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

Journal: Scientific Reports

Article Title: Heatstroke-induced late-onset neurological deficits in mice caused by white matter demyelination, Purkinje cell degeneration, and synaptic impairment in the cerebellum

doi: 10.1038/s41598-022-14849-9

Figure Lengend Snippet: Expression of postsynaptic density-95 and synaptophysin around Purkinje cells. To estimate synaptic connections, pre- (synaptophysin, green) and postsynaptic (PSD95, red) markers were co-stained in the cerebellum and were observed with higher magnification images. Both postsynaptic density 95 and synaptophysin immunoreactions in the Purkinje cell layer appeared to have attenuated 3 weeks after heatstroke and intensified again after 9 weeks.

Article Snippet: After heat-induced antigen retrieval and blocking according to the protocol as mentioned above, the sections were incubated with either monoclonal rabbit anti-calbindin antibody (1:100, Cell Signaling, USA) or polyclonal rabbit anti-synaptophysin antibody (1:200, Proteintech, USA) with monoclonal mouse anti-postsynaptic density 95 (PSD95) antibody (1:400, BD, USA) overnight at 4 °C.

Techniques: Expressing, Staining

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Effect of Tetramethylpyrazine on Neuroplasticity after Transient Focal Cerebral Ischemia Reperfusion in Rats

doi: 10.1155/2021/1587241

Figure Lengend Snippet: Pearson's correlation test. (a) Correlation between mNSS and SYP expression at 28 d after MCAO. (b) Correlation between mNSS and GAP-43 expression at 28 d after MCAO.

Article Snippet: Then, the specimens were incubated in endogenous peroxidase blocking solution (Maixin Technology Co, Fuzhou, Fujian, China) for 10 min. After incubated with normal rabbit serum (Maixin Technology Co, Fuzhou, Fujian, China), the mouse anti-SYP primary antibody (1 : 200, Boster, Wuhan, Hubei, China) and mouse anti-GAP-43 primary antibody (1 : 200, Boster, Wuhan, Hubei, China) were added and incubated, respectively, with samples at 4°C overnight.

Techniques: Expressing

Journal: The journal of physiological sciences : JPS

Article Title: Time-course investigation of blood-brain barrier permeability and tight junction protein changes in a rat model of permanent focal ischemia.

doi: 10.1007/s12576-016-0516-6

Figure Lengend Snippet: Fig. 3 The expression of SYP and PSD95 in penumbra at different time points after MCAO. Expression levels of SYP at 8, 12, 24, 72, and 120 h after ischemia and of PSD95 at 12, 24, 72, and 120 h after were significantly reduced compared to the sham group. Data represent mean ± SEM, n = 3. **p \ 0.01, ***p \ 0.001 vs. sham group

Article Snippet: Membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated overnight at 4 C with anti-Occludin antibody (1:500, Santa Cruz, USA), anti-Claudin-5 antibody (1:500, Santa Cruz, USA), anti-SYP antibody (1:1000, Santa Cruz, USA), anti-PSD95 antibody (1:1000, Santa Cruz, USA), anti-GFAP antibody (1:500, Boster, China), or anti-b-actin antibody (1:1000, Santa Cruz, USA).

Techniques: Expressing