Background and aimsAutomated recording of laboratory animal’s home cage behavior is receiving increasing attention since such non-intruding surveillance will aid in the unbiased understanding of animal cage behavior potentially improving animal experimental reproducibility.Material and methodsHere we investigate activity of group held female C57BL/6J mice (mus musculus) housed in standard Individually Ventilated Cages across three test-sites: Consiglio Nazionale delle Ricerche (CNR, Rome, Italy), The Jackson Laboratory (JAX, Bar Harbor, USA) and Karolinska Insititutet (KI, Stockholm, Sweden). Additionally, comparison of female and male C57BL/6J mice was done at KI. Activity was recorded using a capacitive-based sensor placed non-intrusively on the cage rack under the home cage collecting activity data every 250 msec, 24/7. The data collection was analyzed using non-parametric analysis of variance for longitudinal data comparing sites, weekdays and sex.ResultsThe system detected an increase in activity preceding and peaking around lights-on followed by a decrease to a rest pattern. At lights off, activity increased substantially displaying a distinct temporal variation across this period. We also documented impact on mouse activity that standard animal handling procedures have, e.g. cage-changes, and show that such procedures are stressors impacting in-cage activity.These key observations replicated across the three test-sites, however, it is also clear that, apparently minor local environmental differences generate significant behavioral variances between the sites and within sites across weeks. Comparison of gender revealed differences in activity in the response to cage-change lasting for days in male but not female mice; and apparently also impacting the response to other events such as lights-on in males. Females but not males showed a larger tendency for week-to-week variance in activity possibly reflecting estrous cycling.ConclusionsThese data demonstrate that home cage monitoring is scalable and run in real time, providing complementary information for animal welfare measures, experimental design and phenotype characterization.

In amyotrophic lateral sclerosis (ALS), immune cells and glia contribute to motor neuron (MN) degeneration. We report the presence of NK cells in post-mortem ALS motor cortex and spinal cord tissues, and the expression of NKG2D ligands on MNs. Using a mouse model of familial-ALS, hSOD1 G93A , we demonstrate NK cell accumulation in the motor cortex and spinal cord, with an early CCL2-dependent peak. NK cell depletion reduces the pace of MN degeneration, delays motor impairment and increases survival. This is confirmed in another ALS mouse model, TDP43 A315T . NK cells are neurotoxic to hSOD1 G93A MNs which express NKG2D ligands, while IFNγ produced by NK cells instructs microglia toward an inflammatory phenotype, and impairs FOXP3 + /Treg cell infiltration in the spinal cord of hSOD1 G93A mice. Together, these data suggest a role of NK cells in determining the onset and progression of MN degeneration in ALS, and in modulating Treg recruitment and microglia phenotype.

Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3‐CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX‐induced synaptic changes were absent in Cx3cr1−/− mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses.

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action.Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies.