aav constructs  (Addgene inc)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: A Non-Invasive and DNA-free Approach to Upregulate Mammalian Voltage-Gated Calcium Channels and Neuronal Calcium Signaling via Terahertz Stimulation.

doi: 10.1002/advs.202405436

Figure Lengend Snippet: Figure 4. The 42.5 THz wave impact endogenous CaV1 and neuronal Ca2+ signaling. A) Detail of the experimental protocol for THz stimulation on endogenous CaV1 channels in cultured cortical neurons. TTX (Tetrodotoxin), sodium channel blocker, final concentration: 0.5 μm. NBQX, AMPA receptor blocker, 10 μm. APV, NMDA receptor blocker, 10 μm. Isradipine, CaV1 channel blocker, 10 μm. B) A schematic to illustrate the working mechanism of the genetically-encoded calcium integrator CaMPARI2. Coincidental Ca2+ and PC light drive the irreversible green-to-red conversion of CaMPARI2. Statistical summary of normalized Fred/Fgreen is shown to quantify the total calcium influx of neurons under different stimuli labeled below (n = 15, 31, 101, 75, 100, 58, and 100 cells for 7 columns from left to right). C) Statistical summary of the pCREB signals (n = 20, 20, 35, 12, 12, 28, and 12 cells for 7 columns from left to right). Representative images of merged channels with CaMPARI2 fluorescence (green) and pCREB immunofluorescence (red), and hoechst (blue) for nuclear staining are shown. D) Statistical summary of CREB signals (n = 20, 20, 20, 20, 20, 15 and 15 cells for 7 columns from left to right). Representative images of merged channels with CaMPARI2 (green) and CREB (red), and Hoechst channel (blue) are shown. Values are presented as mean±SEM. One-way ANOVA followed by Tukey for post hoc tests were used (***p < 0.001; n.s., not significant, p > 0.05).

Article Snippet: [34] The sequence of CaMPARI2 is extracted from the addgene (Plasmid #101060).

Techniques: Cell Culture, Concentration Assay, Labeling, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: A Non-Invasive and DNA-free Approach to Upregulate Mammalian Voltage-Gated Calcium Channels and Neuronal Calcium Signaling via Terahertz Stimulation.

doi: 10.1002/advs.202405436

Figure Lengend Snippet: Figure 5. Applications of 42.5 THz-based channel/neuro-modulation in vitro and in vivo. A) The traces of calcium images for 3 representative cultured cortical neurons virally-expressing CaMPARI2 under 3 conditions: THz stimuli under physiological condition (traces in red), THz stimuli under isradipine treatment (traces in black), and THz stimuli post washout of isradipine (traces in yellow). The orange bars mark the duration of the THz stimuli. B) Statistical summary of the peak amplitude of neurons virally-expressing CaMPARI2 under the stimuli in (A). C) Statistical summary of total calcium influx of neurons virally-expressing CaMPARI2 under 3 conditions: basal/physiological condition (n = 10 cells), THz stimuli (n = 131 cells), and THz stimuli under isradipine treatment (n = 36 cells). The normalized Fred/Fgreen of CaMPARI2 serves as the index. D) Statistical summary of pCREB signals (n = 20, 59, and 32 cells) and CREB signals (n = 22, 22, and 20 cells) of neurons under the identical 3 conditions. All signals are immunofluorescent signals in neurons. E) Statistical summary of c-Fos immunofluorescent signals of neurons (n = 809, 959, and 1039 cells) under the identical 3 conditions. F) Diagram detailing THz stimulation applied to the right motor cortex in anesthetized mice, with the left cortex as a control. G) Representative images of c-Fos immunofluorescences in brain slices from the same mice under two conditions: left brain without the THz stimulation (left images), and right brain with the THz stimulation (right images). DAPI is used for nuclear staining and cell counting. H) c-Fos immunofluorescence images of brain slices under two conditions: left brain without THz stimulation (left images), and right brain of the same mice with isradipine pre-treatment before THz exposure (right images). DAPI is used for nuclear staining and cell counting. I,J) Statistical summaries of c-Fos immunofluorescences in brain slices from the same three mice, corresponding to conditions described in (G) and (H), respectively. Values are presented as mean±SEM. Student’s unpaired t-test (I, J), one-way ANOVA followed by Tukey for post hoc tests (B–D), and Kruskal–Wallis test (nonparametric test) (E) were used (*p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant, p > 0.05).

Article Snippet: [34] The sequence of CaMPARI2 is extracted from the addgene (Plasmid #101060).

Techniques: In Vitro, In Vivo, Cell Culture, Expressing, Control, Staining, Cell Counting

Journal: Cell reports

Article Title: Retrosplenial inputs drive visual representations in the medial entorhinal cortex

doi: 10.1016/j.celrep.2024.114470

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For CaMPARI 2.0 tagging of visually responsive cells, 200nL AAV1-hSyn-NES-his-CaMPARI2-WPRE-SV40 (2.5e12 gc/mL, Addgene) was injected into the left MEC.

Techniques: Virus, Recombinant, Staining, Plasmid Preparation, Mouse Assay, Software

Journal: bioRxiv

Article Title: HDAC4 Inhibits NMDA Receptor-Mediated Stimulation of Neurogranin Expression

doi: 10.1101/2024.07.09.602724

Figure Lengend Snippet: A. Hippocampal neurons grown on glass-bottomed gridded plates were transduced at DIV3 with CaMPARI2 AAVs and analyzed for in vivo photoconversion at DIV15. Following photoconversion, neurons were processed for IF with anti-Ng antibodies. Ng content and CaMPARI2 photoconversion were measured in individual neurons. The image set highlights two neurons with similar CaMPARI2 expression (ex488, Pre-ex405) but markedly distinct levels of CaMPARI2 photoconversion and Ng. The graph below compares CaMPARI2 photoconversion and Ng levels in individual neurons, with the fitted exponential curve indicating a direct correlation between neuronal activity (CaMPARI2 photoconversion) and Ng expression. B. Hippocampal neurons cultured in NB and NB+ media were processed for IF with anti-cFos and anti-Ng antibodies at DIV16. The colocalization of both proteins was analyzed. Images acquired with a 25X objective (Zeiss Axiovert 200M). The upper histogram represents the percentage of cells expressing Ng, cFos and their colocalization in neurons cultured in NB and NB+ media (mean ± SEM, n=3). I_Blue and I_Forest lookup tables from https://github.com/cleterrier/ChrisLUTs were used.

Article Snippet: All AAV constructs derived from the original vector pAAV_hsyn_NES-his-CaMPARI2-F391W-WPRE-SV40, Addgene #101061.

Techniques: In Vivo, Expressing, Activity Assay, Cell Culture