Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Infusion of virus expressing Nrxn3 shRNA reduced the number of Nrxn3α positive cells. The central amygdala of Long Evans rats was infused with AAV1 virus containing the construct AAV1-GFP-mNRXN3-shRNA or a scrambled shRNA. Four weeks after infusion the whisker pad was injected with MeWo cells without varicella zoster virus (no VZV) or MeWo cells containing varicella zoster virus. Six weeks after infusion the brain was isolated and the sections imaged. ( A ) is a low magnification image of a brain slice indicating green GFP fluorescence from expression of the virus construct in the central amygdala region (white oval). A high magnification image of the GFP positive cells (green) within the central amygdala is shown in ( B ). Bar = 20 micrometers. ( C ) shows a histogram for Nrxn3 expression within the central amygdala after infusion of AAV1 and injection of the whisker pad. Each point is from an individual animal. An asterisk indicates a significant difference of α=0.05.

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: Virus, Expressing, shRNA, Construct, Whisker Assay, Injection, Isolation, Slice Preparation, Fluorescence

Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Synaptophysin was expressed in GABAergic cells of the central amygdala. The central amygdala of Gad1-iCre Long Evans rats was infused with AAV1 containing pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby. During this same surgery the central amygdala was infused with the shRNA viral construct. Image is from a representative rat infused with control shRNA and injected with no VZV. ( A ) Six weeks after infusion mRuby fluorescent signal (red) was detected within the central amygdala (CeA). White dotted line shows the borders of the central amygdala. Arrow points to the injection site ( A and B ). Enlarged image of synaptophysin positive cell (red, arrow) within the central amygdala is shown in ( C ). Hoechst 33342 stain of the nuclei from the same cell (arrow) is shown in blue ( D ). Bar= 100 µm.

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: shRNA, Construct, Control, Injection, Staining

Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Synaptophysin positive terminals colocalize with excitable cells in the lateral parabrachial nucleus. Atlas image of coronal brain section from a rat with the lateral parabrachial region outlined with a black dotted line in ( A ). The central amygdala of Gad1-iCre Long Evans rats was infused with AAV1 containing pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby. Excitable cells within the lateral parabrachial nucleus were labeled by infusing the lateral parabrachial nucleus with AAV5 virus containing pAAV-CaMKIIa-EGFP. Six weeks after infusion EGFP positive cells (green, ( B ) were present within the lateral parabrachial region (white dotted line). In ( B ), a low magnification image is shown on the left and a higher magnification image is shown on the right. EGFP positive cells (green) are shown in ( C, F, I and L ) and synaptophysin (red) is shown in ( C (through ( N )). Cell nuclei are labeled blue with Hoechst 33342 stain in ( B–D, F, G, I, J, L and M ). ( F ) through ( N ) are images through the z plane of the lateral parabrachial tissue. ( F, I and L ) are three different z plane cross sections, respectively, through the cell in ( C ) (open arrow). ( F–H ) show one z plane slice through the cell, ( I–K ) show the second slice and ( L–N ) are the third slice through the cell. Synaptophysin labeled puncta on the CaMKII positive cell was indicated by small white arrows in ( I–N ). Bar = 50 µm in ( B ) and 5 µm in ( C ).

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: Labeling, Virus, Staining

Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Synaptophysin and CaMKII expression in the lateral parabrachial nucleus. In these rats the central amygdala was infused with virus expressing a scrambled shRNA or a Nrxn3 shRNA. The central amygdala of Gad1-iCre Long Evans rats was infused with AAV1 containing pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby. Excitable cells within the lateral parabrachial nucleus were labeled by infusing this nucleus with AAV5 virus containing pAAV-CaMKIIa-EGFP. After four weeks post-surgery the whisker pad of the infused rats were injected with MeWo cells without varicella zoster virus (no VZV) or MeWo cells containing VZV. Two weeks after injection the brain was isolated and imaged. ( A ) shows the average number of synaptophysin terminals or puncta localized to each CaMKII positive cell within the lateral parabrachial nucleus. Representative images of rats treated with control shRNA/no VZV ( B–E ) or control shRNA/VZV ( F–I ) or Nrxn3 shRNA/no VZV ( J–M ) or Nrxn3 shRNA/VZV ( N–Q ) are shown. Hoechst 33342 nuclear stain ( B, F, J and N ) and CaMKII stain ( C, G, K and O ) and synaptophysin stain ( D, H, L and P ) is represented in several cells . Individual CaMKII positive cells (green) are outlined with a white dotted line. Arrows point to synaptophysin positive puncta (red, D, H, L and P ) colocalizing with CaMKII staining ( E, I, M and Q ). Bar = 10 µm. Panel R shows the number of CaMKII positive cells in the lateral parabrachial nucleus that colocalized with synaptophysin. Each point is from an individual animal in panels A and R. An asterisk indicates a significant difference of α=0.05. Representative images of rats treated with control shRNA/no VZV ( S ) or control shRNA/VZV ( T ) or Nrxn3 shRNA/no VZV ( U ) or Nrxn3 shRNA/VZV ( V ) show cells with synaptophysin stain colocalizing with CaMKII stain (yellow, arrows). Bar = 50 µm.

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: Expressing, Virus, shRNA, Labeling, Whisker Assay, Injection, Isolation, Control, Staining

Journal: Journal of Pain Research

Article Title: Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain

doi: 10.2147/JPR.S441706

Figure Lengend Snippet: Prodynorphin cells within the lateral parabrachial nucleus. The central amygdala of Gad1-iCre Long Evans rats was infused with AAV1 containing pAAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby. Excitable cells within the lateral parabrachial nucleus were labeled by infusing this nucleus with AAV5 virus containing pAAV-CaMKIIa-EGFP. Four weeks after infusion the whisker pad was injected with either no VZV or VZV. Six weeks after infusion brain sections of the treated rats were immunostained for prodynorphin. In ( A and B ) multiple prodynorphin positive (red) cells were imaged in the lateral parabrachial nucleus (LPB). Prodynorphin is a marker for neurons involved in pain. Cell nuclei are labeled blue with Hoechst 33342 in ( A – C ). ( B ) shows only the prodynorphin cells (red) from the same region and ( C ) shows only the EGFP positive cells (green). A higher magnification image of the LPB is shown in ( D ) and cells that colocalize prodynorphin and EGFP are yellow. Insert in ( D ) is an image through the z plane of the lateral parabrachial nucleus after staining for prodynorphin. Prodynorphin is red, EGFP is green and synaptophysin terminals are in yellow for the insert image in ( D ). Images are from a representative rat that was treated with Nrxn3 shRNA and VZV. scp = superior cerebellar peduncle. Bar= 20 µm. The histogram in ( E ) shows the number of EGFP/prodynorphin positive cells that colocalized with synaptophysin in the lateral parabrachial nucleus after knockdown of Nrxn3α in the central amygdala. Each point is from an individual animal. An asterisk indicates a significant difference of α=0.05. Representative images of rats treated with control shRNA/no VZV ( F ) or control shRNA/VZV ( G ) or Nrxn3 shRNA/no VZV ( H ) or Nrxn3 shRNA/VZV ( I ) show cells with synaptophysin stain colocalizing with prodynorphin stain (yellow, arrows). Bar = 50 µm.

Article Snippet: In a separate group of Long Evans rats, the central amygdala was infused with 1 µL of 1 × 10 13 TU/mL AAV1 containing a scrambled shRNA sequence (AAV1-GFP-U6-shRNA, Vector Biolabs) mixed 1 to 1 with the synaptophysin construct.

Techniques: Labeling, Virus, Whisker Assay, Injection, Marker, Staining, shRNA, Knockdown, Control

Journal: Cell metabolism

Article Title: Targeting Peripheral CB 1 Receptors Reduces Ethanol Intake via a Gut-Brain Axis

doi: 10.1016/j.cmet.2019.04.012

Figure Lengend Snippet: (A) Time course and rate of increase in oleate-driven respiratory activity in MGN3–1 cells. Cells were treated with 10 or 100 nM JD5037, 2.5 M of FCCP (positive control) or vehicle in the presence of the fluorescent extracellular O2 consumption reagent. Oxidative respiration was recorded every 90 sec and the slope of the initial linear increase was calculated. Points and bars are means ± SEM from n = 11–15 experiments, as indicated. *P < 0.05 compared to vehicle. (B) Verification of Cnr1 knockdown by rtPCR in cells used in panel C. Cellular uptake of the constructs was verified by fluorescent microscopy (20x magnification) and the degree of knockdown was determined by rt-PCR, *P < 0.05, n = 4. (C) Oleate-driven oxidative activity in MGN3–1 cells with shRNA-mediated knockdown of Cnr1 expression and their mock-transfected controls, n = 3–8 experiments, as indicated, *P < 0.05.

Article Snippet: Scramble shRNA with GFP Adenovirus (Ad-scramble-shRNA) , Vector Biolabs , 1122.

Techniques: Activity Assay, Positive Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Construct, Microscopy, shRNA, Expressing, Transfection

Journal: Hypertension research : official journal of the Japanese Society of Hypertension

Article Title: Histone deacetyltransferase inhibitors Trichostatin A and Mocetinostat differentially regulate MMP9, IL-18 and RECK expression, and attenuate Angiotensin II-induced cardiac fibroblast migration and proliferation.

doi: 10.1038/hr.2016.54

Figure Lengend Snippet: Figure 1 Silencing HDAC1 and HDAC2 inhibits Ang-II-induced cardiac fibroblast proliferation and migration. (a) Expression of HDAC1 and HDAC2 at basal conditions was analyzed by immunoblotting. CF1, CF2: two independent cultures of primary cardiac fibroblasts. Cell extracts from human epithelial cells (HeLa) and mouse embryonic fibroblasts (NIH3T3) served as positive controls (n = 3). (b) Effect of Ang-II dose on activation of HDAC1 and HDAC2 in quiescent CF as analyzed by a colorimetric assay using nuclear protein extracts (100 μg) immunoprecipitated with HDAC1- or HDAC2-specific antibodies. Deacetylase activity was calculated in pmoles min −1 mg−1. Results are expressed as fold change from controls (n = 6). (c) Silencing HDAC1 or HDAC2 attenuates Ang-II-induced CF proliferation. CF transduced with lentiviral HDAC1 or HDAC2 shRNA (moi 0.5 for 24) were treated with Ang-II (10 −7 M) for 48 h, and proliferation analyzed by CyQUANT assay. (d) Silencing HDAC1 or HDAC2 inhibits Ang-II (10 −7 M for 12 h) -induced CF migration. Migration was analyzed using BioCoat Matrigel invasion chambers and MTT assay. (e) Knockdown of HDAC1 and HDAC2 was confirmed by immunoblotting (n = 3). Akt served as an off-target. (f) Silencing HDAC1 or HDAC2 failed to induce cell death. Forty-eight hours after lentiviral transduction, cell death was analyzed by an ELISA that quantifies mono and oligonucleosomal fragmented DNA in cytoplasmic extracts. (b, c, d, f) *Poat least 0.01 vs. untreated, †Poat least 0.05 vs. Ang-II (n = 6-12).

Article Snippet: Anti-vimentin (#sc-373717) antibodies used in fibroblast characterization, anti-caspase-3 (35 kDa, #sc-136219) antibodies and lentiviral GFP shRNA (#sc-45924-V) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Migration, Expressing, Western Blot, Activation Assay, Colorimetric Assay, Immunoprecipitation, Histone Deacetylase Assay, Activity Assay, Transduction, shRNA, CyQUANT Assay, MTT Assay, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: Intra‐epithelial non‐canonical Activin A signaling safeguards prostate progenitor quiescence

doi: 10.15252/embr.202154049

Figure Lengend Snippet: Western blot analysis for Smad4 in mouse prostate organoids upon short‐hairpin RNA mediated knockdown (sh‐Smad4) and in control conditions (sh‐Ctrl). Representative stereoscopic images of control (sh‐Ctrl) and Smad4 knocked down (sh‐Smad4) mouse prostate organoids in normal growth condition (ENRAD) and following A83‐01 withdrawal (‐A83‐01). Scale bar = 1 mm. Source data are available online for this figure.

Article Snippet: The following plasmids were used: pLenti‐AIB‐EGFP (kindly donated by Massimo Pizzato), pSUPER‐retro‐puro‐Smad4 (Addgene #89829), and pSUPER‐retro‐puro‐GFP shRNA (Addgene #30519), MISSION ® shRNA Lentiviral Transduction Particles (AcvrIb, Merck SHCLNV‐TRCN0000022578, SHCLNV‐TRCN0000345027).

Techniques: Western Blot, shRNA, Knockdown, Control