Multiplexed neuropeptide mapping in ant brains integrating microtomography and 3D mass spectrometry imaging

Neuropeptides are important regulators of animal physiology and behavior. Hitherto large-scale localization of neuropeptides mainly relied on immunohistochemical methods requiring the availability of antibody panels, while another limiting factor has been the brain’s opacity for subsequent light or fluorescence microscopy. To address these limitations, we integrated 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 3D distributions of eight conserved neuropeptides throughout the brain micro-anatomy. 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 TK1 and TK4, were widely distributed in many brain areas of both ant species, whereas others, for instance myosuppressin was 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 . Our approach provides the basis for investigating fundamental neurobiological processes by visualizing the unbiased 3D neurochemistry in its complex anatomic environment. Significance statement Until recently, the inability to detect entire molecules such as neuropeptides within their spatial biological context and simultaneously link their occurrence to anatomically and physiologically relevant areas has limited our understanding of complex neurochemical processes. This situation has now changed dramatically with the optimization of a new multiplexed imaging method based on mass spectrometry, which enables us to study previously invisible processes at the microscopic scale. With the marriage of mass spectrometry imaging and microtomography, we show that it has become possible to build high-resolution maps of neuropeptides in complex anatomical structures as small as ant brains. These maps, embedded in the 3D neuroanatomy, expand the understanding of the spatial organization of brain chemistry and provide a baseline for neurobiological and neurochemical studies. ### Competing Interest Statement J.O is employed at Bruker Daltonics GmbH & Co. KG, a vendor for mass spectrometry instruments and solutions.