Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 1. (A–C) Representative sections illustrate immunohistochemical staining for synaptophysin (Syp) in the cerebral cortex and hippocampus (A), cerebellum (B), and corpus callosum (C) of untreated control mice. (D) Accumulation of synaptophysin- positive vesicles ¼ spheroids/ovoids/bulbs in the corpus callosum of a cuprizone-treated mouse. Arrows indicate the different sizes of synaptophysin-positive-spheroids; small [S],<2 mm (as in panel C); middle-sized [M], 2–7.5 mm; large [L], 8–15 mm, pathological size. (E, F) Graphs show numbers of different sized synaptophysin-positive spheroids in the corpus callosum during acute (5 weeks, E) and chronic (12 weeks, F) demyelination followed by remyelination after stopping cuprizone. Significant effects between different time points were calculated separately for each type/size of bulbs (*p <0.05, **p < 0.01, **p < 0.001). Significant effects for different cuprizone time treatments in comparison to the untreated control: #p< 0.05, ##p < 0.01, ###p < 0.001), n ¼ 4–6. (G, H) Representative images show synaptophysin-positive bulbs in the demyelinated hippocampus (G) and internal capsule (H). (I-K) Representative images show that synaptophysin does not colocalize with the oligodendroglial marker Nogo-A (I), the astrocytic marker GFAP (J), or with the microglial marker Iba-1 (K). Scale bar in (K)¼ 100 mm, applies to (I) and (J).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Immunohistochemical staining, Staining, Control, Comparison, Marker

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 2. (A, B) Graphs represent the quantitative analysis of APP, synaptophysin (Syp), and double-positive spheroids in the corpus callosum of cuprizone-treated mice with acute demyelination (A) and chronic demyelination (B). Two-way ANOVA analysis identify that there were no significant differences between numbers of APP- and synaptophysin-positive spheroids during

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques:

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 3. Immunohistochemical staining of the corpus callosum evaluating phosphorylation of neurofilaments and their colocalization with synaptophysin (Syp) during cuprizone-induced demyelination. (A–H) SMI-32 detecting non-phosphorylated heavy neurofilaments are shown in panels (A, C, E, G). SMI-312 detecting phosphorylated neurofilaments H and M are

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Immunohistochemical staining, Staining, Phospho-proteomics

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 5. (A-P) Representative sections show immunostaining in the corpus callosum for synaptophysin/Iba-1 (A-F) and synaptophysin/ PLP (G–L) during acute and chronic cuprizone induced demyelination and subsequent remyelination. Inserts in (A–F) show the morphology/shape of microglia identifying their activation state. Inserts in (G–L) show higher magnification of synaptophysin accumulations. Panels (M) and (N) are confocal images from RCA-1 (microglia activation marker, green) and synaptophysin (red) during inflammation in cuprizone-treated mice. Panels (O) and (P) show remyelinated axons (PLP, green) with internal synaptophysin accumulation (red).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Immunostaining, Activation Assay, Marker

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 6. The appearance of synaptophysin (Syp)-positive pathological (medium-and large-sized) spheroids was compared and correlated with the extent of demyelination (proteolipid protein: PLP, myelin basic protein: MBP) and microglia infiltration (Iba-1) induced by cuprizone feeding (acute or chronic). (A, B) Graphs show the extent of microglia accumulation, appearance of pathological synaptophysin bulbs and the extent of de- and remyelination as judged by scoring of MBP and PLP immunostaining. Score of 3 represents complete myelination; score of 0 represents complete demyelination. (C–H) Correlation analyses of synaptophysin-positive bulbs with the extent of de- and remyelination and microglia numbers (n ¼ 5–6).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Immunostaining

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 8. (A–C) Graphs show microglia/macrophage infiltration (A) and the course of demyelination (B) during Theiler’s virus induced encephalomyelitis in the thoracic segment of murine spinal cord. (C) Panel shows the appearance of pathologic accumulation of synaptophysin (Syp). Significant effects between different time points were indicated as: *p < 0.05, **p < 0.01, **p < 0.001. Significant effects for different time treatments in comparison to the untreated control are indicated as: # p < 0.05, ## p < 0.01, ### p < 0.001), n ¼ 4–6. (D–H) Representative images of thoracic spinal cord segment illustrate infiltration of microglia/macrophage at different days after the injection of Theiler’s murine encephalomyelitis virus. (I–U) Panel (I) shows the appearance of synaptophysin-positive bulbs exclusively in the demyelinated area; (J) and (K) illustrate the appearance of synaptophysin-positive spheroids surrounded by activated microglia/macrophages. (L–N) Colocalization of synaptophysin and APP in the spheroid structures. (O–U) Serial staining of the same spinal cord area during Theiler’s virus induced encephalomyelitis (O, synaptophysin/PLP double staining; P, synaptophysin/Iba-1 double staining; R, synaptophysin/APP double staining; S–U, higher magnification of area shown in R).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Virus, Comparison, Control, Injection, Staining, Double Staining

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 9. Representative images show the expression of synaptophysin (Syp) in CNS tissue of 2 different patients with MS. (A– H) Different immunohistochemical stainings from a chronic active MS lesion (serial sections). Panels (E) and (F) show the higher magnifications from boxed areas 1 in (C) and (D). Panels (G) and (H) demonstrate higher magnification from boxed areas 2 in (C) and D. (I–K) Representative double immunostaining for synaptophysin and Iba-1 demonstrate microglia accumulation (I) and proving colocalization of APP and synaptophysin (J, K). (L–O) Demyelination/inflammation/axonal damage in another MS lesion. DAB staining in (L) and (M) illustrate the extent of demyelination (PLP) and inflammation (LN3 marks HLA-DR-positive cells: lymphocytes and macrophages) in this MS lesion. Panels N and O show higher magnification of the boxed are from this lesion in (L) and (M) demonstrating double expression of APP and synaptophysin in (N) and double staining of PLP and synaptophysin in (O). (P–U) Corpora amylacea are immunostain-positive for APP (P, S), and NeuN (U), but not synaptophysin (R, T).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Expressing, Immunohistochemical staining, Double Immunostaining, Staining, Double Staining

Journal: Neurobiology of Disease

Article Title: Early glial activation, synaptic changes and axonal pathology in the thalamocortical system of Niemann–Pick type C1 mice

doi: 10.1016/j.nbd.2011.12.027

Figure Lengend Snippet: Altered distribution of presynaptic markers in Npc1 −/− mice. a Immunohistochemical staining for synaptophysin (Syp) at the end stage of disease revealed aggregates of immunoreactivity, most prominently in the internal capsule (X), reticular (+) and ventral posterior (*) nucleus of the thalamus in Npc1 −/− mice. b Higher power pictures from the ventral posterior nucleus (VPM/VPL) and internal capsule demonstrate these large aggregates to be immunoreactive for three presynaptic markers; synaptophysin, VAMP2 and SNAP25. c Dual channel immunofluorescence staining for the presynaptic marker synaptophysin and the GABAergic marker GAD65/67 reveal partial co-localisation of synaptic aggregates with GABAergic axonal spheroids in the VPM (ventral medial posterior nucleus of the thalamus) of Npc1 −/− mice. Yellow arrows indicate aggregates immunoreactive for both the synaptic marker and GAD65/67, red arrows indicate aggregates only immunoreactive for synaptophysin/VAMP2 and green arrows indicate aggregates only immunoreactive for GAD65/67. (Scale bar in a = 200 μm; in b and c = 10 μm).

Article Snippet: Gels were blotted onto PVDF membranes, blocked with Odyssey blocking buffer (Li-COR Biosciences, Lincoln, NE) 1:1 PBS-Tween-20 (referred to as blocking buffer) for 1 h at room temperature and then incubated with the following primary antibodies diluted in blocking buffer at 4 °C overnight: mouse anti-synaptophysin (Cambridge Bioscience 1:1000), mouse anti-synaptobrevin (VAMP2) (Synaptic systems 1:10 000).

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Marker

Journal: Neurobiology of Disease

Article Title: Early glial activation, synaptic changes and axonal pathology in the thalamocortical system of Niemann–Pick type C1 mice

doi: 10.1016/j.nbd.2011.12.027

Figure Lengend Snippet: No change in the level of expression of synaptic markers. Western blots on enriched synaptic fractions from cortical and subcortical forebrain indicate no change in the level of expression of synaptic markers. Actin was used as a loading control. Syp; Synaptophysin.

Article Snippet: Gels were blotted onto PVDF membranes, blocked with Odyssey blocking buffer (Li-COR Biosciences, Lincoln, NE) 1:1 PBS-Tween-20 (referred to as blocking buffer) for 1 h at room temperature and then incubated with the following primary antibodies diluted in blocking buffer at 4 °C overnight: mouse anti-synaptophysin (Cambridge Bioscience 1:1000), mouse anti-synaptobrevin (VAMP2) (Synaptic systems 1:10 000).

Techniques: Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation

doi: 10.1523/JNEUROSCI.2468-18.2019

Figure Lengend Snippet: PACAP significantly increased the number of function synapse in the primary cultured hippocampal neurons. A, Representative fluorescence images of the primary hippocampal neuronal dendrites at DIV21. The cells were fixed at DIV21 and immunostained using anti-synaptophysin (magenta) and anti-PSD95 (green) antibodies. Magnification of the area marked with a white dot box. Scale bar, 10 μm. B, Primary hippocampal neurons were treated for 2 d with 1, 10, or 100 nm PACAP. Scale bar, 10 μm. C, Quantification of the number of PSD-95-positive spines that were apposed to synaptophysin-positive presynaptic terminals. Values indicate mean ± SEM on 60 neurons from three independent experiments. **p < 0.01 versus vehicle (one-way ANOVA followed by Tukey–Kramer test). D, Immunoblot of cell surface biotinylated α1 sodium potassium ATPase, NR1, and NR2B from the primary cultured hippocampal neurons at DIV14. Surface biotinylation assay was performed after 2 d treatment with vehicle or 10 nm PACAP. E, Quantification of the cell surface expression levels of NR1 and NR2B were normalized to the levels of α1 sodium potassium ATPase. Values indicate mean ± SEM from five independent experiments. *p < 0.05 versus vehicle (Student's t test). F, Representative immunoblots showing expression of pERK1/2 and ERK2. Synaptic NMDAR activity in the primary cultured hippocampal neurons at DIV14 were treated with vehicle or 500 μm 4-AP and 50 μm bicuculline (bic) for 5 or 30 min after 2 d treatment with vehicle or 10 nm PACAP. 4-AP is a potassium channel blocker, and bicuculline is a GABAA receptor blocker. G, Quantification of the expression levels of pERK2 were normalized to the levels of ERK2. Values indicate mean ± SEM from four independent experiments. *p < 0.05 versus vehicle or 0 min (two-way ANOVA followed by Tukey–Kramer test).

Article Snippet: For predendritic and postdendritic spine labeling, the primary antibodies were used a rabbit polyclonal anti-PSD-95 antibody (1:500; catalog #3450S, RRID: AB_2292883 , Cell Signaling Technology) and a mouse monoclonal anti-synaptophysin antibody (1:2000; catalog #611880, RRID: AB_399360 , BD Biosciences) diluted in 0.2% Triton X-100 in PBS supplemented with 3% BSA.

Techniques: Cell Culture, Fluorescence, Western Blot, Surface Biotinylation Assay, Expressing, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation

doi: 10.1523/JNEUROSCI.2468-18.2019

Figure Lengend Snippet: Viral expression of miR-132 increased dendritic spine density in the primary hippocampal neurons from PACAP−/− mice. A, Representative fluorescence images of the primary hippocampal neuronal dendrites at DIV21. The cells were fixed at DIV21 and immunostained using anti-synaptophysin (magenta) and anti-PSD95 (green) antibodies. Scale bar, 10 μm. B, Quantification of the number of PSD-95-positive spines that were apposed to synaptophysin-positive presynaptic terminals. Values indicate mean ± SEM on 80 neurons from four independent experiments. *p < 0.05 versus vehicle (one-way ANOVA followed by Tukey–Kramer test). C, Expression levels of miR-132 in WT and PACAP−/− mice primary hippocampal neurons were analyzed by qRT-PCR. Primary hippocampal neurons were harvested at DIV20. Values indicate mean ± SEM from nine independent experiments. *p < 0.05 versus WT (one-way ANOVA followed by Tukey-kramer test). D, Representative fluorescence images of primary hippocampal neuron dendrites at DIV21. Primary hippocampal neurons were transduced with miR-132 Venus or control-Venus lentivirus at DIV14 and treated with 10 nm PACAP at DIV19. The cells were fixed at DIV21 and immunostained with anti-GFP (green) and anti-PSD95 (magenta) antibodies at DIV21. Scale bar, 10 μm. E–G, Quantification of the number of total spines (E), PSD-95-positive spines (F), and -negative spines (G). Values indicate mean ± SEM of 60 neurons from three independent experiments. **p < 0.01 versus Venus or WT (two-way ANOVA followed by Tukey–Kramer test).

Article Snippet: For predendritic and postdendritic spine labeling, the primary antibodies were used a rabbit polyclonal anti-PSD-95 antibody (1:500; catalog #3450S, RRID: AB_2292883 , Cell Signaling Technology) and a mouse monoclonal anti-synaptophysin antibody (1:2000; catalog #611880, RRID: AB_399360 , BD Biosciences) diluted in 0.2% Triton X-100 in PBS supplemented with 3% BSA.

Techniques: Expressing, Fluorescence, Quantitative RT-PCR, Transduction