Multiplexed neuropeptide mapping in ant brains integrating microtomography and three-dimensional mass spectrometry imaging

Neuropeptides are important regulators of animal physiology and behavior. Hitherto the gold standard for the localization of neuropeptides have been immunohistochemical methods that require the synthesis of antibody panels, while another limiting factor has been the brain's opacity for subsequent in situ light or fluorescence microscopy. To address these limitations, we explored the integration of high-resolution mass spectrometry imaging (MSI) with microtomography for a multiplexed mapping of neuropeptides in two evolutionary distant ant species, Atta sexdens and Lasius niger. For analyzing the spatial distribution of chemically diverse peptide molecules across the brain in each species, the acquisition of serial mass spectrometry images was essential. As a result, we have comparatively mapped the three-dimensional (3D) distributions of eight conserved neuropeptides throughout the brain microanatomy. We demonstrate that integrating the 3D MSI data into high-resolution anatomy models can be critical for studying organs with high plasticity such as brains of social insects. Several peptides, like the tachykinin-related peptides (TK) 1 and 4, were widely distributed in many brain areas of both ant species, whereas others, for instance myosuppressin, were restricted to specific regions only. Also, we detected differences at the species level; many peptides were identified in the optic lobe of L. niger, but only one peptide (ITG-like) was found in this region in A. sexdens. Building upon MS imaging studies on neuropeptides in invertebrate model systems, our approach leverages correlative MSI and computed microtomography for investigating fundamental neurobiological processes by visualizing the unbiased 3D neurochemistry in its complex anatomic environment. Significance Statement Mass spectrometry imaging (MSI) has enabled label-free mapping of molecules in situ and without prior knowledge. Consequently, MSI holds incredible potential for elucidating the function of small molecules, such as neuropeptides, in environmental samples including insects and other invertebrates. However, few studies have integrated the chemical maps with the anatomically and physiologically relevant areas, critical for interpreting the complex neurochemical processes. We designed an integrated high-resolution threedimensional (3D) MSI and microtomography workflow to advance the field of correlative MSI and enable researchers to integrate neuropeptide distributions with the detailed 3D anatomy when applying MSI. We used two ant species with drastically different morphology and lifestyles as social insect models to reconstruct how neuropeptides distribute within their unmodified 3D neuroanatomy.