colorless raav2 retro cre  (Addgene inc)

Journal: bioRxiv

Article Title: Encoding social preference by interhemispheric neurons in the Insula

doi: 10.1101/2022.12.15.520538

Figure Lengend Snippet: a ., h ., r ., w . Experimental design. b-f . Representative epifluorescent image of a coronal slice of brain injected with an AAV2-DIO-eif1a-eYFP anterograde virus in the Insula showing the injection site in the insula (b, c) and projections to the contralateral insula (d), the dlBNST (e), the CeA (f). Scale 100 µm for b,d,e,f and 50 µm for c. g. Quantification of the Insula interhemispheric projections in bilateral dlBNST, CeA, and contralateral Insula. i , j . Representative epifluorescent image of a coronal slice of brain injected with a rAAV2-retro-CAG-Cre retrograde virus in the ipsilateral insula in AI9dTomato mouse (i, scale 500 µm) with a high magnification of contralateral labeling in Insula (j, scale 25 µm). k , l . Quantification of contralateral labeling in Insula (homotopic labeling) and other cortical regions (heterotopic labeling). m-p . Immunofluorescence confocal images showing Insula interhemispheric neurons (tomato labeling, m), insula Satb2 staining (yellow labeling, n), insula Ctip2 labeling (green labeling, o), and the overlay at low (top, scale 100 µm) and high magnification (bottom, scale 25 µm). At the bottom, white arrows show examples of Tomato and Satb2 colocalizations. q . Quantification of Tomato, Satb2, and Ctip2 colocalization in the Insula. s . Representative traces showing a collision test and a high-frequency stimulation protocol for an Insula interhemispheric neuron projecting to the other Insula . t . Histogram of the onset latency of Insula antidromic responses. u , v . Representative epifluorescent image at low (left, scale bar 500 µm) and high magnification (right, 150 µm) of MBP staining (grey labeling) and Insula interhemispheric neurons (tomato labeling). x , y . Representative image obtained with electron microscopy showing immunogold GFP labeling of unmyelinated interhemispheric Insula axons passing through the corpus callosum (x) or the anterior commissure (y) (green arrow). White arrow shows an example of a myelinated axon (scale 500 nm). dlBNST: dorsolateral bed nucleus of the stria terminalis; ovBNST: oval-BNST; juxta-BNST: juxtacapsular BNST; a . c .: anterior commissure; cc . corpus callosum; BLA: basolateral amygdala; CeA: central amygdala; Ins: Insula; Som: Somatosensory cortex; M1: primary Motor cortex; M2:secondary Motor cortex; mPFC: medial Prefrontal cortex; Rec: recording; MBP: myelin basic protein . n: number of neurons; N: number of mice.

Article Snippet: rAAV2-retro-CAG-Cre (2,8x10 vg/mL; UNC Vector Core, Boyden);AAV2.2-eif1a-DIO-eYFP (3x10 vg/mL; Addgene); AAV2.5-eif1a-DIO-eYFP (1x10 vg/mL; Addgene);AAV2.2-hSyn-eYFP (3x10 vg/mL; 50465-AAV2,Addgene); AAV2.5—eif1a-DIO-eYFP (1x10 vg/mL; 27056-AAV5, Addgene); AAV2.2-hSyn-ChR2(H134R)-eYFP (3.1x10 vg/mL; UNC, AV4384G); AAV5-flex-taCasp3-TEVp (7x10 vg/mL; Addgene); Tetrodotoxin (TTX, 0.5 µM, abcam ab120055); 4 aminopyridine (4AP; 1 mM, ascent scientific, asc-122-100mg)

Techniques: Injection, Labeling, Immunofluorescence, Staining, Electron Microscopy

Journal: The Journal of Comparative Neurology

Article Title: Local targets of T‐stellate cells in the ventral cochlear nucleus

doi: 10.1002/cne.25378

Figure Lengend Snippet: Labeling of T‐stellate cells via viral injection. (a) Schematic of the experimental approach for identifying T‐stellate cells. The rAAV2‐retro‐PKG‐Cre vector was injected into the inferior colliculus (IC) of Ai14 mice to retrogradely infect T‐stellate cells and allow the selective expression of tdTomato in infected neurons. (b) A small volume of injection (plus sign) results in a banding pattern of tdTomato expression (magenta) in the injected IC (outlined with dashed lines). DNA was counterstained with DAPI (blue). The stitched images were placed on black backgrounds for the purpose of visualizing the images all at the same orientation as the schematic in (a). (c) Distribution of tdTomato‐labeled T‐stellate cells in the contralateral cochlear nucleus (CN) from one experiment. Four coronal sections spanning the CN posterior‐anterior axis showing tdTomato‐labeled T‐stellate cells (magenta) distributed along a band within both the posterior ventral CN (pVCN) and anterior VCN (outlined with white dashed lines). DNA was counterstained with DAPI (blue). Axons of T‐stellate cells exit the CN via the ventral acoustic stria (asterisks). Scale bars in (b) = 500 μm, (c) = 200 μm. CB, cerebellum

Article Snippet: A total of 60–80 nl of AAV vector (rAAV2‐retro‐pkg‐Cre, Addgene (Watertown, MA) 24593‐AAVrg; Tervo et al., ) or 400 nl of BDA (3000 MW, Invitrogen, 10% in sodium citrate‐HCl, pH = 3) was loaded into a glass pipette with a tip diameter of 10–15 μm, and pressure was injected into the IC at a rate of 1 nl/s.

Techniques: Labeling, Injection, Plasmid Preparation, Expressing, Infection