Extended Data Figure 4: Real-time imaging of neural ensembles in three-dimensional hippocampal volumes: extraction of neural sources and identification of significant transients.

a, Head-fixed virtual reality setup. Mice run on an axially fixed track ball³¹ while movements and licking behaviour are measured through an optical mouse and a lickometer, respectively, both interfaced with the virtual-reality gaming software. For contextual fear conditioning, water-restricted mice were exposed to two contexts with distinct visual, olfactory, tactile and auditory cues (day 1), and provided with aversive air puffs in one context (fear context), but not the other (neutral context) (day 2). Fear memory retrieval in the two contexts was quantified (days 3, 30) by lick suppression. b, Sample mean intensity z projections from raw videos (scale bar: 50 μm), with extracted neural sources (segmented cells) from CA3 for each of the optical sections, along with the first 50 time-series traces. Scale: 300% ΔF/F, 30 s. c, Identification of significant transients in ΔF/F traces. Histogram showing the distribution of events occurring at amplitude 2σ above noise (noise calculated on a per-cell basis), over a range of event duration in seconds. The number of negative-going transients at each amplitude and duration are plotted in red to the left of the ordinate, and positive-going transients at each amplitude and duration are plotted in blue to the right. d, e, The above analysis is repeated for events that occur at an amplitude of 3σ (d) and 4σ (e). f, False positive rates for 2-, 3-, and 4-σ events (pooled across all neurons in all mice over all FOVs). False positive rate curves were calculated for each σ level by dividing the number of negative events at that level by the number of positive events at that level (Methods). Event onset was defined as the time corresponding to ΔF/F exceeding 2σ, and offset as the time corresponding to ΔF/F falling below 0.5σ. A decaying exponential was fit by least-squares to the false positive rate values, allowing for the determination of a minimum transient duration at each σ level for different confidence levels.