We describe an all-optical strategy for simultaneously manipulating and recording the activity of multiple neurons with cellular resolution in vivo. Concurrent two-photon optogenetic activation and calcium imaging is enabled 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 optogenetic activation, while simultaneous fast calcium imaging provides high-resolution network-wide readout of the manipulation with negligible optical crosstalk. 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 mammalian brain 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 tailor and deliver 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.

Many theories of brain function assume that information is encoded and behaviour is controlled through sparse, distributed patterns of activity. It is therefore crucial to place a lower bound on the amount of neural activity that can drive behaviour and to understand how neuronal networks operate within these constraints. We use an all-optical approach to test this lower limit by driving behaviour with targeted two-photon optogenetic activation of small ensembles of L2/3 pyramidal neurons in mouse barrel cortex while using two-photon calcium imaging to record the impact on the local network. By precisely titrating the number of neurons in activated ensembles we demonstrate that the lower bound for detection of cortical activity is ~14 pyramidal neurons. We show that there is a very steep sigmoidal relationship between the number of activated neurons and behavioural output, saturating at only ~37 neurons, and that this relationship can shift with learning. By simultaneously measuring activity in the local network, we show that the activation of stimulated ensembles is balanced by the suppression of neighbouring neurons. This surprising behavioural sensitivity in the face of potent network suppression supports the sparse coding hypothesis and suggests that perception of cortical activity balances a trade-off between minimizing the impact of noise while efficiently detecting relevant signals.

Highlights d Two-photon optogenetics in VR enables targeted manipulation of place cell ensembles d Activating specific place cell ensembles drives their spatially associated behavior d Place cell stimulation inhibits endogenous place code expression and triggers remapping d Direct evidence for a causal role of place cells in spatial navigation

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Correspondence to Michael Häusser: m.hausser@ucl.ac.uk Our understanding of the link between neural activity and perception remains incomplete. Microstimulation and optogenetic experiments have shown that manipulating cortical activity can influence sensory-guided behaviour or elicit artificial percepts. And yet, some perceptual tasks can still be solved when sensory cortex is silenced or removed, suggesting that cortical activity may not always be essential. Reconciling these findings, and providing a quantitative framework linking cortical activity and behaviour, requires knowledge of the identity of the cells being activated during the behaviour, the engagement of the local and downstream networks, and the cortical and behavioural state. Here, we performed two-photon population calcium imaging in L2/3 primary visual cortex (V1) of headfixed mice performing a visual detection task while simultaneously activating specific groups of neurons using targeted two-photon optogenetics during low contrast visual stimulation. Only activation of groups of cells with similar tuning to the relevant visual stimulus led to a measurable bias of detection behaviour. Targeted photostimulation revealed signatures of centre-surround, predominantly inhibitory and like-to-like connectivity motifs in the local network which shaped the visual stimulus representation and partially explained the change in stimulus detectability. Moreover, the behavioural effects depended on overall performance: when the task was challenging for the mouse, V1 activity was more closely linked to performance, and cortical stimulation boosted perception. In contrast, when the task was easy, V1 activity was less informative about performance and cortical stimulation suppressed stimulus detection.Altogether, we find that both the selective routing of information through functionally specific circuits, and the prevailing cortical state, make similarly large contributions to explaining the behavioural response to photostimulation. Our results thus help to reconcile contradictory findings about the involvement of primary sensory cortex in behavioural tasks, suggesting that the influence of cortical activity on behaviour is dynamically reassigned depending on the demands of the task.Understanding the relationship between cortical activity and perception remains one of the most fundamental

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 impact of functionally defined neurons on neural circuit function and behavioural output with new levels of precision. This greatly increases the 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 labelled populations of neurons than current voltage imaging-based implementations. This protocol describes the experimental workflow for all-optical

Spontaneous bacterial peritonitis (SBP) is a severe complication of liver disease. A significant proportion of patients have culture-negative ascites, despite having similar signs, symptoms and mortality to those with SBP. Therefore, empirical antibiotic treatment for infection is often started without knowledge of the causative organisms. Here, we investigated the potential of molecular techniques to provide rapid and accurate characterisation of the bacteria present in ascitic fluid. Ascites samples were obtained from 29 cirrhotic patients undergoing clinically indicated therapeutic paracentesis. Bacterial content was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis, quantitative polymerase chain reaction (PCR) and 16S ribosomal clone sequence analysis. Bacterial signal was detected in all samples, compared to three out of ten using standard methods. Bacterial loads ranged from 5.5 × 10 2 to 5.4 × 10 7 cfu/ml, with a mean value of 1.9 × 10 6 cfu/ml (standard deviation± 9.6 × 10 6 cfu/ml). In all but one instance, bacterial species identified by culture were also confirmed by molecular analyses. Preliminary data presented here suggests that culture-independent, molecular analyses could provide rapid characterisation of the bacterial content of ascites fluid, providing a basis for the investigation of SBP development and allowing early and targeted antibiotic intervention.