Phenotyping spontaneous locomotor activity in inbred and outbred mouse strains by using Digital Ventilated Cages

Mouse strains differ markedly in all behaviors, independently of their genetic background. We undertook this study to disentangle the diurnal activity and feature key aspects of three non-genetically altered mouse strains widely used in research, C57BL/6NCrl (inbred), BALB/cAnNCrl (inbred) and CRL:CD1(ICR) (outbred). With this aim, we conducted a longitudinal analysis of the spontaneous locomotor activity of the mice during a 24-h period for 2 months, in two different periods of the year to reduce the seasonality effect. Mice (males and females) were group-housed in Digital Ventilated Cages (Tecniplast), mimicking standard housing conditions in research settings and avoiding the potential bias provided in terms of locomotor activity by single housing. The recorded locomotor activity was analyzed by relying on different and commonly used circadian metrics (i.e., day and night activity, diurnal activity, responses to lights-on and lights-off phases, acrophase and activity onset and regularity disruption index) to capture key behavioral responses for each strain. Our results clearly demonstrate significant differences in the circadian activity of the three selected strains, when comparing inbred versus outbred as well as inbred strains (C57BL/6NCrl versus BALB/cAnNCrl). Conversely, males and females of the same strain displayed similar motor phenotypes; significant differences were recorded only for C57BL/6NCrl and CRL:CD1(ICR) females, which displayed higher average locomotor activity from prepuberty to adulthood. All strain-specific differences were further confirmed by an unsupervised machine learning approach. Altogether, our data corroborate the concept that each strain behaves under characteristic patterns, which needs to be taken into consideration in the study design to ensure experimental reproducibility and comply with essential animal welfare principles

Mouse Panx1 Is Dispensable for Hearing Acquisition and Auditory Function

Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of Panx1 on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the Panx1−/− mouse strain, the first with a reported global ablation of Panx1, were scrutinized. Male and female homozygous (Panx1−/−), hemizygous (Panx1+/−) and their wild type (WT) siblings (Panx1+/+) were used for this study. Successful ablation of Panx1 was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of Panx1−/− mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in Panx1−/− cochleae. Hearing sensitivity, outer hair cell-based “cochlear amplifier” and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in Panx1+/− and Panx1−/− mice. In addition, we determined that global deletion of Panx1 impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca2+ signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by Panx1 ablation. Therefore, our results provide strong evidence that, in mice, Panx1 is dispensable for hearing acquisition and auditory function

Manuale per la gestione integrata degli stabulari. Principi e indicazioni per la protezione degli animali e la sicurezza dei lavoratori

Il volume è l’edizione aggiornata del “Manuale per la gestione integrata degli stabulari” - principi e indicazioni per la protezione degli animali e la sicurezza dei lavoratori – (2012), divenuta necessaria in seguito alla emanazione della Direttiva 2010/63/UE, revisione della Direttiva 86/609, recepita in Italia con il Decreto Legislativo 26/2014 il 4 marzo 2014. Il Manuale è redatto secondo un approccio integrato che considera contestualmente sia gli aspetti riguardanti la sperimentazione animale sia quelli di sicurezza e salute degli operatori e dei ricercatori

Natural killer cells modulate motor neuron-immune cell cross talk in models of Amyotrophic Lateral Sclerosis.

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, hSOD1G93A, 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, TDP43A315T. NK cells are neurotoxic to hSOD1G93A 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 hSOD1G93A 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

Antibiotics treatment promotes vasculogenesis in the brain of glioma-bearing mice

In recent years, several studies described the close relationship between the composition of gut microbiota and brain functions, highlighting the importance of gut-derived metabolites in mediating neuronal and glial cells cross-talk in physiological and pathological condition. Gut dysbiosis may affects cerebral tumors growth and progression, but the specific metabolites involved in this modulation have not been identified yet. Using a syngeneic mouse model of glioma, we have investigated the role of dysbiosis induced by the administration of non-absorbable antibiotics on mouse metabolome and on tumor microenvironment. We report that antibiotics treatment induced: (1) alteration of the gut and brain metabolome profiles; (2) modeling of tumor microenvironment toward a pro-angiogenic phenotype in which microglia and glioma cells are actively involved; (3) increased glioma stemness; (4) trans-differentiation of glioma cells into endothelial precursor cells, thus increasing vasculogenesis. We propose glycine as a metabolite that, in ABX-induced dysbiosis, shapes brain microenvironment and contributes to glioma growth and progression

Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing

In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT

Microglia control glutamatergic synapses in the adult mouse hippocampus

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

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function

Microglia modulates hippocampal synaptic transmission and sleep duration along the light/dark cycle

Microglia, the brain's resident macrophages, actively contributes to the homeostasis of cerebral parenchyma by sensing neuronal activity and supporting synaptic remodeling and plasticity. While several studies demonstrated different roles for astrocytes in sleep, the contribution of microglia in the regulation of sleep/wake cycle and in the modulation of synaptic activity in the different day phases has not been deeply investigated. Using light as a zeitgeber cue, we studied the effects of microglial depletion with the colony stimulating factor-1 receptor antagonist PLX5622 on the sleep/wake cycle and on hippocampal synaptic transmission in male mice. Our data demonstrate that almost complete microglial depletion increases the duration of NREM sleep and reduces the hippocampal excitatory neurotransmission. The fractalkine receptor CX3CR1 plays a relevant role in these effects, because cx3cr1GFP/GFP mice recapitulate what found in PLX5622-treated mice. Furthermore, during the light phase, microglia express lower levels of cx3cr1 and a reduction of cx3cr1 expression is also observed when cultured microglial cells are stimulated by ATP, a purinergic molecule released during sleep. Our findings suggest that microglia participate in the regulation of sleep, adapting their cx3cr1 expression in response to the light/dark phase, and modulating synaptic activity in a phase-dependent manner.Bordeaux Region Aquitaine Initiative for Neuroscienc