Journal: bioRxiv

Article Title: Amygdala GABA Neurons: Gatekeepers of Stress and Reproduction

doi: 10.1101/2025.01.06.631361

Figure Lengend Snippet: Functional connectivity of GABAergic neurons in posterodorsal medial amygdala (MePD) reconfigures in response to optical stimulation of urocortin-3 (UCN3) neurons or acute restraint stress. (A) Schematic diagram of the experimental design for optically stimulating MePD UCN3 neurons and simultaneously imaging calcium activity from MePD GABAergic neurons. Heatmaps of calcium activity (ΔF/F) in response to (left) optical stimulation of UCN3 neurons and (right) during restraint-induced stress. (B-G) Representative photomicrographs of the MePD from a UCN3-Cre::VGAT-Flpo mouse injected with pAAV-Syn-Flex-rc+pAAV-EF1a-fDIO-GCaMP6s. (B&E) Red fluorescence (tdTomoato) labels UCN3 neurons expressing Chrimson-tdTomato, (C&F) green fluorescence labels GABAergic neurons, and (D&G) red and green signals merged. (H&I) Functional connectivity in the GABA populations was quantified using signed lagged cross-correlation (SLxCorr). (J) Functional connectivity matrices show a statistically significant difference between the baseline period and subsequent UCN3 stimulation or restraint stress across animals, assessed via Kolmogorov-Smirnov tests for each animal trial and using Fisher’s method for combining p-values across different animals (see Methods). Distribution of combined p-value obtained from different combinations of trials (one from each animal) is consistently below the 0.05 confidence level. Scale bars: B-D, 200µm; E-G, 50µm.

Article Snippet: Separate mice cohorts received the following viral injection into the MePD: (i) a mixture of pAAV-Syn-Flex-rc[Chrimson-tdTomato] (2.0×10 gc/mL, Serotype:5, #62723, Addgene, Massachusetts, USA) + pAAV-EF1a-fDIO-GCaMP6s (2.3×10 gc/mL; Serotype:8; #105715, Addgene, Massachusetts, USA; final volume 400nl); (ii) a mixture of pAAV-Syn-Flex-rc[Chrimson-tdTomato] (2.0×10 gc/mL, Serotype:5, #62723, Addgene, Massachusetts, USA) + pAAV.CAG.Flex.GCaMP6s.WPRE.SV40 (4.3×10 gc/mL; Serotype:9; # 100842, Addgene, Massachusetts, USA; final volume 400nl); (iii) a mixture of control virus AAV-Ef1a-DIO-EYFP (250nl, 4.4 × 10 gc/mL, Serotype:9, #27056, Addgene, Massachusetts, USA) + pAAV-EF1a-fDIO-GCaMP6s (2.3×10 gc/mL; Serotype:8; #105715, Addgene, Massachusetts, USA; final volume 250nl).

Techniques: Functional Assay, Imaging, Activity Assay, Injection, Fluorescence, Expressing