GoKawiilAnimals
All the mice handling procedures followed the Animal Welfare Act and the German guidelines of the State Agency for Consumer Protection and Nutrition (LAVE) North Rhine-Westphalia. The animals were housed in groups of up to five in individually ventilated cages from the Tecniplast Green Line. The following parameters were applied: 12-h light–12-h dark cycle (6:00–18:00/18:00–6:00), 22 °C temperature and 60% humidity. For environmental enrichment, nestlets for nest building and red-coloured hiding places (houses, tubes) were provided.
Mice homozygous for the loxP-site flanked Actr3 allele (Actr3fl/fl) were generated as previously described48,76,77,78. To specifically knockout Actr3 in the CNS, Actr3fl/fl mice were mated with transgenic Nestin-Cre mice (Nestin-cretg/−, Jackson Laboratory), in which the Cre recombinase is specifically expressed in the CNS from E12.5 (ref. 78). The resulting Nestin-cretg/−Actr3fl/WT mice were then backcrossed with the Actr3fl/fl line. The embryos with the Nestin-cretg/−Actr3fl/fl genotype are referred to as Actr3 KO, and those with the Nestin-cre−/−Actr3fl/fl genotype or embryos of WT C57BL/6 mice (Nestin-cre−/−Actr3WT/WT) are referred to as WT. The genotypes of all mice used in this study were verified by PCR77.
Primary embryonic neuron culture
The procedure was based on a previously described protocol79. Cultured neurons were incubated in a 36.5 °C chamber supplied with 5% CO 2 .
Primary mouse hippocampal neurons were dissected from the brains of E17.5 mice. Tails of embryos were collected for PCR genotyping. Brains from WT and Actr3 KO mice were separated according to the genotype. Neurons of dissected regions were separated and dissociated in trypsin solution (0.05% trypsin-EDTA, 7 mM HEPES pH 7.3) at 37 °C for 15 min. After washing once in HBSS supplemented with DNase and twice in HBSS, the cells were dissociated in MEM-HS (1× MEM, 5% horse serum, 0.22% NaHCO 3 , 0.6% glucose, 2 mM glutamine, 1× essential and non-essential amino acids, pH 7.3). After mechanical dissociation with fire-polished Pasteur glass pipettes, we counted and adjusted the neuron density.
For immunocytochemistry, neurons were plated on PLL-coated 15-mm round glass coverslips immersed in MEM-HS (VWR, Marienfeld 630-1597) at a density of around 70 cells per mm2. After 2 h of incubation in the culture chamber, MEM-HS was replaced with glia-conditioned N2 medium (1× MEM, 1 mM sodium pyruvate, 1% Neuropan2 supplement (Pan-Biotech), 0.22% NaHCO 3 , 0.6% glucose, 2 mM L-glutamine and 2% B27 supplement).
For live-cell imaging of hippocampal neurons, 5 × 105 cells and 3 µg plasmid DNA encoding the gene of interest fused with a selected fluorescent protein were transferred to a nucleofection cuvette for electroporation (built-in program 0-005) with an Amaxa Nucleofector II (Lonza). After electroporation, 7.5 × 104 cells were plated on PLL-coated glass-bottom 8-well or 4-well chamber slides (Ibidi). After 2 h of incubation, MEM-HS was replaced with astrocyte-conditioned N2 medium. We acquired the images immediately after exchange of the medium or after an additional 16 h of incubation.
Neuron cultures in 3D collagen matrix were conducted in accordance with our previously published protocol71. In brief, the matrix solution (3.4 mg ml–1 collagen and 1× MEM, 0.3% NaHCO 3 ) was prepared on ice to keep the solution in liquid phase. After mixing with a suspension of neurons (matrix solution to neuron suspension ratio of 3:1), a 40-µl drop of matrix–cell mix with a neuron density of 0.75–1.5 × 106 cells per ml was directly applied to the bottom of each well of 8-well glass bottom chamber slides (Ibidi). After incubation in a neuron culture chamber for 20 min, conditioned N2 medium was added to fully cover the solidified matrix droplet.
For experiments manipulating the neuronal cytoskeleton, chemical stock solutions were diluted in glia-conditioned N2 medium to achieve the indicated working concentrations. DMSO of the same volume of the added stock solution was used as controls. Treatment of cultured neurons was initiated after exchange with MEM-HS after 2 h of seeding. On the basis of published works56,59,65,80, the final concentration of each chemical was as follows: 20 µM blebbistatin, 40 µM para-blebbistatin, 100–200 µM CK-666, 5 nM taxol and 75 nM nocodazole. For the washout experiments, we replaced the medium containing the indicated chemicals with fresh conditioned N2 medium at 24 h after plating.
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