchon was localized primarily to the membrane within 30-45 μm