A latent pool of neurons silenced by sensory-evoked inhibition can be recruited to enhance perception.

To investigate which activity patterns in sensory cortex are relevant for perceptual decision-making, we combined two-photon calcium imaging and targeted two-photon optogenetics to interrogate barrel cortex activity during perceptual discrimination. We trained mice to discriminate bilateral whisker deflections and report decisions by licking left or right. Two-photon calcium imaging revealed sparse coding of contralateral and ipsilateral whisker input in layer 2/3, with most neurons remaining silent during the task. Activating pyramidal neurons using two-photon holographic photostimulation evoked a perceptual bias that scaled with the number of neurons photostimulated. This effect was dominated by optogenetic activation of non-coding neurons, which did not show sensory or motor-related activity during task performance. Photostimulation also revealed potent recruitment of cortical inhibition during sensory processing, which strongly and preferentially suppressed non-coding neurons. Our results suggest that a pool of non-coding neurons, selectively suppressed by network inhibition during sensory processing, can be recruited to enhance perception.

The influence of cortical activity on perception depends on behavioral state and sensory context.

The mechanistic link between neural circuit activity and behavior remains unclear. While manipulating cortical activity can bias certain behaviors and elicit artificial percepts, some tasks can still be solved when cortex is silenced or removed. Here, mice were trained to perform a visual detection task during which we selectively targeted groups of visually responsive and co-tuned neurons in L2/3 of primary visual cortex (V1) for two-photon photostimulation. The influence of photostimulation was conditional on two key factors: the behavioral state of the animal and the contrast of the visual stimulus. The detection of low-contrast stimuli was enhanced by photostimulation, while the detection of high-contrast stimuli was suppressed, but crucially, only when mice were highly engaged in the task. When mice were less engaged, our manipulations of cortical activity had no effect on behavior. The behavioral changes were linked to specific changes in neuronal activity. The responses of non-photostimulated neurons in the local network were also conditional on two factors: their functional similarity to the photostimulated neurons and the contrast of the visual stimulus. Functionally similar neurons were increasingly suppressed by photostimulation with increasing visual stimulus contrast, correlating with the change in behavior. Our results show that the influence of cortical activity on perception is not fixed, but dynamically and contextually modulated by behavioral state, ongoing activity and the routing of information through specific circuits.

All-optical interrogation of neural circuits in behaving mice.

Recent advances combining two-photon calcium imaging and two-photon optogenetics with computer-generated holography now allow us to read and write the activity of large populations of neurons in vivo at cellular resolution and with high temporal resolution. Such 'all-optical' techniques enable experimenters to probe the effects of functionally defined neurons on neural circuit function and behavioral output with new levels of precision. This greatly increases flexibility, resolution, targeting specificity and throughput compared with alternative approaches based on electrophysiology and/or one-photon optogenetics and can interrogate larger and more densely labeled populations of neurons than current voltage imaging-based implementations. This protocol describes the experimental workflow for all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system (~3 h), the preparation of an indicator and opsin-expressing and task-performing animal (~3-6 weeks), the characterization of functional and photostimulation responses (~2 h per field of view) and the design and implementation of an all-optical experiment (achievable within the timescale of a normal behavioral experiment; ~3-5 h per field of view). We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy in three brain areas by using different experimental setups. This approach can in principle be adapted to any brain area to probe functional connectivity in neural circuits and investigate the relationship between neural circuit activity and behavior.

Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo.

We describe an all-optical strategy for simultaneously manipulating and recording the activity of multiple neurons with cellular resolution in vivo. We performed simultaneous two-photon optogenetic activation and calcium imaging by coexpression of a red-shifted opsin and a genetically encoded calcium indicator. A spatial light modulator allows tens of user-selected neurons to be targeted for spatiotemporally precise concurrent optogenetic activation, while simultaneous fast calcium imaging provides high-resolution network-wide readout of the manipulation with negligible optical cross-talk. Proof-of-principle experiments in mouse barrel cortex demonstrate interrogation of the same neuronal population during different behavioral states and targeting of neuronal ensembles based on their functional signature. This approach extends the optogenetic toolkit beyond the specificity obtained with genetic or viral approaches, enabling high-throughput, flexible and long-term optical interrogation of functionally defined neural circuits with single-cell and single-spike resolution in the mouse brain in vivo.