Neuronal recordings in head-fixed and freely-moving mole-rats

Abstract Mole-rats are subterranean rodents that have evolved remarkable sensory adaptations to life in underground tunnel systems, yet their neural mechanisms remain largely unexplored. Here, we present a protocol for in vivo electrophysiological recordings in awake, head-fixed, and freely moving African mole-rats ( Fukomys anselli/micklemi ), overcoming unique challenges of studying the neurobiology of subterranean species. For example, we find that mole-rat brain physiology impacts survival after surgeries, with higher carbon dioxide concentrations required for recovery compared to other rodents, likely due to a mutation in the chloride-potassium symporter KCC2. Having addressed the challenges, we used tetrodes and Neuropixels probes to record single-unit activity and local field potentials (LFP) across several cortical and subcortical regions for several weeks. We observed single units responsive to auditory and visual stimuli in the superior colliculus, and hippocampal recordings in freely moving mole-rats revealed prominent theta rhythms at frequencies lower than those observed in any other rodent species to date. Finally, we performed integrated three-dimensional and two-dimensional probe-track analysis within the same brain using tissue clearing, light sheet imaging, rehydration, and vibratome sectioning, and we present a newly developed stereotaxic brain atlas for implantation and histological alignment. The established methodology will guide future studies in comparative rodent neurobiology, providing further insights into neurobiological adaptations to subterranean environments. Given their phylogenetic and ecological similarities, we expect our protocols to be transferable to other subterranean species, including the widely studied naked mole-rat ( Heterocephalus glaber ). Highlights Protocols for chronic and acute electrode implantations in mole-rats Stereotaxic brain atlas for the Ansell’s mole-rat ( https://doi.org/10.17617/3.UNDKRO ) Neuropixels and tetrode single-unit recordings in head-fixed and freely moving mole-rats Integrated 3D (tissue clearing) and 2D (histology) probe-track analysis within the same brain Discovery of low-frequency hippocampal theta rhythm in Ansell’s mole-rats