Journal: Nature Medicine

Article Title: Bilateral gene therapy in children with autosomal recessive deafness 9: single-arm trial results

doi: 10.1038/s41591-024-03023-5

Figure Lengend Snippet: Interferon-gamma was detected by ELISpot assay in patients 1 ( a ), 2 ( b ), 3 ( c ), 4 ( d ), and 5 ( e ). T cell responses to the AAV1 capsid were negative in 5 patients at baseline and 6 weeks after AAV1-hOTOF gene therapy. SFU, spot-forming unit. NC: negative control; PC: positive control.

Article Snippet: The AAV1-hOTOF, containing the functional human OTOF coding sequence packaged by dual-AAV vectors, was produced by PackGene Biotechnology and stored at ≤−65 °C.

Techniques: Enzyme-linked Immunospot, Negative Control, Positive Control

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 Reports

Article Title: Acute changes in systemic glycemia gate access and action of GLP-1R agonist on brain structures controlling energy homeostasis

doi: 10.1016/j.celrep.2022.111698

Figure Lengend Snippet: The access of Exendin-4 in the ARC under hypoglycemia involves tanycyte-borne VEGF (A) Quantification of hypothalamic Exendin-4_VT 750 (120 nmol/kg) fluorescent signal normalized to vehicle and (B) glycemic changes in response to VEGF (0.1 mg/kg, i.p.), insulin (240 nmol/kg, i.p. ) + VEGF, VEGF receptor antagonist Axitinib (25 mg/kg, i.p.), Axitinib alone, or the prostacyclin analog sodium beraprost (BPS 1 mg/kg, i.p.). n = 5–7/group. ∗ p < 0.05, vehicle versus insulin. Data are expressed as mean ± SEM. (C) Model for tanycyte-restricted invalidation of Vegfa in Tanycyte ΔVegfa mice (Vegfa lox/lox ; third ventricle injection of TAT-CRE or AAV-CRE-GFP) and experimental setup for concomitant injection of vehicle or 2-DG (250 mg/kg) together with Ex-4_Cy3 (120 nmol/kg) ∼15 min before sacrifice. (D and E) (D) Representative photomicrographs for Ex-4_Cy3 fluorescent distribution and (E) signal quantification in the dorsal (green circles) and ventral part (red circles) of the arcuate nucleus ∼15 min after 2-DG injection. Signal quantification was acquired on four to six brain sections from each animal, N = 2–5/group. Data are expressed as mean ± SEM. ∗ p < 0.05. (F) Glycemic change after saline (black, red) or insulin (0.75 U/kg, gray, orange) in control (black, gray) and Tanycyte ΔVegfa mice (red, orange). (G) 3D fluorescent signal quantification in the ARC in normoglycemic (NG) and hypoglycemic (HG) conditions. (H) Representative 2D planes from whole-brain light-sheet scanning to visualize fluorescent signal of peripherally injected Exendin-4_VT 750 (120 nmol/kg) in the ME/ARC region of control and Tanycyte ΔVegfa mice. (I) Signal quantification of fluorescent Exendin-4_VT 750 in the dorsal (green circles) and ventral part (red circles) of the arcuate nucleus 60 min after insulin (0.75 U/kg) injection. Signal quantification was acquired on three brain sections from each animal, n = 2–4/group. Data are expressed as mean ± SEM. ∗ p value < 0.05, insulin versus vehicle. For statistical details, see .

Article Snippet: Tanycytic specific knockdown of VEGFa was performed in isoflurane-anesthetized 8-weeks old Vegfa loxP/ loxP or tdTomato loxP−STOP-loxP Vegfa loxP/ loxP male mice by stereotactic injection of either TAT-Cre (Experimental group in C–4E) or AAV 1/2 -GFP (AAV 1/2 -CAG-eGFP; serotype 1:2 chimeric; titer = 1.2 x 10 13 GC/mL; Vector Biolabs) to produce control or AAV 1/2 -CRE-GFP (AAV 1/2 -CAG-iCre/eGFP; serotype 1:2 chimeric; titer = 2.8 x 10 13 ; Vector Biolabs) or AAV1/2 Dio2:Cre (serotype 1:2 chimeric, 0.5 × 10 10 genomic particles μl −1 , produce as previously described ( )) to produce Tanycyte ΔVegfa mice.

Techniques: Injection, Saline, Control

Journal: Cell Reports

Article Title: Acute changes in systemic glycemia gate access and action of GLP-1R agonist on brain structures controlling energy homeostasis

doi: 10.1016/j.celrep.2022.111698

Figure Lengend Snippet: Metabolic action of Exendin-4 involves tanycyte-borne VEGF (A) Experimental schedule for the characterization of metabolic efficiency in controls (Vegfa lox/lox ; ventricular injection of AAV-GFP) and Tanycyte ΔVegfa mice (Vegfa lox/lox ; ventricular injection of AAV-CRE-GFP) in response to daily i.p. saline injection (baseline, gray) followed by a 3-day treatment period consisting of a daily injection (2:00 p.m.) of Exendin-4 (120 nmol/kg, red), followed by insulin (20 nmol/kg, blue), and 3 days of mix of insulin + Exendin-4 (20 nmol/kg, 120 nmol/kg, green). Control and Tanycyte ΔVegfa mice were then exposed to a 3-week high-fat feeding regimen and reevaluated for their response to Exendin-4 or insulin + Exendin-4. Graphs represent averaged values for (B and C) cumulative food intake, (D and E) body weight change, (F and G) fat oxidation, and (H and I) food intake on chow diet. Three-day averaged cumulative food intake (J and K) and body weight change (L and M) through i.p. saline injection (black) followed by a 3-day treatment period consisting of a daily injection (2:00 p.m.) of Exendin-4 (120 nmol/kg, red) and 3 days of mix of insulin + Exendin-4 (20 nmol/kg, 120 nmol/kg, green) of control and Tanycyte ΔVegfa mice after exposure to high-fat diet. n = 8–5/group. Data are expressed as mean ± SEM. ∗ p < 0.05. $p < 0.05, insulin versus vehicle. £p < 0.05, Ex-4 versus vehicle. #p < 0.05, insulin + Ex-4 versus vehicle. &p < 0.05, insulin + Ex-4 versus Ex-4. For statistical details, see .

Article Snippet: Tanycytic specific knockdown of VEGFa was performed in isoflurane-anesthetized 8-weeks old Vegfa loxP/ loxP or tdTomato loxP−STOP-loxP Vegfa loxP/ loxP male mice by stereotactic injection of either TAT-Cre (Experimental group in C–4E) or AAV 1/2 -GFP (AAV 1/2 -CAG-eGFP; serotype 1:2 chimeric; titer = 1.2 x 10 13 GC/mL; Vector Biolabs) to produce control or AAV 1/2 -CRE-GFP (AAV 1/2 -CAG-iCre/eGFP; serotype 1:2 chimeric; titer = 2.8 x 10 13 ; Vector Biolabs) or AAV1/2 Dio2:Cre (serotype 1:2 chimeric, 0.5 × 10 10 genomic particles μl −1 , produce as previously described ( )) to produce Tanycyte ΔVegfa mice.

Techniques: Injection, Saline, Control