Jason. N. D. Kerr

Capturing the fine details of behavior when animals perform complex tasks, such as prey pursuit and capture, in natural environments is a challenging endeavor. For birds of prey, commonly used tracking approaches, such as lightweight GPS and inertial measurement units, cannot simultaneously track the bird’s position and the fine details of body part motion or moving external objects. Here we designed a head-mounted, tetherless, 4π camera system which, combined with a high-resolution environmental scan, enabled detailed three-dimensional measurement of both head position and orientation in freely flying Harris’s hawks ( Parabuteo unicinctus ) performing aerial pursuit in the wild. This stable head-mounted imaging approach enabled reconstruction of flight trajectories in natural environments, detailed quantification of head motion, optic flow and target position, while the bird tracked and captured airborne targets.

Head-mounted multiphoton microscopes enable imaging of activity from neuronal populations spread throughout the cortical layers in freely moving mice but so far have been restricted to recording from one cortical layer at a time. Here, combining two- and three-photon-based excitation delivered through multiple fibers, we built a head-mounted multiplane microscope enabling near-simultaneous imaging of neuronal activity from five vertically separated planes, spread across multiple cortical layers. Both excitation pathways had remote focusing mechanisms for fine axial adjustments, enabling activity recordings from the same neuronal populations over weeks in freely behaving mice. The lightweight microscope utilized an onboard, two-channel detection system designed to enable activity recordings from neuronal populations spread across visual-cortex layers in both lit and dark conditions as well as imaging activity across posterior parietal cortex layers during complex gap-crossing behaviors. We show that during gap-crossing tasks, layer 5 and 2/3 neuronal subpopulations in the posterior parietal cortex have differential pattern sequences during free decision-making.

Head-mounted multiphoton microscopes enable imaging of activity from neuronal populations spread throughout the cortical layers in freely moving mice, but so far have been restricted to recording from one cortical layer at a time. Here, combining 2- and 3-photon based excitation delivered through multiple fibers, we built a head-mounted multiplane microscope enabling near simultaneous imaging (8ns between planes) of neuronal activity from five vertically separated planes, spread across multiple cortical layers. Both excitation pathways had remote focusing mechanisms for fine axial adjustments enabling activity recordings from the same neuronal populations over weeks in freely behaving mice. The lightweight microscope utilized an onboard, 2-channel detection system designed to enable activity recordings from neuronal populations spread across visual-cortex layers in both lit and dark conditions as well as imaging activity across posterior parietal cortex layers during complex gap-crossing behaviors. We show that during gap- crossing tasks, layer 5 and 2/3 neuronal subpopulations in posterior parietal cortex have differential pattern sequences during free decision making. Editorial summary: A light-weight head-mounted multiplane microscope allows simultaneous imaging from >1800 neurons spread across the cortical layers in freely moving mice performing complex behavioral tasks, sampled over weeks.

Understanding brain function and pathology requires observation of cellular dynamics within intact neural circuits. Although two-photon microscopy revolutionized mammalian in vivo brain imaging, its limitation to upper cortical layers has restricted access to many important brain regions. Three-photon microscopy overcomes this constraint, enabling minimally invasive yet high-resolution visualization of the deep cortical and subcortical structures that are crucial for higher-order brain functions. This emerging technology opens new avenues for investigating fundamental aspects of neuroscience, from circuit dynamics to disease mechanisms. Here, we examine how three-photon microscopy has started to transform our ability to investigate neural circuits, glial biology, and oncological and neuroimmune interactions in previously inaccessible brain regions, primarily in the mouse, but also in other model organisms. We discuss current technical challenges, recent innovations and future applications that promise to bring us greater understanding of the living brain.

In the mouse retina, sustained ON alpha (sONα) retinal ganglion cells (RGCs) have different dendritic and receptive field sizes along the nasotemporal axis, with temporal sONα RGCs likely playing a role in visually guided hunting. Thus, we hypothesized that this cell type also exhibits regional adaptations in dendritic signal processing and that these adaptations are advantageous for prey capture. Here, we measured dendritic signals from individual sONα RGCs at different retinal locations. We measured both postsynaptic Ca ²⁺ signals at dendrites and presynaptic glutamate signals from bipolar cells (BCs). We found that temporal sONα RGCs exhibit, in addition to sustained-ON signals with only weak surrounds, signals with strong surround suppression, which were not present in nasal sONα RGCs. This difference was also present in the presynaptic inputs from BCs. Last, using population models in an encoder-decoder paradigm, we showed that these adaptations might be beneficial for detecting crickets in hunting behavior.

Various retinal ganglion cells (RGCs) show regional adaptations, for instance, to increase visual acuity. However, for many RGC types, it is not known how they vary in their signalprocessing properties across the retina. In the mouse retina, sustained ON alpha (sONα) RGCs were found to have differences in morphology and receptive field sizes along the nasotemporal axis, and temporal sONα RGCs are likely to play a role in visually guided hunting. Thus, we hypothesised that this cell type also exhibits regional adaptations on the level of dendritic signal processing and that these adaptations are advantageous for prey capture. Here, we measured dendritic signals from individual sONα RGCs at different locations in the exvivo whole-mount mouse retina using two-photon microscopy. We measured both postsynaptic Ca ²⁺ signals at the dendrites of individual RGCs and presynaptic glutamate signals from bipolar cells (BCs). We found that temporal sONα RGC dendrites exhibit, in addition to the expected sustained-ON signals with only weak surrounds, signals with strong surround suppression, which were not present in nasal sONα RGCs. This difference was also present in the excitatory presynaptic inputs from BCs, suggesting a presynaptic origin. Finally, using population models in an encoder-decoder paradigm, we showed that these adaptations might be beneficial for detecting crickets in hunting behaviour.

Imaging in the freely moving animal gives unparalleled access to circuit activity as the animal interacts with its environment in a self-guided way. Over the past few years, new imaging technologies have enabled the interrogation of neuronal populations located at any depth of the cortex in freely moving mice while preserving the animal’s behavioral repertoire. This commentary gives an updated overview of the recent advances that have enabled the link between behavior and the underlying neuronal activity to be explored.

Advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2-g, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. The microscope had on-board photon detectors, robust to environmental light, and the arena lighting was timed to the end of each line-scan, enabling functional imaging of activity from cortical layer 4 and layer 6 neurons expressing jGCaMP7f in mice roaming a fully lit or dark arena. By comparing the neuronal activity measured from populations in these layers we show that activity in cortical layer 4 and layer 6 is differentially modulated by lit and dark conditions during free exploration.

Forming a complete picture of the relationship between neural activity and skeletal kinematics requires quantification of skeletal joint biomechanics during free behavior; however, without detailed knowledge of the underlying skeletal motion, inferring limb kinematics using surface-tracking approaches is difficult, especially for animals where the relationship between the surface and underlying skeleton changes during motion. Here we developed a videography-based method enabling detailed three-dimensional kinematic quantification of an anatomically defined skeleton in untethered freely behaving rats and mice. This skeleton-based model was constrained using anatomical principles and joint motion limits and provided skeletal pose estimates for a range of body sizes, even when limbs were occluded. Model-inferred limb positions and joint kinematics during gait and gap-crossing behaviors were verified by direct measurement of either limb placement or limb kinematics using inertial measurement units. Together we show that complex decision-making behaviors can be accurately reconstructed at the level of skeletal kinematics using our anatomically constrained model.