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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.

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.

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

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