SMART: An Open-Source Extension of WholeBrain for Intact Mouse Brain Registration and Segmentation

Abstract

Mapping immediate early gene (IEG) expression across intact mouse brains allows for unbiased identification of brainwide activity patterns underlying complex behaviors. Accurate registration of sample brains to a common anatomic reference is critical for precise assignment of IEG-positive ("active") neurons to known brain regions of interest (ROIs). While existing automated voxel-based registration methods provide a high-throughput solution, they require substantial computing power, can be difficult to implement and fail when brains are damaged or only partially imaged. Additionally, it is challenging to cross-validate these approaches or compare them to any preexisting literature based on serial coronal sectioning. Here, we present the open-source R package SMART (Semi-Manual Alignment to Reference Templates) that extends the WholeBrain R package framework to automated segmentation and semi-automated registration of intact mouse brain light-sheet fluorescence microscopy (LSFM) datasets. The SMART package was created for novice programmers and introduces a streamlined pipeline for aligning, registering, and segmenting LSFM volumetric datasets across the anterior-posterior (AP) axis, using a simple "choice game" and interactive menus. SMART provides the flexibility to register whole brains, partial brains or discrete user-chosen images, and is fully compatible with traditional sectioned coronal slice-based analyses. We demonstrate SMART's core functions using example datasets and provide step-by-step video tutorials for installation and implementation of the package. We also present a modified iDISCO1 tissue clearing procedure for uniform immunohistochemical labeling of the activity marker Fos across intact mouse brains. The SMART pipeline, in conjunction with the modified iDISCO1 Fos procedure, is ideally suited for examination and orthogonal cross-validation of brain-wide neuronal activation datasets.

Fluorescent transgenic mouse models for whole-brain imaging in health and disease

“…Simultaneous with the development of imaging technologies, the need for image processing tool sets for terabyte-sized datasets has emerged. The development of a common atlas of the mouse brain (Common Coordinate Framework v3; Wang et al, 2020), combined with image stitching (Bria and Iannello, 2012;Wang et al, 2020), transformation to multi-resolution image formats (Bria et al, 2016), and spatial registration (Tward et al, 2020;Chandrashekhar et al, 2021;Jin et al, 2022;Qu et al, 2022), allows the quantification of cell densities (Renier et al, 2016), axonal projections (Ye et al, 2016), vasculature (Kirst et al, 2020), and the reconstruction of full single neurons (Winnubst et al, 2019;Peng et al, 2021;Gao et al, 2022).…”

UNRAVELing the synergistic effects of psilocybin and environment on brain-wide immediate early gene expression in mice

“…To study the effect of environmental context on psychedelic-elicited states in mice, we mapped, at cellular resolution, neural activities related to context, psilocybin, and the interaction between these factors. To capture experience-dependent brain activity, we performed immunofluorescent labeling of c-Fos, an immediate early gene (IEG) product and transcription factor that is transiently expressed after neural activation (Sagar et al, 1988), followed by brain clearing via iDISCO+ (Renier et al, 2016) and light sheet fluorescent microscopy (LSFM) (Davoudian et al, 2023; Hansen et al, 2021; Jin et al, 2022; Renier et al, 2016). Psilocybin or saline was administered to mice in two distinct contexts, a familiar home cage or an enriched environment (EE) known to promote neural and behavioral plasticity (Nithianantharajah & Hannan, 2006).…”

Incubation of palatable food craving is associated with brain-wide neuronal activation in mice

“…To address this knowledge gap, we leveraged recent developments in brain-wide activity mapping approaches, including whole mouse brain immunofluorescent staining and clearing ( 30 – 35 ), light sheet fluorescence microscopy (LSFM) ( 36 , 37 ), and open-source analysis tools ( 38 – 41 ) to investigate changes in brain-wide activation patterns during incubation of palatable food seeking in mice.…”

“…We trained food-sated CD-1 male mice to self-administer palatable high-carbohydrate food pellets ( 42 , 43 ) for 7 d and then tested them for relapse to food seeking after 1, 15, or 60 abstinence days. We perfused and extracted their brains 90 min after the relapse tests (or directly from homecage as a baseline activity control), labeled “active” Fos+ nuclei across intact mouse brains using an optimized iDISCO+ Fos immunofluorescent staining protocol ( 41 ) and imaged Fos immunofluorescence at single-cell resolution using LSFM. We used the ClearMap pipeline ( 39 , 40 ) for unbiased mapping of “incubation-associated” neural activation patterns across the entire anterior–posterior (AP) axis of the mouse brain.…”

“…We used the ClearMap pipeline ( 39 , 40 ) for unbiased mapping of “incubation-associated” neural activation patterns across the entire anterior–posterior (AP) axis of the mouse brain. We also updated the SMART analysis package ( 41 ) to conduct targeted analysis of neural activation patterns within LSFM coronal subvolumes and used SMART2 to cross-validate and extend our ClearMap findings within a subset of corticostriatal and thalamocortical brain regions and subdivisions.…”