Journal: Neuron

Article Title: Visual intracortical and transthalamic pathways carry distinct information to cortical areas

doi: 10.1016/j.neuron.2021.04.017

Figure Lengend Snippet:

Article Snippet: For the experiments involving optogenetic manipulations ( , , and ), AAV1.Syn.DIO.ChrimsonR.tdTomato (120 nl, 3.9 × 10 12 vg/mL, 1:5 dilution in saline solution, UNC vector core, Addgene plasmid # 62723, from Edward Boyden) or AAV1.CAG.DIO.tdTomato (control, 120 nl, 2.6 × 10 13 vg/mL, diluted 1:5 in saline, Addgene viral prep # 28306-AAV1, from Edward Boyden) was injected into AL, PM (single injection) or V1 (5 to 7 injections).

Techniques: Plasmid Preparation, Recombinant, Software

Journal: Cell reports

Article Title: The neurexin gene family regulates olfactory glomerular formation

doi: 10.1016/j.celrep.2025.116125

Figure Lengend Snippet: (A) Schematic of the in vitro microfluidics-based axonal isolation chamber assay. Dissociated embryonic cortical neurons were cultured in the somal compartment for 3 days. Axons in the axonal compartment were exposed to gradients of His-tagged recombinant proteins to analyze their effect on axonal attraction or repulsion. (B) Representative images of axons exposed to various neurexin ligand recombinant proteins. NLGN1, neuroligin1; LPHN2, latrophilin2; LRRTM2, leucin-rich repeat transmembrane protein2; NXPH1, neurexophilin1; CLSTN3, calsyntenin3. Scale bar, 100 μm. (C) Quantitative analysis of axonal fluorescence intensities at different distances from the wall following exposure to gradients of different neurexin ligands (left). Statistical comparison of the area under the curve (AUC) of the graphs on the left (right). Fluorescence intensities in the axon compartment were normalized to those in the somal compartments and further normalized to AUC values of fluorescence graphs from the His-Fc ctrl group (which was set at the value of 1). n = 33 for His-Fc, 13 for NXPH1-His-Fc, 17 for LRRTM2-His-Fc, 14 for LPHN2-His-Fc, 12 for NLGN1-His-Fc, and 11 for CLSTN3-His-Fc. All data were collected from at least 3 independent days. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, Fisher’s LSD test following one-way ANOVA. (D) Nucleic acid sequence differences between neuroligin1 and neuroligin1B (top), leading to reduced binding affinity between neurexin and neuroligin1B (bottom). (E) Representative images of axons exposed to neuroligin1 and neuroligin1B recombinant proteins. Scale bar, 100 μm. (F) Quantitative analysis of axonal fluorescence intensities upon exposure to recombinant neuroligin1 proteins (left) and statistical comparison of the AUC of the graphs on the left (right). Fluorescence intensities in the axonal compartment were normalized to those in the somal compartments and further normalized to AUC values of fluorescence graphs from the His-Fc ctrl group. n = 9 for His-Fc, 8 for NLGN1-His-Fc, and 8 for NLGN1B-His-Fc. All data were collected from 3 independent days. Data are presented as mean ± SEM. * p < 0.05, Fisher’s LSD test following one-way ANOVA. (G) Representative images of WT and Nrxn1α KO axons exposed to ctrl or neuroligin1 recombinant proteins. Scale bar, 100 μm. (H) Quantitative analysis of axonal fluorescence intensities in response to exposure to gradients of neuroligin1 recombinant proteins in WT (left) and Nrxn1α KO neurons (right). Fluorescence intensities in the axonal compartments were normalized to those in the somal compartments and further normalized to AUC values of fluorescence graphs from the His-Fc ctrl group. n = 12 for His-Fc in WT, 11 for NLGN1-His-Fc in WT, 11 for His-Fc in Nrxn1α KO, and 11 for NLGN1-His-Fc in Nrxn1α KO. All data were collected on at least 3 independent days. Data are presented as mean ± SEM. * p < 0.05, unpaired Student’s t test.

Article Snippet: Signal peptide and extracellular domain sequences of Nxph1 (NM_00875.5), Lphn1 ( NM_181039.2 ), and Clstn3 ( NM_153508.4 ) were synthesized by GENEWIZ, while sequences of Nlgn1 and LRRTM2 were amplified from pCAG-NL1(−) (Peter Scheiffele, Addgene, #15260) and FSW-HRP-V5-LRRTM2 (Alice Ting, Addgene, #82537) plasmids, respectively.

Techniques: In Vitro, Isolation, Boyden Chamber Assay, Cell Culture, Recombinant, Fluorescence, Comparison, Sequencing, Binding Assay

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