L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

26 octobre 19201920/10/26 (A21,N7255)

NRTN gene transfection increase spine density and total number of intersections in MSN.

<p><b>A</b>. Sholl diagram for the morphological study of dendrites and dendritic spines. <b>B</b>. Representative micrograph of a medium spiny neuron (MSN) of a healthy striatum. The arrow shows the 50 μm segment (primary dendrite) where dendritic spines were analyzed. Upper calibration bar = 25 μm. The small panels show different types of spines (thin, mushroom, stubby and wide, from left to right) indicated by the arrow. Calibration bar = 5 μm. The transfections of pGreenLantern-1 (GFP) and pTracer-mNRTN-His (NRTN) plasmids were made at week 12 after lesion and the Sholl analysis was performed at the end of the study (12 weeks after transfection or 24 weeks after lesion). UT = untransfected rats with lesion. <b>C.</b> Sholl analysis of total number of intersections along dendritic trees in MSNs at all distances in 200-μm radius from the soma. <b>D.</b> Plot of mean spine density analyzed per 50-μm primary dendrite from cell body (proximal segment, upper arrow in <b>B</b>) from six neurons per rat. All values represent the mean ± SEM (<i>n</i> = 6 independent rats in each experimental condition). Statistical analysis (<b>C</b> and <b>D</b>) was performed using one-way ANOVA and Tukey <i>post-hoc</i> test.</p

NRTN gene transfection increase spine density and total number of intersections in MSN.

<p><b>A</b>. Sholl diagram for the morphological study of dendrites and dendritic spines. <b>B</b>. Representative micrograph of a medium spiny neuron (MSN) of a healthy striatum. The arrow shows the 50 μm segment (primary dendrite) where dendritic spines were analyzed. Upper calibration bar = 25 μm. The small panels show different types of spines (thin, mushroom, stubby and wide, from left to right) indicated by the arrow. Calibration bar = 5 μm. The transfections of pGreenLantern-1 (GFP) and pTracer-mNRTN-His (NRTN) plasmids were made at week 12 after lesion and the Sholl analysis was performed at the end of the study (12 weeks after transfection or 24 weeks after lesion). UT = untransfected rats with lesion. <b>C.</b> Sholl analysis of total number of intersections along dendritic trees in MSNs at all distances in 200-μm radius from the soma. <b>D.</b> Plot of mean spine density analyzed per 50-μm primary dendrite from cell body (proximal segment, upper arrow in <b>B</b>) from six neurons per rat. All values represent the mean ± SEM (<i>n</i> = 6 independent rats in each experimental condition). Statistical analysis (<b>C</b> and <b>D</b>) was performed using one-way ANOVA and Tukey <i>post-hoc</i> test.</p

NRTN-His levels measured with ELISA.

<p>NRTN-His levels measured with ELISA.</p

Neurotrophic effect of NRTN gene transfection on TH(+) cells and neurites of the substantia nigra with chronic 6-OHDA lesion.

<p><b>A.</b> Representative micrographs of TH immunohistochemistry. Headings = Anterior-posterior coordinates of Paxinos and Watson Rat Atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188239#pone.0188239.ref054" target="_blank">54</a>]. 24WLUT = Untransfected rats with 24 weeks of 6-OHDA lesion. 12WL4WT = 12 weeks after lesion and 4 weeks after transfection, 12WL12WT = 12 weeks after lesion and 12 weeks after transfection. The scale bar of 300 μm is common for slices at different levels and of 50 μm for details. <b>B.</b> TH(+) neuron counting. <b>C.</b> Densitometry of TH(+) relative area. 4WT and 12 WT = 4 and 12 weeks after transfection. 24WL = untransfected rats with 24 weeks of lesion. 12WL = 12 weeks after lesion. The transfections of pGreenLantern-1 (GFP) and pTracer-mNRTN-His (NRTN) plasmids were made at week 12 after lesion. All values are the mean ± SEM (<i>n</i> = 3 independent rats for each experimental condition). One-way ANOVA and Newman-Keuls post-test. * <i>P</i> < 0.001, ** <i>P</i> < 0.0001. ns = no statistical significance, <i>P</i> > 0.05.</p

NRTN gene transfection increases NTSR1 levels in the substantia nigra of rats with chronic 6-OHDA lesion.

<p><b>A.</b> Representative micrographs of double immunofluorescence staining against NTSR1 and TH. 24WLUT = Untransfected rats with 24 weeks of 6-OHDA lesion. 12WL4WT = 12 weeks after lesion and 4 weeks after transfection, 12WL12WT = 12 weeks after lesion and 12 weeks after transfection. The scale is common for all micrographs. <b>B.</b> immunofluorescence area density (IFAD) for NTSR1 and TH was determined using ImageJ software v.1.46r (National Institutes of Health; Bethesda, MD). NRTN4WT and NRTN412WT = 4 and 12 weeks after transfection. UT = untransfected rats. The transfections of pGreenLantern-1 (GFP) and pTracer-mNRTN-His (NRTN) plasmids were made at week 12 after lesion. All values are the mean ± SEM (<i>n</i> = 3 independent rats for each experimental condition). Two-way ANOVA and Bonferroni post-test. ns = no statistical significance.</p

Neuronal cytoskeleton restoration after NRTN-His NPs transfection.

<p>Representative micrographs of double immunofluorescence to TH and β-III-tubulin. 24WLUT = Untransfected rats with 24 weeks of 6-OHDA lesion. 12WL4WT = 12 weeks after lesion and 4 weeks after transfection, 12WL12WT = 12 weeks after lesion and 12 weeks after transfection. The transfections of pGreenLantern-1 (GFP) and pTracer-mNRTN-His (NRTN) plasmids were made at week 12 after lesion. The scale bar is common for all micrographs.</p

NRTN gene transfection recovers dopamine levels in the substantia nigra (SN) and striatum (St) of rats with chronic 6-OHDA lesion.

<p>HPLC measurements of dopamine levels were made at weeks 4 (<b>A</b>) and 12 (<b>B</b>) after pTracer-mNRTN-His (NRTN) transfection made at week 12 after the lesion. All values represent mean ± SEM (<i>n</i> = 4 independent rat in each experimental condition). One-way ANOVA and Newman-Keuls post-test. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. ns = non statistical significance, <i>P</i> > 0.05.</p

Effect of NRTN gene transfection on circling behavior of rats with chronic 6-OHDA lesion.

<p>Ipsilateral circling behavior (<b>A and C</b>) was activated by methamphetamine (8 mg/kg; i.p.), and contralateral circling behavior (<b>B and D</b>) was activated by apomorphine (0.5 mg/kg; i.p.) at weeks 0, 4 and 12 after transfection. The transfections of pGreenLantern-1 (GFP) and pTracer-mNRTN-His (NRTN) plasmids were made at week 12 after lesion (Week 0). All values represent the mean ± SEM (<i>n</i> = 4 independent rats in each experimental condition). One-way ANOVA and Newman-Keuls post-test. *<i>P</i> <0.0005, **<i>P</i> < 0.0001.</p

Proportional density of the different types of spines in medium spiny neurons from the rats of the four groups studied.

<p>Proportional density of the different types of spines in medium spiny neurons from the rats of the four groups studied.</p