Long-term mesoscale imaging of 3D intercellular dynamics across a mammalian organ.

A comprehensive understanding of physio-pathological processes necessitates non-invasive intravital three-dimensional (3D) imaging over varying spatial and temporal scales. However, huge data throughput, optical heterogeneity, surface irregularity, and phototoxicity pose great challenges, leading to an inevitable trade-off between volume size, resolution, speed, sample health, and system complexity. Here, we introduce a compact real-time, ultra-large-scale, high-resolution 3D mesoscope (RUSH3D), achieving uniform resolutions of 2.6 × 2.6 × 6 µm3 across a volume of 8,000 × 6,000 × 400 µm3 at 20 Hz with low phototoxicity. Through the integration of multiple computational imaging techniques, RUSH3D facilitates a 13-fold improvement in data throughput and an orders-of-magnitude reduction in system size and cost. With these advantages, we observed premovement neural activity and cross-day visual representational drift across the mouse cortex, the formation and progression of multiple germinal centers in mouse inguinal lymph nodes, and heterogeneous immune responses following traumatic brain injury-all at single-cell resolution, opening up a horizon for intravital mesoscale study of large-scale intercellular interactions at the organ level.

An entorhinal-visual cortical circuit regulates depression-like behaviors.

Major depressive disorder is viewed as a 'circuitopathy'. The hippocampal-entorhinal network plays a pivotal role in regulation of depression, and its main sensory output, the visual cortex, is a promising target for stimulation therapy of depression. However, whether the entorhinal-visual cortical pathway mediates depression and the potential mechanism remains unknown. Here we report a cortical circuit linking entorhinal cortex layer Va neurons to the medial portion of secondary visual cortex (Ent→V2M) that bidirectionally regulates depression-like behaviors in mice. Analyses of brain-wide projections of Ent Va neurons and two-color retrograde tracing indicated that Ent Va→V2M projection neurons represented a unique population of neurons in Ent Va. Immunostaining of c-Fos revealed that activity in Ent Va neurons was decreased in mice under chronic social defeat stress (CSDS). Both chemogenetic inactivation of Ent→V2M projection neurons and optogenetic inactivation of the projection terminals induced social deficiency, anxiety- and despair-related behaviors in healthy mice. Chemogenetic inactivation of Ent→V2M projection neurons also aggravated these depression-like behaviors in CSDS-resilient mice. Optogenetic activation of Ent→V2M projection terminals rapidly ameliorated depression-like phenotypes. Optical recording using fiber photometry indicated that elevated neural activity in Ent→V2M projection terminals promoted antidepressant-like behaviors. Thus, the Ent→V2M circuit plays a crucial role in regulation of depression-like behaviors, and can function as a potential target for treating major depressive disorder.

Maintenance of persistent activity in a frontal thalamocortical loop.

Persistent neural activity maintains information that connects past and future events. Models of persistent activity often invoke reverberations within local cortical circuits, but long-range circuits could also contribute. Neurons in the mouse anterior lateral motor cortex (ALM) have been shown to have selective persistent activity that instructs future actions. The ALM is connected bidirectionally with parts of the thalamus, including the ventral medial and ventral anterior-lateral nuclei. We recorded spikes from the ALM and thalamus during tactile discrimination with a delayed directional response. Here we show that, similar to ALM neurons, thalamic neurons exhibited selective persistent delay activity that predicted movement direction. Unilateral photoinhibition of delay activity in the ALM or thalamus produced contralesional neglect. Photoinhibition of the thalamus caused a short-latency and near-complete collapse of ALM activity. Similarly, photoinhibition of the ALM diminished thalamic activity. Our results show that the thalamus is a circuit hub in motor preparation and suggest that persistent activity requires reciprocal excitation across multiple brain areas.

Prolonged chronic social defeat stress promotes less resilience and higher uniformity in depression-like behaviors in adult male mice.

Chronic social defeat stress (CSDS) is widely applied to study of depression in rodents. 10-day CSDS was a most commonly employed paradigm but with high resilience ratio (∼30%), producing potential variation in depression-like behavioral symptoms. Whether prolonged period (21 days) of CSDS would promote less resilience and reduce behavioral variability remains unknown. We applied 10-day and 21-day CSDS paradigms to induce mouse model of depression and compared their resilience ratio and behavioral phenotypes. Mice under 21-day CSDS had significantly lower resilience ratio and greater changes in behavioral indicators relative to mice under 10-day CSDS. Behavioral indicators from 21-day CSDS paradigm had higher correlations and better prediction for susceptibility which indicating higher uniformity in behavioral phenotypes. Furthermore, a subset of behavioral indicators in 21-day CSDS had high prediction efficacy and should be first applied to screen susceptibility of CSDS. Thus, our study demonstrates that 21-day CSDS is a more robust paradigm inducing reliable depression-like behaviors relative to 10-day CSDS, and should be preferentially used in rodent studies of depression.

A motor cortex circuit for motor planning and movement.

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.

Controlling interneuron activity in Caenorhabditis elegans to evoke chemotactic behaviour.

Animals locate and track chemoattractive gradients in the environment to find food. With its small nervous system, Caenorhabditis elegans is a good model system in which to understand how the dynamics of neural activity control this search behaviour. Extensive work on the nematode has identified the neurons that are necessary for the different locomotory behaviours underlying chemotaxis through the use of laser ablation, activity recording in immobilized animals and the study of mutants. However, we do not know the neural activity patterns in C. elegans that are sufficient to control its complex chemotactic behaviour. To understand how the activity in its interneurons coordinate different motor programs to lead the animal to food, here we used optogenetics and new optical tools to manipulate neural activity directly in freely moving animals to evoke chemotactic behaviour. By deducing the classes of activity patterns triggered during chemotaxis and exciting individual neurons with these patterns, we identified interneurons that control the essential locomotory programs for this behaviour. Notably, we discovered that controlling the dynamics of activity in just one interneuron pair (AIY) was sufficient to force the animal to locate, turn towards and track virtual light gradients. Two distinct activity patterns triggered in AIY as the animal moved through the gradient controlled reversals and gradual turns to drive chemotactic behaviour. Because AIY neurons are post-synaptic to most chemosensory and thermosensory neurons, it is probable that these activity patterns in AIY have an important role in controlling and coordinating different taxis behaviours of the animal.

Cross-hemispheric communication: Insights on lateralized brain functions.

On the surface, the two hemispheres of vertebrate brains look almost perfectly symmetrical, but several motor, sensory, and cognitive systems show a deeply lateralized organization. Importantly, the two hemispheres are connected by various commissures, white matter tracts that cross the brain's midline and enable cross-hemispheric communication. Cross-hemispheric communication has been suggested to play an important role in the emergence of lateralized brain functions. Here, we review current advances in understanding cross-hemispheric communication that have been made using modern neuroscientific tools in rodents and other model species, such as genetic labeling, large-scale recordings of neuronal activity, spatiotemporally precise perturbation, and quantitative behavior analyses. These findings suggest that the emergence of lateralized brain functions cannot be fully explained by largely static factors such as genetic variation and differences in structural brain asymmetries. In addition, learning-dependent asymmetric interactions between the left and right hemispheres shape lateralized brain functions.

Optical interrogation of neural circuits in Caenorhabditis elegans.

The nematode Caenorhabditis elegans has a compact nervous system with only 302 neurons. Whereas most of the synaptic connections between these neurons have been identified by electron microscopy serial reconstructions, functional connections have been inferred between only a few neurons through combinations of electrophysiology, cell ablation, in vivo calcium imaging and genetic analysis. To map functional connections between neurons, we combined in vivo optical stimulation with simultaneous calcium imaging. We analyzed the connections from the ASH sensory neurons and RIM interneurons to the command interneurons AVA and AVD. Stimulation of ASH or RIM neurons using channelrhodopsin-2 (ChR2) resulted in activation of AVA neurons, evoking an avoidance behavior. Our results demonstrate that we can excite specific neurons expressing ChR2 while simultaneously monitoring G-CaMP fluorescence in several other neurons, making it possible to rapidly decipher functional connections in C. elegans neural circuits.

Lateralization of short-term memory in the frontal cortex.

Despite essentially symmetric structures in mammalian brains, the left and right hemispheres do not contribute equally to certain cognitive functions. How both hemispheres interact to cause this asymmetry remains unclear. Here, we study this question in the anterior lateral motor cortex (ALM) of mice performing five versions of a tactile-based decision-making task with a short-term memory (STM) component. Unilateral inhibition of ALM produces variable behavioral deficits across tasks, with the left, right, or both ALMs playing critical roles in STM. Neural activity and its encoding capability are similar across hemispheres, despite that only one hemisphere dominates in behavior. Inhibition of the dominant ALM disrupts encoding capability in the non-dominant ALM, but not vice versa. Variable behavioral deficits are predicted by the influence on contralateral activity across sessions, mice, and tasks. Together, these results reveal that the left and right ALM interact asymmetrically, leading to their differential contributions to STM.

Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X.

Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2-3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

A cortico-basal ganglia-thalamo-cortical channel underlying short-term memory.

Persistent activity underlying short-term memory encodes sensory information or instructs specific future movement and, consequently, has a crucial role in cognition. Despite extensive study, how the same set of neurons respond differentially to form selective persistent activity remains unknown. Here, we report that the cortico-basal ganglia-thalamo-cortical (CBTC) circuit supports the formation of selective persistent activity in mice. Optogenetic activation or inactivation of the basal ganglia output nucleus substantia nigra pars reticulata (SNr)-to-thalamus pathway biased future licking choice, without affecting licking execution. This perturbation differentially affected persistent activity in the frontal cortex and selectively modulated neural trajectory that encodes one choice but not the other. Recording showed that SNr neurons had selective persistent activity distributed across SNr, but with a hotspot in the mediolateral region. Optogenetic inactivation of the frontal cortex also differentially affected persistent activity in the SNr. Together, these results reveal a CBTC channel functioning to produce selective persistent activity underlying short-term memory.

Sparse imaging and reconstruction tomography for high-speed high-resolution whole-brain imaging.

Understanding brain functions requires detailed knowledge of long-range connectivity through which different areas communicate. A key step toward illuminating the long-range structures is to image the whole brain at synaptic resolution to trace axonal arbors of individual neurons to their termini. However, high-resolution brain-wide imaging requires continuous imaging for many days to sample over 10 trillion voxels, even in the mouse brain. Here, we have developed a sparse imaging and reconstruction tomography (SMART) system that allows brain-wide imaging of cortical projection neurons at synaptic resolution in about 20 h, an order of magnitude faster than previous methods. Analyses of morphological features reveal that single cortical neurons show remarkable diversity in local and long-range projections, with prefrontal, premotor, and visual neurons having distinct distribution of dendritic and axonal features. The fast imaging system and diverse projection patterns of individual neurons highlight the importance of high-resolution brain-wide imaging in revealing full neuronal morphology.

Multi-Scale Light-Sheet Fluorescence Microscopy for Fast Whole Brain Imaging.

Whole-brain imaging has become an increasingly important approach to investigate neural structures, such as somata distribution, dendritic morphology, and axonal projection patterns. Different structures require whole-brain imaging at different resolutions. Thus, it is highly desirable to perform whole-brain imaging at multiple scales. Imaging a complete mammalian brain at synaptic resolution is especially challenging, as it requires continuous imaging from days to weeks because of the large number of voxels to sample, and it is difficult to acquire a constant quality of imaging because of light scattering during in toto imaging. Here, we reveal that light-sheet microscopy has a unique advantage over wide-field microscopy in multi-scale imaging because of its decoupling of illumination and detection. Based on this observation, we have developed a multi-scale light-sheet microscope that combines tiling of light-sheet, automatic zooming, periodic sectioning, and tissue expansion to achieve a constant quality of brain-wide imaging from cellular (3 µm × 3 µm × 8 µm) to sub-micron (0.3 µm × 0.3 µm × 1 µm) spatial resolution rapidly (all within a few hours). We demonstrated the strength of the system by testing it using mouse brains prepared using different clearing approaches. We were able to track electrode tracks as well as axonal projections at sub-micron resolution to trace the full morphology of single medial prefrontal cortex (mPFC) neurons that have remarkable diversity in long-range projections.

Mapping Functional Connectivity from the Dorsal Cortex to the Thalamus.

Neural activity in the corticothalamic network is crucial for sensation, memory, decision, and action. Nevertheless, a systematic characterization of corticothalamic functional connectivity has not been achieved. Here, we developed a high throughput method to systematically map functional connections from the dorsal cortex to the thalamus in awake mice by combing optogenetic inactivation with multi-channel recording. Cortical inactivation resulted in a rapid reduction of thalamic activity, revealing topographically organized corticothalamic excitatory inputs. Cluster analysis showed that groups of neurons within individual thalamic nuclei exhibited distinct dynamics. The effects of inactivation evolved with time and were modulated by behavioral states. Furthermore, we found that a subset of thalamic neurons received convergent inputs from widespread cortical regions. Our results present a framework for collecting, analyzing, and presenting large electrophysiological datasets with region-specific optogenetic perturbations and serve as a foundation for further investigation of information processing in the corticothalamic pathway.

Spatiotemporal constraints on optogenetic inactivation in cortical circuits.

Optogenetics allows manipulations of genetically and spatially defined neuronal populations with excellent temporal control. However, neurons are coupled with other neurons over multiple length scales, and the effects of localized manipulations thus spread beyond the targeted neurons. We benchmarked several optogenetic methods to inactivate small regions of neocortex. Optogenetic excitation of GABAergic neurons produced more effective inactivation than light-gated ion pumps. Transgenic mice expressing the light-dependent chloride channel GtACR1 produced the most potent inactivation. Generally, inactivation spread substantially beyond the photostimulation light, caused by strong coupling between cortical neurons. Over some range of light intensity, optogenetic excitation of inhibitory neurons reduced activity in these neurons, together with pyramidal neurons, a signature of inhibition-stabilized neural networks ('paradoxical effect'). The offset of optogenetic inactivation was followed by rebound excitation in a light dose-dependent manner, limiting temporal resolution. Our data offer guidance for the design of in vivo optogenetics experiments.

Flow of cortical activity underlying a tactile decision in mice.

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.

Procedures for behavioral experiments in head-fixed mice.

The mouse is an increasingly prominent model for the analysis of mammalian neuronal circuits. Neural circuits ultimately have to be probed during behaviors that engage the circuits. Linking circuit dynamics to behavior requires precise control of sensory stimuli and measurement of body movements. Head-fixation has been used for behavioral research, particularly in non-human primates, to facilitate precise stimulus control, behavioral monitoring and neural recording. However, choice-based, perceptual decision tasks by head-fixed mice have only recently been introduced. Training mice relies on motivating mice using water restriction. Here we describe procedures for head-fixation, water restriction and behavioral training for head-fixed mice, with a focus on active, whisker-based tactile behaviors. In these experiments mice had restricted access to water (typically 1 ml/day). After ten days of water restriction, body weight stabilized at approximately 80% of initial weight. At that point mice were trained to discriminate sensory stimuli using operant conditioning. Head-fixed mice reported stimuli by licking in go/no-go tasks and also using a forced choice paradigm using a dual lickport. In some cases mice learned to discriminate sensory stimuli in a few trials within the first behavioral session. Delay epochs lasting a second or more were used to separate sensation (e.g. tactile exploration) and action (i.e. licking). Mice performed a variety of perceptual decision tasks with high performance for hundreds of trials per behavioral session. Up to four months of continuous water restriction showed no adverse health effects. Behavioral performance correlated with the degree of water restriction, supporting the importance of controlling access to water. These behavioral paradigms can be combined with cellular resolution imaging, random access photostimulation, and whole cell recordings.

Neural coding during active somatosensation revealed using illusory touch.

Active sensation requires the convergence of external stimuli with representations of body movements. We used mouse behavior, electrophysiology and optogenetics to dissect the temporal interactions among whisker movement, neural activity and sensation of touch. We photostimulated layer 4 activity in single barrels in a closed loop with whisking. Mimicking touch-related neural activity caused illusory perception of an object at a particular location, but scrambling the timing of the spikes over one whisking cycle (tens of milliseconds) did not abolish the illusion, indicating that knowledge of instantaneous whisker position is unnecessary for discriminating object locations. The illusions were induced only during bouts of directed whisking, when mice expected touch, and in the relevant barrel. Reducing activity biased behavior, consistent with a spike count code for object detection at a particular location. Our results show that mice integrate coding of touch with movement over timescales of a whisking bout to produce perception of active touch.