Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse
"> Figure 1
<p>Frontal sections of an adult mouse cerebellum immunostained with p75NTR, CaBP and HSP25 at P28. The lobules are shown by Roman numerals. (<b>A</b>,<b>B</b>) The cerebellar cortex immunoperoxidase stained by p75NTR shows that the Pc somata in the Purkinje cell layer and dendrites in the molecular layer are immunoreactive (arrow; black in A, and white in B). Some Pc somata expressing p75NTR are weak (A, arrowhead) or absent (B, arrowhead); (<b>C</b>–<b>E</b>) Double immunostaining of a frontal section at lobule IX and X using p75NTR (red) and CaBP (green). Stripes of immunoreactive Pcs are clear in the vermis; (<b>F</b>) Immunostaining of a frontal section at the cerebellum using p75NTR. Immunoreactivity is not present in the AZ and PZ. Stripes of immunoreactive Pcs are clear in the vermis of the CZ (VII) and NZ (X); (<b>G</b>) Immunostaining of a frontal section through the lobule VII using HSP25. Stripes of immunoreactive Pcs are clear in the vermis of the CZ. Abbreviations: ml, molecular layer; Pcl, Purkinje cell layer; gl, granular layer.Scale bars: <b>A</b> = 100 µm; <b>B</b> = 20 µm; <b>E</b> = 250 μm (applies to <b>C</b>–<b>E</b>); <b>F</b> = 500 µm; <b>G</b> = 250 µm.</p> "> Figure 2
<p>Sagittal sections of P4 wt<sup>+/+</sup> (<b>A</b>–<b>C</b>) and <span class="html-italic">nax</span><sup>−/−</sup> (<b>D</b>–<b>I</b>) and sagittal sections of P6 wt (<b>J</b>) and <span class="html-italic">nax</span> (<b>K</b>) cerebella immunostained with p75NTR, CaBP and Pax6. The lobules are shown by Roman numerals. (<b>A</b>–<b>C</b>) The wt cerebellum shows normal lobules (<b>A</b>) with expression of p75NTR (red) in the entire egz (external germinal zone)and dispersing through the Pc layers. A higher magnification of “A” shown in “B” and Pax6 immunostaining is indicated egz in “C”; (<b>D</b>–<b>F</b>) The <span class="html-italic">nax</span> cerebellum shows a small cerebellum with underdeveloped lobulation (<b>B</b>). P75NTR expression (red) is present in the underdeveloped egz and in the developing Pcl (<b>B</b>). Immunostaining with Pax6 shows the thin layer of the egz in the <span class="html-italic">nax</span> cerebellum (<b>F</b>); (<b>G</b>–<b>I</b>) Magnified views of white box in E showing CaBP (<b>G</b>), p75NTR (<b>H</b>), and merged image (<b>I</b>); (<b>J</b>–<b>K</b>) The sagittal sections immunoperoxidase stained by p75NTR shows immunoreactivity of the Pc in wt (<b>J</b>) and <span class="html-italic">nax</span> (<b>K</b>) cerebella. Abbreviations: egz, external germinal zone; Pcl, Purkinje cell layer. Scale bars: <b>D</b> = 500 µm (applies to <b>A</b> and <b>D</b>); <b>E</b> = 50 µm (applies to <b>B</b> and <b>E</b>); <b>F</b> = 40 μm (applies to <b>C</b> and <b>F</b>); <b>J</b> = 50 µm; <b>K</b> = 50 µm.</p> "> Figure 3
<p>Frontal sections at the cerebellum of wt (<b>A</b>,<b>B</b>) and <span class="html-italic">nax</span> (<b>C</b>,<b>D</b>) at P6 immunoperoxidase stained with p75NTR. (<b>A</b>,<b>B</b>) The wt cerebellum shows normal vermis (v) and hemisphere (h) lobulation (<b>A</b>) with uniform expression of p75NTR in the entire cerebellar cortex. A higher magnification of black box (b) in “<b>A</b>” indicates localization of p75NTR expression in Pcl and EGZ; (<b>C</b>,<b>D</b>) Underdeveloped <span class="html-italic">nax</span> cerebellum exhibits uniform p75NTR expression in putative vermis and in the hemisphere. Magnified views of black box (d) in “<b>C</b>” showing p75NTR in multilayer Pc and thin or lack of EGZ. The p75NTR expression shows the outline of cerebellar nuclei (cn) both in wt (<b>A</b>) and <span class="html-italic">nax</span> (<b>C</b>) sections. Abbreviations: egz, external germinal zone; Pcl, Purkinje cell layer; h, hemisphere; cn, cerebellar nuclei; gl, granular layer. Scale bars: <b>A</b> and <b>C</b> = 500 µm; <b>D</b> = 100 µm (applies to <b>B</b> and <b>D</b>).</p> "> Figure 4
<p>Transverse sections of <span class="html-italic">nax</span><sup>−/−</sup> (<b>A</b>–<b>D</b>) and wt sibling cerebella at P22 immunostained with p75NTR, CaBP and HSP25. (<b>A</b>,<b>B</b>) P75NTR immunostaining shows an array of parasagittal stripes in the CZ and NZ of the <span class="html-italic">nax</span> cerebellum, with high magnification of the CZ shown in <b>B</b>; (<b>C</b>,<b>D</b>) HSP25 immunostaining of the <span class="html-italic">nax</span> cerebellum shows parasagittal stripes in the CZ and NZ, <b>D</b> is a higher magnification of <b>C</b>; (<b>E</b>) At a high magnification, p75NTR expression is shown in the CZ of the wt<sup>+/+</sup> sibling. Scale bars: <b>C</b> = 1 mm (applies to <b>A</b> and <b>C</b>); <b>D</b> = 500 µm (applies to <b>B</b> and <b>D</b>); <b>E</b> = 250 μm.</p> "> Figure 5
<p>Western blot analysis of p75NTR expression during cerebellar development at P5, P9 and P18. This experiment was repeated over three different litters for each postnatal days in wt and <span class="html-italic">nax</span> siblings (P5, P9 and P18, wt; <span class="html-italic">n</span> = 9 and <span class="html-italic">nax</span>; <span class="html-italic">n</span> = 9). (<b>A</b>) Immunoblots of total cell lysate from wt sibling and <span class="html-italic">nax</span> mouse cerebellum indicate significant down-regulation in the wt sibling and also in the <span class="html-italic">nax</span> cerebellum from early postnatal development to P18. However, there is no considerable difference in p75NTR expression between the wt and <span class="html-italic">nax</span> cerebellum, despite the higher p75NTR expression in <span class="html-italic">nax</span> compared with wt. (<span class="html-italic">p</span> > 0.05). Protein loading was confirmed using GAPDH (<span class="html-italic">n</span> = 18); (<b>B</b>) The data in the bar graph are presented as the mean ± SEM, and statistical analysis was performed using Kruskal-Wallis test followed by Dunnett’s multiple comparison test using Mann-Whitney test. Abbreviation: ns, not significant.</p> "> Figure 5 Cont.
<p>Western blot analysis of p75NTR expression during cerebellar development at P5, P9 and P18. This experiment was repeated over three different litters for each postnatal days in wt and <span class="html-italic">nax</span> siblings (P5, P9 and P18, wt; <span class="html-italic">n</span> = 9 and <span class="html-italic">nax</span>; <span class="html-italic">n</span> = 9). (<b>A</b>) Immunoblots of total cell lysate from wt sibling and <span class="html-italic">nax</span> mouse cerebellum indicate significant down-regulation in the wt sibling and also in the <span class="html-italic">nax</span> cerebellum from early postnatal development to P18. However, there is no considerable difference in p75NTR expression between the wt and <span class="html-italic">nax</span> cerebellum, despite the higher p75NTR expression in <span class="html-italic">nax</span> compared with wt. (<span class="html-italic">p</span> > 0.05). Protein loading was confirmed using GAPDH (<span class="html-italic">n</span> = 18); (<b>B</b>) The data in the bar graph are presented as the mean ± SEM, and statistical analysis was performed using Kruskal-Wallis test followed by Dunnett’s multiple comparison test using Mann-Whitney test. Abbreviation: ns, not significant.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animal Maintenance and Tissue Processing
2.2. Antibodies
2.3. Immunohistochemistry
2.4. Western Blotting
2.5. Preparation of the Figures
2.6. Statistical Methods
3. Results
3.1. Expression Pattern of p75NTR in Adult Mouse Cerebellum
3.2. Pattern Expression of p75NTR in wt and nax Cerebellum at P4 and P6
3.3. Pattern Expression of p75NTR in wt and nax at P22
3.4. Protein Expression of p75NTR in wt and nax Cerebellum
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Rahimi Balaei, M.; Jiao, X.; Ashtari, N.; Afsharinezhad, P.; Ghavami, S.; Marzban, H. Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse. Int. J. Mol. Sci. 2016, 17, 115. https://doi.org/10.3390/ijms17010115
Rahimi Balaei M, Jiao X, Ashtari N, Afsharinezhad P, Ghavami S, Marzban H. Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse. International Journal of Molecular Sciences. 2016; 17(1):115. https://doi.org/10.3390/ijms17010115
Chicago/Turabian StyleRahimi Balaei, Maryam, Xiaodan Jiao, Niloufar Ashtari, Pegah Afsharinezhad, Saeid Ghavami, and Hassan Marzban. 2016. "Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse" International Journal of Molecular Sciences 17, no. 1: 115. https://doi.org/10.3390/ijms17010115