Fig. 1.
The central amygdala (Amy) γ-aminobutyric acid–mediated (GABAergic) neurons projects onto ventrolateral periaqueductal gray neurons. (A) Schematic diagram of AAV-Cre-eGFP injected into the Amy for anterograde tracing. (B) Left, Viral expression in the Amy of a C57BL/6J mouse. Scale bar, 500 µm; inset, 25 µm. Right, A representative image of the projection site in ipsilateral lateral periaqueductal gray (top), ventrolateral periaqueductal gray, and dorsal raphe nucleus (bottom). Scale bar, 50 µm; inset, 25 µm. (C) Strategy for cell type–specific monosynaptic tracing. (D) Representative images of the viral injection site and expression within the ventrolateral periaqueductal gray. Starter cells (yellow) coexpressing TVA-GFP (green), rabies G and RV-EnvA-ΔG-dsRed (red) in ventrolateral periaqueductal gray of GAD2-Cre mice (top) and CaMKII-Cre (bottom) mice. Scale bar, 500 µm; inset, 50 µm. (E) Representative images of presynaptic cells in central Amy. Rabies-labeled cells within central Amy of GAD2-Cre mice (top) and CaMKII-Cre (bottom) mice. Numbers indicate the distance from bregma. Scale bar, 100 µm. (F) Identification of rabies virus-labeled central Amy neurons. White arrows represent colocalization of rabies virus–discosoma species red fluorescent protein and γ-aminobutyric acid (GABA; top: rabies virus–positive cells and GABA, GAD2-Cre mouse; bottom: rabies virus–positive cells and GABA, CaMKII-Cre mouse). Scale bar, 25 µm. (G) Quantitation of GABA–expressing rabies virus–positive cells that projection onto ventrolateral periaqueductal gray GABAergic and glutamatergic neurons (77.7% ± 5.4% and 77.7% ± 4.5%; n = 6 slices from three mice per group). (H) Strategy for cell type–specific anterograde tracing. (I) Schematic showing the ChR2-expressing cells in central Amy and terminals in ventrolateral periaqueductal gray of GAD2-Cre mice. Scale bars, 200 µm and 100 µm, respectively. Aq, aqueduct; AP, anterior-posterior; BLA, basolateral amygdala; CeA, central amygdala; DRN, dorsal raphe nucleus; ChR2, channelrhodopsin-2; DsRed, discosoma species red fluorescent protein; lPAG, lateral periaqueductal gray; rabies G, rabies virus glycoprotein; RV, rabies virus; TVA, avian tumor virus receptor A; vlPAG, ventrolateral periaqueductal gray; 4n, trochlear nerve.