Journal: Neural Regeneration Research

Article Title: Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning

doi: 10.4103/1673-5374.285003

Figure Lengend Snippet: Effects of HPC on the degradation of spectrin in HT22 cells. (A) The protein levels of 140/145 kDa spectrin and 120 kDa spectrin were measured by western blot assay. HPC decreased the levels of necrotic and excitotoxic neuronal death (associated with 140/145 kDa spectrin). (B) Semi-quantitative analysis of the changes in 140/145 kDa spectrin protein levels. (C) Semi-quantitative analysis of changes in the protein levels of 120 kDa spectrin. * P < 0.05, vs. C group; # P < 0.05, vs . H group. The grey densitometric values of western blot assays were normalized against the loading control (β-actin). Data are expressed as the mean ± SD ( n = 3; one-way analysis of variance followed by Tukey's honest significant difference post hoc test). The experiment was conducted in triplicate. C: Control; H: hypoxia; HPC: hypoxic preconditioning.

Article Snippet: The membranes were incubated with primary antibodies at 4°C for 12 hours as follows: rabbit anti-mouse DNMT1, DNMT3A, and DNMT3B polyclonal antibodies (1:1000; all from Novus Biologicals, Littleton, CO, USA), rabbit anti-mouse β-actin monoclonal antibody (1:1000; Sigma), rabbit anti-mouse caspase-3 polyclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-mouse spectrin polyclonal antibody (1:1000; Cell Signaling Technology).

Techniques: Western Blot

Journal: Science Advances

Article Title: An axon initial segment is required for temporal precision in action potential encoding by neuronal populations

doi: 10.1126/sciadv.aau8621

Figure Lengend Snippet: ( A ) The axon cytoskeleton in the AIS is highly regular. Tetramers of α- and β-spectrin serve as 190-nm-long spacers between rings of actin (green) and adducin (blue) ( , ). ( B ) Na V and K V channels are anchored to AnkG (light blue), which binds to βIV-spectrin and the lipid membrane (blue double layer). ( C ) The qv 3J mutation affects the very C-terminal portion of βIV-spectrin. We hypothesized that this might lead to a reduction in the number of intact tetramers, and hence, a reduction of AnkG and ion channels bound to it, while other channels (green) might be unaffected. The relative sizes of cytoskeletal proteins follow measures from electron microscopy . ( D ) We used a compacted version of a detailed biophysical model of pyramidal neurons to study AP initiation under reduced AIS channel densities. The conductance densities of two Na channel subtypes in the model are displayed here (continuous lines, somatic; dashed lines, axonal channel variant with more hyperpolarized activation curve). All other conductances were also scaled down proportionally but are omitted here for clarity. Color code in (D) to (I) is identical, and steps are identical in (E) and (F). Note that for a 90% reduction (red), the somatic conductance density is higher than the axonal one; for 95% reduction (orange), the soma/axon density ratio is 3:1. ( E ) The voltages occurring during AP onset in the soma and 50 μm into the axon are plotted against each other. Even when axonal channel density is lower than somatic, the AP still started in the axon. Only for a 99% reduction of channel densities (soma/axon density ratio, 15:1), no sign of axonal initiation could be detected. ( F ) Phase plots, plotting the first temporal derivative of the somatic membrane voltage dV m / dt against V m , also showed a gradual change in the properties of the AP onset, as the axonal channel densities were reduced. Threshold was shifted by 20 mV, and the initial lateral current into the soma was less pronounced; in particular, the initial slope in the phase plot, called onset rapidness, decreased from 29.8 to 4.5 ms −1 . In contrast, the second phase of the AP waveform remained largely unchanged. Biphasic phase plots, indicating axonal initiation of APs, were obtained for density reduction as severe as 95%. ( G ) AP threshold (squares) and onset rapidness (circles) plotted against the degree of axonal channel density reduction. ( H ) Dynamic gain curves were calculated from 10 6 APs for each condition (see Materials and Methods). Dynamic gain curves showed a reduced bandwidth when the channel densities in the axonal compartment were reduced to 10% in six exponentially spaced steps. Loss of axonal initiation (yellow) reduced the bandwidth further. ( I ) Cutoff frequencies, defined as the frequencies at which the gain reaches 60% of maximum, drop as the axonal channel density is reduced, although for all densities probed, the AP starts in the soma, not in the axon.

Article Snippet: The primary antibodies that were used in this study were as follows: mouse monoclonal anti-sodium channel (pan-Na V ) (1:900, clone K58/35; Sigma-Aldrich, S8809-1MG), goat polyclonal anti–βIV-spectrin antibody (1:200, OriGene, TA317365, targeting N-terminal sequence, amino acids 2 to 14), rabbit polyclonal anti–βIV-spectrin antibody (1:200, Atlas, HPA043370, targeting a centrally located sequence), rabbit polyclonal anti-AnkG antibody (1:200, Santa Cruz Biotechnology, sc-28561, targeting C-terminal sequence), mouse monoclonal anti-AnkG antibody (1:200, Santa Cruz Biotechnology, sc-12719, targeting N-terminal sequence), chicken polyclonal anti-Map2 (1:2000, Abcam, ab5392), and mouse monoclonal anti–βII-spectrin (1:200, Santa Cruz Biotechnology, sc-136074).

Techniques: Membrane, Mutagenesis, Electron Microscopy, Variant Assay, Activation Assay

Journal: Science Advances

Article Title: An axon initial segment is required for temporal precision in action potential encoding by neuronal populations

doi: 10.1126/sciadv.aau8621

Figure Lengend Snippet: ( A ) dSTORM images of AISs of control (top) and mutant (bottom) neurons, labeled with antibodies against AnkG (N terminus) at 13 to 14 DIV (for 10, 11, and 19 DIV, see fig. S6). Scale bars, 1 μm. ( B ) Power spectra analysis of AnkG immunofluorescence profile along the AIS of control, n = 28 (3, 3), and mutant, n = 53 (3, 3), demonstrating periodic pattern in both populations, with a periodic length of about 190 nm. Examples of individual immunofluorescence profiles along 1-μm segments are shown in the inset. ( C and D ) Same as (A) and (B) but with antibodies against Na V , demonstrating periodic pattern with 190-nm periodicity in control and mutant (see fig. S6C for reduced periodicity in qv 3J at 19 DIV). n control = 33 (3, 2), n mutant = 29 (4, 3). Error bars represent SEM. Replication numbers refer to cells (animals, preps). ( E ) Antibodies against AnkG (C terminus) label AIS (gray) in control and mutant cells (11 to 13 DIV). In the framed regions, βII-spectrin dSTORM imaging was performed (red). ( F ) Power spectra of βII-spectrin immunofluorescence profiles along the AIS show 190-nm periodicity in mutant, n = 14 (1, 1), and control cells, n = 25 (3, 2). βII-spectrin structural organization appears unaffected by the qv 3J mutation. Error bars represent SEM. ( G and H ) Cultures of 8 DIV control (G) and qv 3J mutant (H) neurons double labeled with antibodies against βII-spectrin (magenta) and AnkG (green) (C terminus) and 4′,6-diamidino-2-phenylindole (DAPI; nuclei, blue) (separate images in fig. S6). ( I ) βII-spectrin expression in control and qv 3J mutant neurons at three maturation stages. Fluorescence intensity of βII-spectrin label was averaged over 50.0 μm into the AIS. The results are similar for mutant and control and show no rescue effect for the βIV-spectrin deficiency. βII-spectrin expression was reduced with development. n control = 36 (3, 2), 22 (2, 1), and 27 (2, 2); n mutant = 32 (2, 1), 11 (1, 1), and 22 (2, 2). Error bars represent SEM. Replication numbers refer to cells (animals, preps).

Article Snippet: The primary antibodies that were used in this study were as follows: mouse monoclonal anti-sodium channel (pan-Na V ) (1:900, clone K58/35; Sigma-Aldrich, S8809-1MG), goat polyclonal anti–βIV-spectrin antibody (1:200, OriGene, TA317365, targeting N-terminal sequence, amino acids 2 to 14), rabbit polyclonal anti–βIV-spectrin antibody (1:200, Atlas, HPA043370, targeting a centrally located sequence), rabbit polyclonal anti-AnkG antibody (1:200, Santa Cruz Biotechnology, sc-28561, targeting C-terminal sequence), mouse monoclonal anti-AnkG antibody (1:200, Santa Cruz Biotechnology, sc-12719, targeting N-terminal sequence), chicken polyclonal anti-Map2 (1:2000, Abcam, ab5392), and mouse monoclonal anti–βII-spectrin (1:200, Santa Cruz Biotechnology, sc-136074).

Techniques: Control, Mutagenesis, Labeling, Immunofluorescence, Imaging, Expressing, Fluorescence