Fig. 1: Visual stimulus detection is modulated by behavioral state.

a Experiment schematic. Mice coexpress GCaMP6s and C1V1-Kv2.1 in excitatory neurons of L2/3 V1 enabling simultaneous two-photon calcium imaging and two-photon holographic stimulation. Mice are head-fixed and trained to perform a visual stimulus detection task. Pupil size is recorded with a camera. b Timeline of animal preparation, training, and experiment. c Behavioral trial structure. After withholding licks for a randomized interval (4 ± 3 s) a stimulus (visual and/or optogenetic) is presented to the mouse. When a visual stimulus is presented the mouse has 1.5 s to lick the lickometer in order to receive a water reward. Neural analysis is performed in the 1 s immediately following stimulus offset (to avoid photostimulation artifact) using the 1 s immediately prior to the stimulus as baseline. The state of the animal is measured in the 4 s preceding the delivery of the stimulus. d Behavioral session structure. 12 different trial types are presented to the mouse in a pseudorandom blocked structure. Visual-only trials (40% of session) of varying contrast (1%, 2%, 5%, 10% and 100%) are interleaved with Visual+Photostimulation trials (40% of session) where a 1 s 20 Hz photostimulation is delivered coincident with the visual stimulus. Catch trials (20% of session) with and without photostimulation are delivered to assess chance licking probabilities. Any trial with a visual stimulus is rewarded if the mouse responds during the response window. e Example lick raster plot. Trials were delivered pseudorandomized but are shown sorted by stimulus contrast. Gray dots indicate lick responses, with the first lick (reaction time) highlighted in black. Right: the simultaneously recorded pre-trial pupil size, neuronal synchrony and running speed are shown for each trial. Orange indicates large values, blue indicates small values. f Pupillometry is performed throughout the behavioral session. Large and small pupil sizes are seen (left) reflecting different behavioral states. Stimulus triggered average pupil traces are averaged across all hit or miss trials of threshold stimuli, and then averaged across sessions (middle). On visual-only threshold trials hits (mice licked within response window) are associated with a larger pupil prior to the stimulus delivery (right). Error bars show mean ± SEM across sessions, n = 28 sessions, 12 mice. g Neuronal synchrony in the pre-trial period is computed as the average pairwise correlation between all pairs of cells’ deconvolved activity traces. Periods of low and high synchrony are seen (left) reflecting different behavioral states. Stimulus triggered average correlation traces are averaged across all hit or miss threshold trials, and then averaged across sessions. Note the traces are made with a 4 s sliding window hence the synchrony appears to increase before the stimulus is delivered (middle). On threshold contrast visual-only trials hits are associated with a lower level of synchrony prior to the stimulus delivery (right). Error bars show mean ± SEM across sessions, n = 28 sessions, 12 mice. h Each session is median-split into two collections of interspersed trials, based on pupil size (normalized relative to the median size in the session) and network synchrony (average pairwise Pearson’s correlation coefficient between all recorded neurons) on each trial preceding stimulus delivery. The more engaged state contains trials with the largest pupil sizes and lowest neuronal synchrony. The less engaged state contains trials with the smallest pupil sizes and highest neuronal synchrony. Each dot represents the average pupil size and synchrony for that state of a given session. Gray lines connect the two states in a given session (n = 28 sessions, 12 mice). i The behavior of the mice differs in the two states. The “more engaged” state, the state with large pupil size and low neuronal synchrony, is associated with higher stimulus detection rates, as expressed by higher d-prime values. Error bars show mean ± SEM across sessions. j The psychometric function is steeper (left) and more sensitive (right) when the animal is engaged. Error bars show mean ± SEM across sessions, n = 28 sessions, 12 mice. The insets above the plots illustrate the measurement of psychometric curve width (left) and threshold (right).