Whole-brain c-Fos mapping reveals distinct activation signatures of reference anti-psychiatric compounds

Understanding how centrally acting compounds engage brain circuits at whole-brain scale remains a major challenge in translational neuropsychopharmacology. Drug-evoked neuronal activation can be mapped using c-Fos, but conventional analyses are often restricted to selected regions and do not readily support unbiased comparison across compounds. We aimed to establish a whole-brain framework to define and compare activation signatures induced by reference compounds with distinct pharmacological profiles, using xanomeline, emraclidine and clozapine as exemplars.

Adult male mice (n=8, per group) received acute treatment with xanomeline, emraclidine, clozapine or vehicle. Mice were transcardially perfused and their brains collected 2h after dosing, processed using iDISCO-based immunolabelling for c-Fos, and imaged using light-sheet fluorescence microscopy (LSFM).

c-Fos positive cells are detected and mapped to one of more than 800+ brain regions found in the digital version of Allen’s Brain Atlas. For each region, a negative binomial generalized linear model (GLM) is fitted to the cell count data, and a subsequent Dunnett’s test is performed for multiple comparisons. The model deviance residuals are investigated to see if they align with the assumptions of Normality and homoscedasticity. Cook’s distance is evaluated for each data point to make sure no point is overly influential in the fitted model. If a region does not pass the validation, it will not appear in the final list of significantly regulated brain regions. In case of ambiguity, a region is not removed from the list. Brain regions will undergo another validation step, to ensure the quantified signal originates from the region of interest and not spill-over from a neighboring region. This can be important for regions in areas of the atlas with low contrast region borders.

We developed cFos 2.0, an artificial intelligence (AI)-based analysis pipeline for unbiased whole-brain mapping. The workflow integrates atlas-based regional quantification, voxel-wise statistics, compound-to-compound similarity analysis, and a reference library of drug-evoked activation patterns. To support biological interpretation, we implemented a functional enrichment analysis that maps curated brain functions onto anatomical regions and tests their overlap with each compound-specific activation signature. Whole-brain analysis demonstrated that each reference compound produced a distinguishable activation profile relative to vehicle. Voxel-wise mapping confirmed both shared and compound-selective hotspots and enabled separation of putative direct versus indirect circuit engagement based.

These data support whole-brain immediate early gene mapping as a translationally useful strategy to characterise neuropsychopharmacological compound signatures beyond predefined regions of interest. cFos 2.0 enables unbiased comparison of reference compounds through regional, voxel-wise and similarity-based analyses, while functional enrichment adds a biologically interpretable layer linking drug-induced activity patterns to brain functions. Extending this framework with alternative activity-dependent markers may improve sensitivity for chronic, subtle or plasticity-related drug effects and broaden its utility for mechanism-of-action studies, reference profiling and compound benchmarking in central nervous system drug discovery

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