Neural computations are implemented by activity in spatially distributed neural circuits. Cellular imaging fills a unique niche in linking activity of specific types of neurons to behavior, over spatial scales spanning single neurons to entire brain regions, and temporal scales from milliseconds to months. Imaging may soon make it possible to track activity of all neurons in a brain region, such as a cortical column. We review recent methodological advances that facilitate optical imaging of neuronal populations in vivo, with an emphasis on calcium imaging using protein indicators in mice. We point out areas that are particularly ripe for future developments.

Cortical cells integrate synaptic input from multiple sources, but how these different inputs are distributed across individual neurons is largely unknown. Differences in input might account for diverse responses in neighboring neurons during behavior. We present a strategy for comparing the strengths of multiple types of input onto the same neuron. We developed methods for independent dual-channel photostimulation of synaptic inputs using ChR2 together with ReaChR, a red-shifted channelrhodopsin. We used dual-channel photostimulation to probe convergence of sensory information in the mouse primary motor cortex. Input from somatosensory cortex and thalamus converges in individual neurons. Similarly, inputs from distinct somatotopic regions of the somatosensory cortex are integrated at the level of single motor cortex neurons. We next developed a ReaChR transgenic mouse under the control of both Flp- and Cre-recombinases that is an effective tool for circuit mapping. Our approach to dual-channel photostimulation enables quantitative comparison of the strengths of multiple pathways across all length scales of the brain.

Activity in motor cortex predicts specific movements seconds before they occur, but how this preparatory activity relates to upcoming movements is obscure. We dissected the conversion of preparatory activity to movement within a structured motor cortex circuit. An anterior lateral region of the mouse cortex (a possible homologue of premotor cortex in primates) contains equal proportions of intermingled neurons predicting ipsi- or contralateral movements, yet unilateral inactivation of this cortical region during movement planning disrupts contralateral movements. Using cell-type-specific electrophysiology, cellular imaging and optogenetic perturbation, we show that layer 5 neurons projecting within the cortex have unbiased laterality. Activity with a contralateral population bias arises specifically in layer 5 neurons projecting to the brainstem, and only late during movement planning. These results reveal the transformation of distributed preparatory activity into movement commands within hierarchically organized cortical circuits.

Eyes may be 'the window to the soul' in humans, but whiskers provide a better path to the inner lives of rodents. The brain has remarkable abilities to focus its limited resources on information that matters, while ignoring a cacophony of distractions. While inspecting a visual scene, primates foveate to multiple salient locations, for example mouths and eyes in images of people, and ignore the rest. Similar processes have now been observed and studied in rodents in the context of whisker-based tactile sensation. Rodents use their mechanosensitive whiskers for a diverse range of tactile behaviors such as navigation, object recognition and social interactions. These animals move their whiskers in a purposive manner to locations of interest. The shapes of whiskers, as well as their movements, are exquisitely adapted for tactile exploration in the dark tight burrows where many rodents live. By studying whisker movements during tactile behaviors, we can learn about the tactile information available to rodents through their whiskers and how rodents direct their attention. In this primer, we focus on how the whisker movements of rats and mice are providing clues about the logic of active sensation and the underlying neural mechanisms.

GABAergic terminals of chandelier cells exclusively innervate the axon initial segment (AIS) of excitatory neurons. Although the anatomy of these synapses has been well-studied in several brain areas, relatively little is known about their physiological properties. Using vesicular γ-aminobutyric acid transporter-channelrhodopsin 2-enhanced yellow fluorescence protein (VGAT-ChR2-YFP)-expressing mice and a novel fibreoptic 'laserspritzer' approach that we developed, we investigated the physiological properties of axo-axonic synapses (AASs) in brain slices from the piriform cortex (PC) of mice. AASs were in close proximity to voltage-gated Na(+) (NaV) channels located at the AIS. AASs were selectively activated by a 5 μm laserspritzer placed in close proximity to the AIS. Under a minimal laser stimulation condition and using whole-cell somatic voltage-clamp recordings, the amplitudes and kinetics of IPSCs mediated by AASs were similar to those mediated by perisomatic inhibitions. Results were further validated with channelrhodopsin 2-assisted circuit mapping (CRACM) of the entire inhibitory inputs map. For the first time, we revealed that the laserspritzer-induced AAS-IPSCs persisted in the presence of TTX and TEA but not 4-AP. Next, using gramicidin-based perforated patch recordings, we found that the GABA reversal potential (EGABA) was -73.6 ± 1.2 mV when induced at the AIS and -72.8 ± 1.1 mV when induced at the perisomatic site. Our anatomical and physiological results lead to the novel conclusions that: (1) AASs innervate the entire length of the AIS, as opposed to forming a highly concentrated cartridge, (2) AAS inhibition suppresses action potentials and epileptiform activity more robustly than perisomatic inhibitions, and (3) AAS activation alone can be sufficient to inhibit action potential generation and epileptiform activities in vitro.

Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.

Understanding brain function requires monitoring and interpreting the activity of large networks of neurons during behavior. Advances in recording technology are greatly increasing the size and complexity of neural data. Analyzing such data will pose a fundamental bottleneck for neuroscience. We present a library of analytical tools called Thunder built on the open-source Apache Spark platform for large-scale distributed computing. The library implements a variety of univariate and multivariate analyses with a modular, extendable structure well-suited to interactive exploration and analysis development. We demonstrate how these analyses find structure in large-scale neural data, including whole-brain light-sheet imaging data from fictively behaving larval zebrafish, and two-photon imaging data from behaving mouse. The analyses relate neuronal responses to sensory input and behavior, run in minutes or less and can be used on a private cluster or in the cloud. Our open-source framework thus holds promise for turning brain activity mapping efforts into biological insights.

During many natural behaviors the relevant sensory stimuli and motor outputs are difficult to quantify. Furthermore, the high dimensionality of the space of possible stimuli and movements compounds the problem of experimental control. Head fixation facilitates stimulus control and movement tracking, and can be combined with techniques for recording and manipulating neural activity. However, head-fixed mouse behaviors are typically trained through extensive instrumental conditioning. Here we present a whisker-based, tactile virtual reality system for head-fixed mice running on a spherical treadmill. Head-fixed mice displayed natural movements, including running and rhythmic whisking at 16 Hz. Whisking was centered on a set point that changed in concert with running so that more protracted whisking was correlated with faster running. During turning, whiskers moved in an asymmetric manner, with more retracted whisker positions in the turn direction and protracted whisker movements on the other side. Under some conditions, whisker movements were phase-coupled to strides. We simulated a virtual reality tactile corridor, consisting of two moveable walls controlled in a closed-loop by running speed and direction. Mice used their whiskers to track the walls of the winding corridor without training. Whisker curvature changes, which cause forces in the sensory follicles at the base of the whiskers, were tightly coupled to distance from the walls. Our behavioral system allows for precise control of sensorimotor variables during natural tactile navigation.

We report learning-related structural plasticity in layer 1 branches of pyramidal neurons in the barrel cortex, a known site of sensorimotor integration. In mice learning an active, whisker-dependent object localization task, layer 2/3 neurons showed enhanced spine growth during initial skill acquisition that both preceded and predicted expert performance. Preexisting spines were stabilized and new persistent spines were formed. These findings suggest rapid changes in connectivity between motor centers and sensory cortex guide subsequent sensorimotor learning.

Perceptual decisions involve distributed cortical activity. Does information flow sequentially from one cortical area to another, or do networks of interconnected areas contribute at the same time? Here we delineate when and how activity in specific areas drives a whisker-based decision in mice. A short-term memory component temporally separated tactile "sensation" and "action" (licking). Using optogenetic inhibition (spatial resolution, 2 mm; temporal resolution, 100 ms), we surveyed the neocortex for regions driving behavior during specific behavioral epochs. Barrel cortex was critical for sensation. During the short-term memory, unilateral inhibition of anterior lateral motor cortex biased responses to the ipsilateral side. Consistently, barrel cortex showed stimulus-specific activity during sensation, whereas motor cortex showed choice-specific preparatory activity and movement-related activity, consistent with roles in motor planning and movement. These results suggest serial information flow from sensory to motor areas during perceptual decision making.