SummarySensory information is encoded within the brain in distributed spatiotemporal patterns of neuronal activity. Understanding how these patterns influence behavior requires a method to measure and to bidirectionally perturb with high spatial resolution the activity of the multiple neuronal cell types engaged in sensory processing. Here, we combined two-photon holography to stimulate neurons expressing blue light-sensitive opsins (ChR2 and GtACR2) with two-photon imaging of the red-shifted indicator jRCaMP1a in the mouse neocortex in vivo. We demonstrate efficient control of neural excitability across cell types and layers with holographic stimulation and improved spatial resolution by opsin somatic targeting. Moreover, we performed simultaneous two-photon imaging of jRCaMP1a and bidirectional two-photon manipulation of cellular activity with negligible effect of the imaging beam on opsin excitation. This all-optical approach represents a powerful tool to causally dissect how activity patterns in specified ensembles of neurons determine brain function and animal behavior.

Large scale transitions between active (up) and silent (down) states during quiet wakefulness or NREM sleep regulate fundamental cortical functions and are known to involve both excitatory and inhibitory cells. However, if and how inhibition regulates these activity transitions is unclear. Using fluorescence-targeted electrophysiological recording and cell-specific optogenetic manipulation in both anesthetized and non-anesthetized mice, we found that two major classes of interneurons, the parvalbumin and the somatostatin positive cells, tightly control both up-to-down and down-to-up state transitions. Inhibitory regulation of state transition was observed under both natural and optogenetically-evoked conditions. Moreover, perturbative optogenetic experiments revealed that the inhibitory control of state transition was interneuron-type specific. Finally, local manipulation of small ensembles of interneurons affected cortical populations millimetres away from the modulated region. Together, these results demonstrate that inhibition potently gates transitions between cortical activity states, and reveal the cellular mechanisms by which local inhibitory microcircuits regulate state transitions at the mesoscale.DOI: http://dx.doi.org/10.7554/eLife.26177.001

Genetically encoded calcium indicators and optogenetic actuators can report and manipulate the activity of specific neuronal populations. However, applying imaging and optogenetics simultaneously has been difficult to establish in the mammalian brain, even though combining the techniques would provide a powerful approach to reveal the functional organization of neural circuits. Here, we developed a technique based on patterned two-photon illumination to allow fast scanless imaging of GCaMP6 signals in the intact mouse brain at the same time as single-photon optogenetic inhibition with Archaerhodopsin. Using combined imaging and electrophysiological recording, we demonstrate that single and short bursts of action potentials in pyramidal neurons can be detected in the scanless modality at acquisition frequencies up to 1 kHz. Moreover, we demonstrate that our system strongly reduces the artifacts in the fluorescence detection that are induced by single-photon optogenetic illumination. Finally, we validated our technique investigating the role of parvalbumin-positive (PV) interneurons in the control of spontaneous cortical dynamics. Monitoring the activity of cellular populations on a precise spatiotemporal scale while manipulating neuronal activity with optogenetics provides a powerful tool to causally elucidate the cellular mechanisms underlying circuit function in the intact mammalian brain.

Fluorescence is a powerful mean to probe information processing in the mammalian brain [1]. However, neuronal tissues are highly heterogeneous and thus opaque to light. A wide set of noninvasive or invasive techniques for scattered light rejection, optical sectioning or localized excitation, have been developed, but non-invasive optical recording of activity through highly scattering layer beyond the ballistic regime is to date impossible. Here, we show that functional signals from fluorescent time-varying sources located below an highly scattering tissue can be retrieved efficiently, by exploiting matrix factorization algorithms to demix this information from low contrast fluorescence speckle patterns.In the last decades novel light-enabled tools established new paradigms in neuroscience [1][2][3], and among them the emergence of fluorescence functional indicators revolutionized the way to monitor information processing through the brain of different animal models, with unprecedented combination of contrast, resolution and specificity [4,5]. With this approach, optical resolution is often not paramount, and in general only a coarse (cell) resolution is needed [6]. Furthermore, when the location of neurons is known, it is possible to avoid slow rasterscanning techniques and image only the needed location at high frame-rate [6-10].However, brain tissues are usually opaque, and light emitted or delivered at depth in the brain is often quickly subject to multiple scattering events. This results in a loss of directionality after few scattering lengths, corresponding to a few hundred microns, and ultimately means that all wide field or scanning microscopy techniques fails at depth. While the brains of simple organisms are sufficiently small and/or transparent so they can be imaged in totality, for instance C.Elegans, drosophila or zebrafish[5], mammalian brain, starting with its most common animal model, the mouse, is too large and too scattering to image in full. When imaging is performed in superficial layers, it is possible to implement wide field recording with multi-site multiphoton excitation [10,11], or with wide-field excitation and a-posteriori demixing, exploiting the few forward scattered or ballistic photons as a seed to separate the individual neuron contributions [12][13][14]. However, observing neuronal activity beyond a millimeter in the cortex or through the skull, is to date extremely challenging. In this depth range, in the multiple scattering regime, several techniques have been introduced to focus light and image using wavefront shaping [15,16]. Fluorescence is conventionally considered very incoherent, so these techniques based on coherence do not straightforwardly apply. However, it has been shown that it is still possible to reconstruct a fluorescent object hidden behind a scattering medium, by * claudio.moretti@lkb.ens.fr † sylvain.gigan@lkb.ens.fr analyzing spatial correlation within a single low contrast fluorescent speckle [17][18][19][20][21]. These techniques require thin media (with the ...