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.

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.

How many neurons are sufficient for perception of cortical activity?

Many theories of brain function propose that activity in sparse subsets of neurons underlies perception and action. To place a lower bound on the amount of neural activity that can be perceived, we used an all-optical approach to drive behaviour with targeted two-photon optogenetic activation of small ensembles of L2/3 pyramidal neurons in mouse barrel cortex while simultaneously recording local network activity with two-photon calcium imaging. By precisely titrating the number of neurons stimulated, we demonstrate that the lower bound for perception of cortical activity is ~14 pyramidal neurons. We find a steep sigmoidal relationship between the number of activated neurons and behaviour, saturating at only ~37 neurons, and show this relationship can shift with learning. Furthermore, activation of ensembles is balanced by inhibition of neighbouring neurons. This surprising perceptual sensitivity in the face of potent network suppression supports the sparse coding hypothesis, and suggests that cortical perception balances a trade-off between minimizing the impact of noise while efficiently detecting relevant signals.

Closed-loop all-optical interrogation of neural circuits in vivo.

Understanding the causal relationship between neural activity and behavior requires the ability to perform rapid and targeted interventions in ongoing activity. Here we describe a closed-loop all-optical strategy for dynamically controlling neuronal activity patterns in awake mice. We rapidly tailored and delivered two-photon optogenetic stimulation based on online readout of activity using simultaneous two-photon imaging, thus enabling the manipulation of neural circuit activity 'on the fly' during behavior.

Stratification of MDD and GAD patients by resting state brain connectivity predicts cognitive bias.

Patients with Generalized Anxiety Disorder (GAD) and Major Depressive Disorder (MDD) show between-group comorbidity and symptom overlap, and within-group heterogeneity. Resting state functional connectivity might provide an alternate, biologically informed means by which to stratify patients with GAD or MDD. Resting state functional magnetic resonance imaging data were acquired from 23 adults with GAD, 21 adults with MDD, and 27 healthy adult control participants. We investigated whether within- or between-network connectivity indices from five resting state networks predicted scores on continuous measures of depression and anxiety. Successful predictors were used to stratify participants into two new groups. We examined whether this stratification predicted attentional bias towards threat and whether this varied between patients and controls. Depression scores were linked to elevated connectivity within a limbic network including the amygdala, hippocampus, VMPFC and subgenual ACC. Patients with GAD or MDD with high limbic connectivity showed poorer performance on an attention-to-threat task than patients with low limbic connectivity. No parallel effect was observed for control participants, resulting in an interaction of clinical status by resting state group. Our findings provide initial evidence for the external validity of stratification of MDD and GAD patients by functional connectivity markers. This stratification cuts across diagnostic boundaries and might valuably inform future intervention studies. Our findings also highlight that biomarkers of interest can have different cognitive correlates in individuals with versus without clinically significant symptomatology. This might reflect protective influences leading to resilience in some individuals but not others.