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The COMBO window: A chronic cranial implant for multiscale circuit interrogation in mice [1]

['Bradley J. Edelman', 'Brain-Wide Circuits For Behavior Research Group', 'Max Planck Institute For Biological Intelligence', 'Planegg', 'Emotion Research Department', 'Max Planck Institute Of Psychiatry', 'Munich', 'Dynamics Of Excitable Cell Networks Research Group', 'Department Of Ophthalmology', 'University Medical Center Göttingen']

Date: 2024-06 

Neuroscientists studying the neural correlates of mouse behavior often lack access to the brain-wide activity patterns elicited during a specific task of interest. Fortunately, large-scale imaging is becoming increasingly accessible thanks to modalities such as Ca 2+ imaging and functional ultrasound (fUS). However, these and other techniques often involve challenging cranial window procedures and are difficult to combine with other neuroscience tools. We address this need with an open-source 3D-printable cranial implant—the COMBO ( C hr O nic M ultimodal imaging and B ehavioral O bservation) window. The COMBO window enables chronic imaging of large portions of the brain in head-fixed mice while preserving orofacial movements. We validate the COMBO window stability using both brain-wide fUS and multisite two-photon imaging. Moreover, we demonstrate how the COMBO window facilitates the combination of optogenetics, fUS, and electrophysiology in the same animals to study the effects of circuit perturbations at both the brain-wide and single-neuron level. Overall, the COMBO window provides a versatile solution for performing multimodal brain recordings in head-fixed mice.

Here, we propose to simplify multimodal imaging across spatial-temporal scales in head-fixed behaving mice using a chronic cranial implant, termed the C hr O nic M ultimodal imaging and B ehavioral O bservation (COMBO) window. The COMBO window aims to combine and expand the advantages of currently available cranial window implants by providing (1) a larger field of view; (2) compatibility with optical and acoustic imaging; (3) integration with local recording and manipulation methods; and (4) unobstructed access to head-fixed behavioral readouts. We first validated modality-specific versions of the COMBO window for awake imaging with both fUS and two-photon Ca 2+ imaging. We then further validated an additional variant of the COMBO window with the combination of fUS, optogenetics, behavioral analyses, and electrophysiology in the same animals. To increase accessibility, we developed all components of the COMBO window and head fixation components for 3D printer or laser cutter production and provide all relevant files ( S1 – S16 Files, https://doi.org/10.5281/zenodo.11092491 ). By doing so, the most appropriate off-the-shelf option can be used for individual applications and can be 3D-printed in-house without the need for significant customization. Overall, the COMBO window provides a unified solution to the requirements of a large variety of neuroscientific experiments that benefit from access to a large portion of the mouse brain during behavioral tasks.

A common feature of some of the most popular techniques used in neuroscience (electrophysiology, wide-field imaging, two-photon microscopy, optogenetics, fUS) is that they are most often applied through a cranial window. This is either because direct access to the brain is needed, e.g., for electrode or fiber implantation (electrophysiology, optogenetics, fiber photometry), or to maximize imaging quality and depth (optical imaging, fUS) [ 10 – 12 ]. Early chronic cranial windows developed specifically for optical imaging reported clear optical access to the brain at cellular resolution for months at a time [ 13 , 14 ]. However, these windows utilize flat glass coverslips that can cause anatomical distortions when placed over large areas of curved tissue. Therefore, such approaches are limited in spatial extent, often covering only 2 to 5 mm 2 of the brain [ 15 ]. A solution to this problem was provided by the “Crystal Skull” implant, a curved glass coverslip giving access to 75 mm 2 of the dorsal cortex and which is commonly used for wide-field imaging [ 16 ]. However, glass windows are not compatible with methods such as electrophysiology and fUS as glass strongly attenuates ultrasound waves and is difficult to penetrate with a probe. Recently, this restriction has been largely overcome with other geometry-based approaches that involve shaping a plastic film to the curvature of the skull and brain [ 17 , 18 ]. Such examples are also accompanied by curved implant frames that can be easily attached to the skull and combined with a lightweight head plate for head-fixation under an imaging apparatus [ 17 , 18 ]. In particular, the “See-Shell” implant designed by Ghanbari and colleagues [ 18 ] provides access to 45 mm 2 of the dorsal cortical surface and can be 3D-printed/laser cut using low-cost machines often found in laboratory settings. Such window designs exhibit robust long-term functionality, minimize the impact on brain tissue, and suggest a standardized surgical procedure. Despite these advantages, such designs have not been tested for acoustic imaging and are still limited in field of view. In parallel, other implants have been proposed to specifically accommodate fUS while being compatible with optical methods [ 19 – 22 ]. However, these implants are designed primarily for single-slice fUS acquisitions and therefore exhibit limited coverage, whereas volumetric fUS can now acquire a much larger portion of the mouse brain in a single acquisition (approximately 1 cm 3 ) [ 23 ]. Finally, the impact of available chronic cranial window implants on mouse behavior has been relatively unexplored even though orofacial movements are increasingly popular readouts of arousal, whisking, sniffing, or emotional states in head-fixed contexts [ 24 , 25 ].

Acquiring large neural activity datasets from distant and interconnected regions is paramount to understanding the neural correlates of behavior [ 1 , 2 ]. For animal models with small brains, such as zebrafish and fruit flies, optical imaging approaches can fulfill this need by acquiring whole-brain activity during behavior at cellular resolution [ 3 , 4 ]. By contrast, in the larger mouse brain, no such optimal technique is currently available. Large-scale recording modalities such as functional MRI (fMRI), wide-field Ca 2+ imaging, and more recently functional ultrasound (fUS) imaging exhibit a relatively low spatiotemporal resolution. On the other hand, techniques with cellular resolution such as two-photon microscopy and electrophysiology suffer from limited brain coverage. Therefore, as no single modality is capable of imaging whole-brain activity in mice at the level of individual neurons, alternative strategies using existing tools must be employed. One recent strategy is to utilize techniques with low brain coverage (e.g., neuropixel recordings) in a highly parallelized manner to acquire large quantities of neuronal recordings across different regions of the brain during the same standardized behavioral task [ 5 – 7 ]. An alternative strategy that is more suitable to non-standardized behaviors is to combine different modalities such that information from multiple spatiotemporal scales is acquired in the same animals. In this case, large-scale recordings can inform which individual regions should be investigated in more detail without a priori knowledge [ 8 ]. Beyond acquiring neural data, it is increasingly popular to manipulate specific neural circuits (e.g., with optogenetics) to determine their causal impact on behavior. Combining optogenetics and large-scale imaging is a powerful approach to identify unexpected regions modulated by specific manipulations and to guide targeted recordings [ 9 ]. However, combining all these techniques in a single animal is technically challenging and therefore relatively rare.

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002664

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