Computational protocol: Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength

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Protocol publication

[…] Total RNA from FACS AS was extracted with Trizol (Invitrogen) and purified using the RNeasy Kit (QIAGEN). cDNA was generated from full-length RNA using the NuGEN RNA-Seq V2 kit which uses the single primer isothermal amplification method to deplete ribosomal RNA, and sheared by Covaris to yield uniform size fragments. The NuGen Ultralow kit was used to add adapters, barcoding, and amplification. Library was purified using Agencourt XP magnetic beads, quality controlled with an agilent bioanalyzer, and quantified by qPCR. Eight libraries were pooled per lane for single end (SE75) sequencing. Over 51M reads were sequenced per sample (range 38-66M reads) using a NextSeq 500.Read quality was assessed using fastqc (version 0.11.4) () and the first 5 bases as well as the last base of each read were trimmed and aligned to the Mus musculus genome (Ensembl GRCm38) () using TopHat2 (version 2.0.11) with bowtie2 (version 2.2.3) (). TopHat2 was run with the following arguments “–no-coverage-search–segment-mismatches 1” with the genes.gtf file from Ensembl GRCm38. Gene counts were created from the alignment files using SAMtools (version 0.1.19) () to generate BAM files and subsequently htseq-count (version 0.6.1p1) with default parameters () to create count files. From raw count files, DESeq2 () was used to detect differentially expressed genes. FPKM values were generated using cuffquant and cuffnorm from the cufflinks suite of software tools (). [...] Motor neuron soma size and counts were performed on maximal intensity projections of 10 μm z stacks with a 2 μm z-step of lumbar (L3-L6) levels on 2-4 sections per animal. Soma size was determined by tracing the ChAT-GFP signal using ImageJ software. Measurements were performed on the optical section with the largest soma area and only on motor neurons that had a DAPI nucleus visible. Similarly, motor neurons were only counted if a DAPI+ nucleus was present. Phopho-S6 fluorescence intensity (IR) was measured using the mean gray value function on ImageJ within a ROI delineating individual fast MMP-9+ MN soma, which assures value normalization according to the size of the selected area (i.e., MN soma). In addition, as immunofluorescence intensity can vary between sets of experiments (for example, depending on tissue fixation), the absolute values of pS6 IR were normalized to the value in their respective internal controls ( =1) for both LOF and GOF experiments. Quantifications were performed on 40x confocal images (12 μm z stack, 1 μm z-step) at the lumbar (L3-L6) spinal cord levels on 5-8 sections per mouse. For the contact/non-contact soma area ratio quantification, MN soma area was quantified on merged images with transduced astrocytes in both groups. A potential limitation to this analysis is the fact that contact was defined based on 16 μm-thick sections. There may still be contact from astrocytes out of the section, which could inflate the observed effect in what we are labeling as non-contacted MNs. All quantifications regarding MN number and soma area were performed under blind conditions for experimental group (genotype, viral vectors injection or treatment groups). [...] For AS Kir4.1 intensity quantification in Aldh1L1-GFP-labeled AS (C), we quantified signal on maximal intensity projections of 10 μm z stacks with a 1 μm z-step using cellular areas centered on Aldh1l1-GFP+ AS somas in ImageJ, since Aldh1l1-GFP signal is restricted to the soma and primary processes. Synaptic area coverage of VGLUT1 and VGLUT2 synapses were similarly obtained from maximal intensity projections of 10 μm z stacks with a 1 μm z-step using the analyze particle function in ImageJ. Thresholds were set identically for each image. For AS Kir4.1 intensity measurements in EAAT2-Td-Tomato-labeled AS (J and 1K), 3D reconstruction was performed on 10–12 μm z -stacks with a 0.5–1 μm z-step using the surface tool in Imaris software, as previously described (). The volume of individual AS can be directly measured from generated 3D domains in Imaris. To quantify Kir4.1 signal within each AS domain, individual domains were masked onto the Kir4.1 channel by setting all pixel signals outside the masked cell domain to zero so that only Kir4.1 signal within the masked AS domain remained. The Kir4.1 signal for each AS domain was quantified using ImageJ software. For AAV transduction quantification, td-Tomato+ or GFP+ area was normalized to the ventral horn total area. The mean fluorescence intensity was also quantified in the ventral spinal cord for the two groups. […]

Pipeline specifications

Software tools ImageJ, Imaris
Application Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Amyotrophic Lateral Sclerosis