Dynamic remodelling of synapses can occur in the absence of the parent cell body-6

<p><b>Copyright information:</b></p><p>Taken from "Dynamic remodelling of synapses can occur in the absence of the parent cell body"</p><p>http://www.biomedcentral.com/1471-2202/8/79</p><p>BMC Neuroscience 2007;8():79-79.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2048966.</p><p></p>urofilament 165 and SV2 (green: axons and terminals), visualised with a Cy2-conjugated secondary antibody and co-stained with TRITC-αBTX (red: muscle endplates), 165 min after a 15 min BzATP pulse (100 μM). Both 'occupied' (a: arrow) and 'unoccupied' (a: arrowhead) endplates were present, as were unoccupied endplates with intact presynaptic axons (b). Many terminals appeared to be in the process of retraction resulting in minor (c: asterisk) or major (d: asterisks) regions of unoccupied endplates. These were classified as 'intermediate' in subsequent data. Control nerve/muscle preparations, which were maintained in physiological saline for up to 300 min, showed no visible signs of degeneration or retraction of nerve terminals (e). Scale bar a-e = 15 μ

Dynamic remodelling of synapses can occur in the absence of the parent cell body-2

<p><b>Copyright information:</b></p><p>Taken from "Dynamic remodelling of synapses can occur in the absence of the parent cell body"</p><p>http://www.biomedcentral.com/1471-2202/8/79</p><p>BMC Neuroscience 2007;8():79-79.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2048966.</p><p></p>urofilament 165 and SV2 (green: axons and terminals), visualised with a Cy2-conjugated secondary antibody and co-stained with TRITC-αBTX (red: muscle endplates), 165 min after a 15 min BzATP (100 μM) pulse. Nerve terminals associated with either fragmented (a), or absent (b) muscle endplates were seen, as were fragmented nerve terminals contacting either apparently normal (c), or fragmented endplates (c, d). Scale bar a, d = 15 μm, b, c = 30 μ

Dynamic remodelling of synapses can occur in the absence of the parent cell body-1

<p><b>Copyright information:</b></p><p>Taken from "Dynamic remodelling of synapses can occur in the absence of the parent cell body"</p><p>http://www.biomedcentral.com/1471-2202/8/79</p><p>BMC Neuroscience 2007;8():79-79.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2048966.</p><p></p>plates present in: 300 min control (open bar); 165 min subsequent to a 15 min BzATP pulse (100 μM: filled bar); 165 min subsequent to a 15 min BzATP pulse (filled bar) in the presence of Brilliant Blue G (a selective P2X7 receptor antagonist: BBG, 1 μM: broad diagonal cross-hatching); 165 min subsequent to a 15 min BzATP pulse in the presence of Reactive Blue 2 (which blocks P2Y and P2X receptor subunits: 100 μM, RB2: narrow diagonal cross-hatching) or 165 min subsequent to a 15 min BzATP pulse in the presence of Suramin (a broad spectrum P2 receptor agonist: 100 μM: stipple). All data is mean ± SEM of multiple repeats (see text for n values)

Dynamic remodelling of synapses can occur in the absence of the parent cell body-8

<p><b>Copyright information:</b></p><p>Taken from "Dynamic remodelling of synapses can occur in the absence of the parent cell body"</p><p>http://www.biomedcentral.com/1471-2202/8/79</p><p>BMC Neuroscience 2007;8():79-79.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2048966.</p><p></p>urofilament 165 and SV2 (green: axons and terminals), visualised with a Cy2-conjugated secondary antibody and co-stained with TRITC-αBTX (red: muscle endplates), 165 min after a 15 min BzATP (100 μM) pulse. Nerve terminals associated with either fragmented (a), or absent (b) muscle endplates were seen, as were fragmented nerve terminals contacting either apparently normal (c), or fragmented endplates (c, d). Scale bar a, d = 15 μm, b, c = 30 μ

Dynamic remodelling of synapses can occur in the absence of the parent cell body-7

<p><b>Copyright information:</b></p><p>Taken from "Dynamic remodelling of synapses can occur in the absence of the parent cell body"</p><p>http://www.biomedcentral.com/1471-2202/8/79</p><p>BMC Neuroscience 2007;8():79-79.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2048966.</p><p></p>plates present in: 300 min control (open bar); 165 min subsequent to a 15 min BzATP pulse (100 μM: filled bar); 165 min subsequent to a 15 min BzATP pulse (filled bar) in the presence of Brilliant Blue G (a selective P2X7 receptor antagonist: BBG, 1 μM: broad diagonal cross-hatching); 165 min subsequent to a 15 min BzATP pulse in the presence of Reactive Blue 2 (which blocks P2Y and P2X receptor subunits: 100 μM, RB2: narrow diagonal cross-hatching) or 165 min subsequent to a 15 min BzATP pulse in the presence of Suramin (a broad spectrum P2 receptor agonist: 100 μM: stipple). All data is mean ± SEM of multiple repeats (see text for n values)

YFP expression activates cell stress responses in neurons <i>in vivo</i>.

<p>A – 3D bar chart showing fold-differences in mRNA expression levels for 84 cell stress related genes comparing spinal cord from YFP-16 mice with wild-type mice (N = 3 samples, each consisting of pooled tissue from 3 mice). Note that we only observed increased expression of cell stress related genes. B – Representative fluorescent western blots for cell stress proteins in the spinal cord of wild-type and YFP-16 mice. Both caspase 1 (Casp1) and CCL3 had increased expression in YFP-expressing tissue, whereas STI1 (a stress protein not on the array) remained at the same levels found in wild-type mice and YFP (FP) was only present in YFP-16 tissue. C - Bar chart (mean ± s.e.m.) showing quantification of protein expression levels in YFP-16 spinal cord (normalised to wild-type: fluorescence intensity ratio of 1 = identical to wild-type), confirming increased expression levels of CCL3 and caspase 1 (N = 3 samples, each consisting of pooled tissue from 3 mice). D/E - Representative fluorescent western blots and bar chart showing caspase 1 expression in the spinal cord of wild-type, YFP-H (low YFP expression) and YFP-16 (high YFP expression) mice. Increased levels of caspase 1 correlated with the amount of YFP present. F-I - Representative confocal micrographs showing caspase 1 immunohistochemistry (red = caspase 1; yellow = YFP; blue = TO-PRO) in the spinal cord of a YFP-H mouse. Increased caspase 1 immunolabelling was restricted to YFP-positive neurons. Scale bar = 20 µm.</p

Mouse SuperArray data showing greater than 1.5 fold cell stress RNA expression changes in the spinal cord of YFP-16 mice compared with wild-type controls (*array cell refers to the location on the 3D bar chart in Fig. 1A).

<p>Mouse SuperArray data showing greater than 1.5 fold cell stress RNA expression changes in the spinal cord of YFP-16 mice compared with wild-type controls (*array cell refers to the location on the 3D bar chart in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017639#pone-0017639-g001" target="_blank">Fig. 1A</a>).</p

YFP expression in motor neurons subtly disrupts normal neuronal morphology and alters responses to dying-back pathology but not Wallerian degeneration.

<p>A–D – Representative confocal micrographs of NMJs in the levator auris longus muscle from a YFP-H mouse labelled for neurofilaments (NF: red) and postsynaptic motor endplates (BTX: blue). Note that all motor endplates were innervated, but only a small proportion of motor axons were YFP-positive in these mice (A). Panels B–D show high power micrographs of NMJs identified in panel A. Note abnormal accumulations of NF only in the motor nerve terminals of YFP-positive NMJs (B = NF accumulation score of 0, C = 3, D = 5). E – Bar chart (mean ± s.e.m.) showing quantification of NF accumulation in motor nerve terminals from YFP-H mice (0 = no accumulation; 5 = large abnormal accumulation), revealing a significant increase in NF accumulation in YFP-positive terminals (N = 5 mice, n = 9 muscles; *** P<0.001 Mann-Whitney test). F–G – Representative confocal micrographs of intramuscular axons supplying the transversus abdominis muscle from YFP-H mice (also labelled for NFs; red), before (F) and 20 hours after (G) intercostal nerve lesion. The presence of YFP did not alter the rate or morphological appearance of Wallerian degeneration after nerve injury, with NF fragmentation occurring in YFP-positive and –negative axons in all nerves examined (N = 6 mice, n = 6 nerves). H–K - Representative confocal micrographs of two NMJs in the levator auris longus muscle from a late-symptomatic (P24) <i>wasted</i>/YFP-H mouse labelled to reveal NFs (red) and postsynaptic motor endplates (BTX: blue). Note how the motor axon with YFP (bottom NMJ) remained intact whereas the motor axon without YFP (top) was undergoing retraction, characteristic of a dying-back pathology. L - Bar chart showing quantification of dying-back pathology at the NMJ in late-symptomatic (P24) <i>wasted</i>/YFP-H mice, revealing a significant attenuation of dying-back pathology in motor nerve terminals where YFP was present (i.e. a retention of endplates fully occupied by overlying NFs and a reduction in the numbers of partially occupied endplates; N = 4 mice, n = 7 muscles; *** P<0.001 Mann-Whitney test). Scale bars = 80 µm (A), 40 µm (B–D), 30 µm (F–G), 50 µm (H–K).</p