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Two-photon calcium imaging has been extensively used to record neural activity in the brain. It has been long used solely with analysis, but the recent efforts began to include closed-loop experiments. Closed-loop experiments pose new challenges because they require fast, real-time image processing without iterative parameter tuning. When imaging awake animals, one of the crucial steps of image analysis is correction of lateral motion artifacts. In most of the closed-loop experiments, this step has not been implemented and ignored due to technical difficulties. We recently reported the first experiments with real-time processing of calcium imaging that included lateral motion correction. Here, we report the details of the implementation of fast motion correction and present performance analysis across several algorithms with different parameters. Additionally, we introduce a novel method to estimate baseline calcium signal using kernel density estimate, which reduces the number of parameters to be tuned. Combined, we propose a novel software pipeline of real-time image processing suited for closed-loop experiments. The pipeline is also useful for rapid image processing.

Axonal projection conveys neural information. The divergent and diverse projections of individual neurons imply the complexity of information flow. It is necessary to investigate the relationship between the projection and functional information at the single neuron level for understanding the rules of neural circuit assembly, but a gap remains due to a lack of methods to map the function to whole‐brain projection. Here an approach is developed to bridge two‐photon calcium imaging in vivo with high‐resolution whole‐brain imaging based on sparse labeling with the genetically encoded calcium indicator GCaMP6. Reliable whole‐brain projections are captured by the high‐definition fluorescent micro‐optical sectioning tomography (HD‐fMOST). A cross‐modality cell matching is performed and the functional annotation of whole‐brain projection at the single‐neuron level (FAWPS) is obtained. Applying it to the layer 2/3 (L2/3) neurons in mouse visual cortex, the relationship is investigated between functional preferences and axonal projection features. The functional preference of projection motifs and the correlation between axonal length in MOs and neuronal orientation selectivity, suggest that projection motif‐defined neurons form a functionally specific information flow, and the projection strength in specific targets relates to the information clarity. This pipeline provides a new way to understand the principle of neuronal information transmission. An approach is established to couple the functional activities with the whole‐brain projection of the same neuron by GCaMP6 sparse labeling and a cross‐modality cell matching. It extends the understanding of structure–function correlations through obtaining functionally relevant projection motifs or projection strengths at the single neuron level.

Many animals maintain an internal representation of their heading as they move through their surroundings. Such a compass representation was recently discovered in a neural population in the Drosophila melanogaster central complex, a brain region implicated in spatial navigation. Here, we use two-photon calcium imaging and electrophysiology in head-fixed walking flies to identify a different neural population that conjunctively encodes heading and angular velocity, and is excited selectively by turns in either the clockwise or counterclockwise direction. We show how these mirror-symmetric turn responses combine with the neurons’ connectivity to the compass neurons to create an elegant mechanism for updating the fly’s heading representation when the animal turns in darkness. This mechanism, which employs recurrent loops with an angular shift, bears a resemblance to those proposed in theoretical models for rodent head direction cells. Our results provide a striking example of structure matching function for a broadly relevant computation.

The nucleus accumbens (NAc) is a canonical reward center that regulates feeding and drinking but it is not known whether these behaviors are mediated by same or different neurons. We employed two-photon calcium imaging in awake, behaving mice and found that during the appetitive phase, both hunger and thirst are sensed by a nearly identical population of individual D1 and D2 neurons in the NAc that respond monophasically to food cues in fasted animals and water cues in dehydrated animals. During the consummatory phase, we identified three distinct neuronal clusters that are temporally correlated with action initiation, consumption, and cessation shared by feeding and drinking. These dynamic clusters also show a nearly complete overlap of individual D1 neurons and extensive overlap among D2 neurons. Modulating D1 and D2 neural activities revealed analogous effects on feeding versus drinking behaviors. In aggregate, these data show that a highly overlapping set of D1 and D2 neurons in NAc detect food and water reward and elicit concordant responses to hunger and thirst. These studies establish a general role of this mesolimbic pathway in mediating instinctive behaviors by controlling motivation-associated variables rather than conferring behavioral specificity.

•Neurons in a local cortical microcircuit are active exclusively in a slow wave event.•Visually evoked slow wave events travel from occipital to frontal cortical areas.•Integrating fast line scanning fMRI with photometry increases the sensitivity of fMRI.•Fast line scanning can resolve propagation dynamics of slow wave events.•Slow wave event-associated BOLD responses propagate continuously over the cortex. Population up-states, also referred to as slow wave events (SWEs), are a hallmark of the slow wave brain state in mammalian cortex, typically occurring during deep sleep or under certain types of anesthesia. We explore the neuronal recruitment and propagation of SWEs on observational scales ranging from single neurons to the entire cortex, intertwining optical and translationally relevant functional MRI (fMRI). By two-photon calcium imaging in mouse visual cortex, we demonstrate that all active cells of the observed local micronetwork participate in a population wide SWE. Implementing an optomagnetic-integration concept, involving simultaneous fiber photometry with fast cortical line-scanning fMRI in rats, we identify and follow propagating SWEs across the cortex. We can demonstrate continuous cortical propagation of a SWE by non-invasive line-scanning fMRI. This opens the door for monitoring the spatiotemporal dynamics of a neurophysiological-defined signal - here SWEs. Non-invasive monitoring of SWE propagation might represent a proxy for the functional integrity of local and global cortical networks in rodents and humans. [Display omitted]

The prefrontal cortex (PFC) plays a critical role in curbing impulsive behavior, but the underlying circuit mechanism remains incompletely understood. Here we show that a subset of dorsomedial PFC (dmPFC) layer 5 pyramidal neurons, which project to the subthalamic nucleus (STN) of the basal ganglia, play a key role in inhibiting impulsive responses in a go/no-go task. Projection-specific labeling and calcium imaging showed that the great majority of STN-projecting neurons were preferentially active in no-go trials when the mouse successfully withheld licking responses, but lateral hypothalamus (LH)-projecting neurons were more active in go trials with licking; visual cortex (V1)-projecting neurons showed only weak task-related activity. Optogenetic activation and inactivation of STN-projecting neurons reduced and increased inappropriate licking, respectively, partly through their direct innervation of the STN, but manipulating LH-projecting neurons had the opposite effects. These results identify a projection-defined subtype of PFC pyramidal neurons as key mediators of impulse control.

Many insects use patterns of polarized light in the sky to orient and navigate. Here, we functionally characterize neural circuitry in the fruit fly, Drosophila melanogaster , that conveys polarized light signals from the eye to the central complex, a brain region essential for the fly’s sense of direction. Neurons tuned to the angle of polarization of ultraviolet light are found throughout the anterior visual pathway, connecting the optic lobes with the central complex via the anterior optic tubercle and bulb, in a homologous organization to the ‘sky compass’ pathways described in other insects. We detail how a consistent, map-like organization of neural tunings in the peripheral visual system is transformed into a reduced representation suited to flexible processing in the central brain. This study identifies computational motifs of the transformation, enabling mechanistic comparisons of multisensory integration and central processing for navigation in the brains of insects.

•Two-photon calcium imaging of neurons’ spontaneous activity in macaque V1 & V2.•Neurons dynamically group into different active ensembles.•Ensembles resemble intrinsic functional architectures including orientation, ocular dominance, and color maps. Neurons’ spontaneous activity provides rich information about the brain. A single neuron's activity has close relationships with the local network. In order to understand such relationships, we studied the spontaneous activity of thousands of neurons in macaque V1 and V2 with two-photon calcium imaging. In V1, the ongoing activity was dominated by global fluctuations in which the activity of majority of neurons were correlated. Neurons’ activity also relied on their relative locations within the local functional architectures, including ocular dominance, orientation, and color maps. Neurons with similar preferences dynamically grouped into co-activating ensembles and exhibited spatial patterns resembling the local functional maps. Different ensembles had different strengths and frequencies. This observation was consistent across all hypercolumn-sized V1 locations we examined. In V2, different imaging sites had different orientation and color features. However, the spontaneous activity in the sampled regions also correlated with the underlying functional architectures. These results indicate that functional architectures play an essential role in influencing neurons’ spontaneous activity.

Neurons in sensory cortex exhibit a remarkable capacity to maintain stable firing rates despite large fluctuations in afferent activity levels. However, sudden peripheral deafferentation in adulthood can trigger an excessive, non-homeostatic cortical compensatory response that may underlie perceptual disorders including sensory hypersensitivity, phantom limb pain, and tinnitus. Here, we show that mice with noise-induced damage of the high-frequency cochlear base were behaviorally hypersensitive to spared mid-frequency tones and to direct optogenetic stimulation of auditory thalamocortical neurons. Chronic two-photon calcium imaging from ACtx pyramidal neurons (PyrNs) revealed an initial stage of spatially diffuse hyperactivity, hyper-correlation, and auditory hyperresponsivity that consolidated around deafferented map regions three or more days after acoustic trauma. Deafferented PyrN ensembles also displayed hypersensitive decoding of spared mid-frequency tones that mirrored behavioral hypersensitivity, suggesting that non-homeostatic regulation of cortical sound intensity coding following sensorineural loss may be an underlying source of auditory hypersensitivity. Excess cortical response gain after acoustic trauma was expressed heterogeneously among individual PyrNs, yet 40% of this variability could be accounted for by each cell’s baseline response properties prior to acoustic trauma. PyrNs with initially high spontaneous activity and gradual monotonic intensity growth functions were more likely to exhibit non-homeostatic excess gain after acoustic trauma. This suggests that while cortical gain changes are triggered by reduced bottom-up afferent input, their subsequent stabilization is also shaped by their local circuit milieu, where indicators of reduced inhibition can presage pathological hyperactivity following sensorineural hearing loss.

The central complex is a highly conserved insect brain region composed of morphologically stereotyped neurons that arborize in distinctively shaped substructures. The region is implicated in a wide range of behaviors and several modeling studies have explored its circuit computations. Most studies have relied on assumptions about connectivity between neurons based on their overlap in light microscopy images. Here, we present an extensive functional connectome of Drosophila melanogaster’s central complex at cell-type resolution. Using simultaneous optogenetic stimulation, calcium imaging and pharmacology, we tested the connectivity between 70 presynaptic-to-postsynaptic cell-type pairs. We identified numerous inputs to the central complex, but only a small number of output channels. Additionally, the connectivity of this highly recurrent circuit appears to be sparser than anticipated from light microscopy images. Finally, the connectivity matrix highlights the potentially critical role of a class of bottleneck interneurons. All data are provided for interactive exploration on a website. Some of the most evocative discoveries in neuroscience have been those of internal representations, such as neural activity patterns that represent which direction an animal is facing and its place in its surroundings. Understanding how neurons connect to one another to form ‘circuits’ is crucial to understanding how these circuits maintain such representations. Many of the design principles that underlie circuit function in the brains of fruit flies apply to other animals. However, fly brains are easier to study because genetic tools can be used on them to selectively activate and image the activity of specific types of neurons. By activating one type of neuron and imaging the activity of another that may be connected to it, we obtain what is called a functional ‘connectome’: a map of neural connectivity that identifies different pathways that information can flow along. A region of the fly brain called the central complex is involved in many important behaviors, including navigation and sleep. Researchers know about the types of neurons in the region and about how the activity of some of them changes during different behaviors. However, obtaining the connectome of the central complex would make it easier to understand how the central complex works. A technique called optogenetics allows specific types of neurons to be activated one at a time by shining light onto them. By imaging the activity of neurons that might be connected to an optogenetically activated neuron, Franconville et al. have now built an extensive – albeit still incomplete – map of the connections within the central complex of fruit flies. The map reveals two key bottlenecks in the central complex circuit. Firstly, a neuron type in a substructure called the protocerebral bridge controls a lot of the information flowing through the circuit. Secondly, the circuit appears to have very few true ‘output’ neuron types – Franconville et al. identified only one. These results suggest that however complicated the computations performed by the central complex circuit might be, the output of the circuit, which likely guides the fly’s actions, may be much simpler. Franconville et al. have compiled the mapping results into an interactive website that makes the neuroscientific data both freely available and easily explorable. As researchers perform more such experiments, the new data can be added to the map. This information can be used to constrain theories and inspire new ideas about how the central complex does what it does.