Estimating the effects of slicing on the electrophysiological properties of spinal motoneurons under normal and disease conditions

Although slice recordings from spinal motoneurons (MNs) are being widely used, the effects of slicing on the measured MN electrical properties under normal and disease conditions have not been assessed. Using high-fidelity cell models of neonatal WT and SOD cells, we examined the effects of slice thickness, soma position within the slice, and slice orientation to estimate the error induced in measured MN electrical properties from spinal slices. Our results show that most MN electrical properties are not adversely affected by slicing, except for cell time constant, cell capacitance, and Ca2+ PIC, which all exhibited large errors, regardless of the slice condition. Among the examined factors, soma position within the slice appears to be the strongest factor in influencing the magnitude of error in measured MN electrical properties. Transverse slices appear to have the least impact on measured MN electrical properties. Surprisingly, and despite their anatomical enlargement, we found that G85R-SOD MNs experience similar error in their measured electrical properties to those of WT MNs, but their errors are more sensitive to the soma position within the slice than WT MNs. Unless in thick and symmetrical slices, slicing appears to reduce motoneuron type differences. Accordingly, slice studies should attempt to record from MNs at the slice center to avoid large and inconsistent errors in measured cell properties and have valid cell measurements' comparisons. Our results, therefore, offer information that would enhance the rigor of MN electrophysiological data measured from the slice preparation under normal and disease conditions.

Microglia perform local protein synthesis at perisynaptic and phagocytic structures

Recent studies have illuminated the importance of several key signaling pathways in regulating the dynamic surveillance and phagocytic activity of microglia. Yet little is known about how these signals result in the assembly of phagolysosomal machinery near targets of phagocytosis, especially in processes distal from the microglial soma. Neurons, astrocytes, and oligodendrocytes locally regulate protein translation within distal processes. Therefore, we tested whether there is regulated local translation within peripheral microglia processes (PeMPs). We show that PeMPs contain ribosomes which engage in de novo protein synthesis, and these associate with a subpool of transcripts involved in pathogen defense, motility, and phagocytosis. Using a live slice preparation, we further show that acute translation blockade impairs the formation of PeMP phagocytic cups, the localization of lysosomal proteins within them, and phagocytosis. Collectively, these data argue for a regulated local translation in PeMPs and indicate a need for new translation to support dynamic microglial function.

Metabolic changes in brain slices over time: a multiplatform metabolomics approach

ABSTRACTBrain slice preparations are widely used for research in neuroscience. However, a high-quality preparation is essential and there is no consensus regarding stable parameters that can be used to define the status of the brain slice preparation after its collection at different time points. Thus, it is critical to establish the best experimental conditions for ex-vivo studies using brain slices for electrophysiological recording. In this study, we used a multiplatform (LC-MS and GC-MS) untargeted metabolomics-based approach to shed light on the metabolome and lipidome changes induced by the brain slice preparation process. We have found significant modifications in the levels of 300 compounds, including several lipid classes and their derivatives, as well as metabolites involved in the GABAergic pathway and the TCA cycle. All these preparation-dependent changes in the brain biochemistry should be taken into consideration for future studies to facilitate non-biased interpretations of the experimental results.