The regular firing pattern exhibited by medial entorhinal (mEC) grid cells of locomoting rodents is hypothesized to provide spatial metric information relevant for navigation. The development of virtual reality (VR) for head-fixed mice confers a number of experimental advantages and has become increasingly popular as a method for investigating spatially-selective cells. Recent experiments using 1D VR linear tracks have shown that some mEC cells have multiple fields in virtual space, analogous to grid cells on real linear tracks. We recorded from the mEC as mice traversed virtual tracks featuring regularly spaced repetitive cues and identified a population of cells with multiple firing fields, resembling the regular firing of grid cells. However, further analyses indicated that many of these were not, in fact, grid cells because: (1) when recorded in the open field they did not display discrete firing fields with six-fold symmetry; and (2) in different VR environments their firing fields were found to match the spatial frequency of repetitive environmental cues. In contrast, cells identified as grid cells based on their open field firing patterns did not exhibit cue locking. In light of these results we highlight the importance of controlling the periodicity of the visual cues in VR and the necessity of identifying grid cells from real open field environments in order to correctly characterize spatially modulated neurons in VR experiments.

8The regular firing pattern exhibited by medial entorhinal (mEC) grid cells of locomoting rodents is 9hypothesized to provide spatial metric information relevant for navigation. The development of virtual reality 10 (VR) for head-fixed mice confers a number of experimental advantages and has become increasingly popular 11 as a method for investigating spatially-selective cells. Recent experiments using 1D VR linear tracks have 12 shown that some mEC cells have multiple fields in virtual space, analogous to grid cells on real linear tracks. 13We recorded from the mEC as mice traversed virtual tracks featuring regularly spaced repetitive cues and 14 identified a population of cells with multiple firing fields, resembling the regular firing of grid cells. However, 15 further analyses indicated that many of these were not, in fact, grid cells because: 1) When recorded in the 16 open field they did not display discrete firing fields with six-fold symmetry; 2) In different VR environments 17 their firing fields were found to match the spatial frequency of repetitive environmental cues. In contrast, cells 18 identified as grid cells based on their open field firing patterns did not exhibit cue locking. In light of these 19 results we highlight the importance of controlling the periodicity of the visual cues in VR and the necessity of 20 identifying grid cells from real open field environments in order to correctly characterise spatially modulated 21 neurons in VR experiments. 22