Journal: Brain structure & function

Article Title: Preservation of KCC2 expression in axotomized abducens motoneurons and its enhancement by VEGF

doi: 10.1007/s00429-023-02635-w

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: ATF3 Used for injured motoneuron identification , Recombinant protein corresponding to aa 1-103 in human ATF3 , Mouse/monoclonal , Novus Clone 1685 NBP2-34489 , AB_2786997 Recognizes the epitope: ASAIVPCLSPPGSL (Manufacturer’s information) Not present in uninjured motoneurons , 1:200.

Techniques: Comparison, Expressing, Recombinant

Journal: Brain structure & function

Article Title: Preservation of KCC2 expression in axotomized abducens motoneurons and its enhancement by VEGF

doi: 10.1007/s00429-023-02635-w

Figure Lengend Snippet: KCC2 immunoreactivity in brainstem oculomotor, trochlear, abducens and facial motoneurons, in control and after axotomy, in the rat. A, C, E, G Confocal images of double immunofluorescence against ChAT (green) and pKCC2 (red) in control oculomotor (A), trochlear (C), abducens (E) and facial (G) motoneurons. B, D, F, H Confocal images of triple immunofluorescence against ChAT (green), KCC2 (red) and ATF3 (white) in the same brainstem nuclei, but after axotomy. Axotomized oculomotor, trochlear and facial motoneurons showed a marked reduction in pKCC2 and ChAT. ATF3 is a general marker of axotomized motoneurons and labels the cell nucleus, as can be observed in axotomized oculomotor (B), trochlear (D) and facial (H) motoneurons (some examples are indicated by white arrows). However, in axotomized abducens motoneurons (F), immunostaining for pKCC2 and ChAT showed a similar appearance to control (E), and ATF3 labeling was absent. I Quantification of KCC2 immunofluorescence in the four nuclei (oculomotor, trochlear, abducens and facial) and in the control and axotomy situation. Asterisks indicate significant (p < 0.001) difference between control and axotomized motoneurons within the same nucleus. Hashtag indicates significant difference (p < 0.001) between axotomized abducens motoneurons and the axotomized motoneurons of the other three nuclei (Two-way ANOVA followed by Holm-Sidak method; n = 35, 36, 33 and 39 for control and n = 33, 35, 44 and 42 for axotomized motoneurons of the oculomotor, trochlear, abducens and facial nuclei, respectively). Data in histograms represent mean ± SEM. Depicted to the right are the Cumming plots of estimated differences after bootstrap resampling with average difference indicated by a dot and 95% CI limit of the distribution by the vertical bars. Oculomotor, trochlear and facial motoneurons all showed significant reduction of 72%, 89% and 87% of the control value, respectively (two-sided permutation t-test p < 0.001 for all comparisons). There were no significant differences when comparing axotomized and control motoneurons in the abducens nucleus. Scale bar = 30 μm in H for A–H

Article Snippet: ATF3 Used for injured motoneuron identification , Recombinant protein corresponding to aa 1-103 in human ATF3 , Mouse/monoclonal , Novus Clone 1685 NBP2-34489 , AB_2786997 Recognizes the epitope: ASAIVPCLSPPGSL (Manufacturer’s information) Not present in uninjured motoneurons , 1:200.

Techniques: Control, Immunofluorescence, Marker, Immunostaining, Labeling

Journal: Brain structure & function

Article Title: Preservation of KCC2 expression in axotomized abducens motoneurons and its enhancement by VEGF

doi: 10.1007/s00429-023-02635-w

Figure Lengend Snippet: KCC2 changes induced by axotomy in cat spinal motoneurons. A–H High magnification single plane confocal images of spinal motoneurons immunostained against ChAT, pKCC2 and ATF3. A–D corresponds to a control motoneuron and E–H to a motoneuron axotomized 21 days previously. Individual immunoreactivities are presented in isolation and then merged (D, H), as indicated in the figure. Axotomized motoneurons expressed ATF3 in the nucleus (arrow in G) and decreased ChAT immunoreactivity (E). They also lacked surface pKCC2 immunoreactivity in the cell body and proximal dendrite surfaces (F). I Axotomized spinal motoneuron immunolabeled for ChAT and ATF3. J KCC2b immunofluorescence of the same axotomized motoneuron as in I illustrating lack of KCC2b labeling in its somatic membrane. K Merge image of I and J. L Comparison of pKCC2 immunofluorescence between control and axotomized spinal motoneurons. Axotomized spinal motoneurons showed a significantly (asterisk) lower level of pKCC2 than control spinal motoneurons (t-test, p ≤ 0.001; n = 96 and 101 control and axotomized motoneurons, respectively). M Swarm dot plots of raw data (individual motoneurons) and differences between control (blue) and axotomy (yellow) shown in Cumming estimation plots. The distribution of differences obtained from bootstrap resampling are shown with the mean difference depicted as a dot and the 95% confidence interval indicated by the ends of the vertical error bars. A 70% significant decrease was detected in axotomized spinal motoneurons (two-sided permutation t-test p < 0.001). N Bar chat illustrating the results of a two-way ANOVA test and Holm-Sidak method comparing the following two factors: experimental condition (control versus axotomy) and type of KCC2 immunoreactivity (pKCC versus KCC2b). Data were gathered from one cat stained in serial sections with pKCC2 and KCC2b (control, n = 29 and 26 motoneurons; axotomy, n = 33 and 27 motoneurons for pKCC2 and KCC2b, respectively). Two-way ANOVA detected significant differences in control vs. axotomized motoneurons (asterisk, p < 0.001), but no difference between pKCC2 and KCC2b (p = 0.099, n.s.), or the interaction between axotomy and type of immunoreactivity (p = 0.334). Post hoc Holm-Sidak pairwise comparisons revealed significant difference between control and injured motoneuron for pKCC2 and KCC2b (#, p < 0.001 for both). O Swarm dot plots of raw data (individual motoneurons) and differences between pKCC2 in control (blue), KCC2b in control (yellow) and pKCC2 after axotomy (green) and KCC2b after axotomy (red) all shown in Cumming estimation plots. The distribution of differences obtained from bootstrap resampling are shown with the mean difference depicted as a dot and the 95% confidence interval indicated by the ends of the vertical error bars. No significant differences were found between KCC2b and pKCC2 in control motoneurons. Axotomized motoneurons showed a 70% significant decrease compared to their respective antibody matched controls (two-sided permutation t-test p < 0.001 in both comparisons). Scale bars = 30 μm in H for A–H; 20 μm in K for I–K

Article Snippet: ATF3 Used for injured motoneuron identification , Recombinant protein corresponding to aa 1-103 in human ATF3 , Mouse/monoclonal , Novus Clone 1685 NBP2-34489 , AB_2786997 Recognizes the epitope: ASAIVPCLSPPGSL (Manufacturer’s information) Not present in uninjured motoneurons , 1:200.

Techniques: Control, Isolation, Immunolabeling, Immunofluorescence, Labeling, Membrane, Comparison, Staining