Figure 2. Two molecularly and anatomically distinct types of Amb^Cardiac neurons.

a, tSNE plot of Amb^Cardiac neuron expression profiles (dots). Nearest neighbor analysis defined two molecularly distinct clusters designated ACP (dark blue) and ACV (light blue) for reasons described later. b, Heat map showing expression of selected genes enriched in ACP neurons (dark blue gene names), ACV neurons (light blue), all Amb^Cardiac neurons (purple), all Amb^Laryngeal neurons (“Larynx”, coral), and all Amb neurons (black). c, Immunostaining for ACP marker Calbindin and ACV marker BChE in rostral Amb. Rostral Amb neurons (arrowheads) labeled by intrapericardial CTB stained positive for Calbindin but expressed low levels of BChE, indicating they were ACP neurons. d, Immunostaining for ACP marker Calbindin and ACV marker BChE in caudal Amb of same sample as panel c. Caudal Amb neurons (arrowheads) labeled by intrapericardial CTB expressed high levels of BChE but stained negative for Calbindin, indicating they were ACV neurons. Bars, 20 μm. e, Map of ACP and ACV neurons in Amb. Sagittal schematic view showing soma of all neurons (circles) in a representative single postnatal day 2 Amb that was retrograde labeled with CTB from the heart (CTB>Heart) and stained for ACP marker Calbindin (dark blue circles) and ACV marker butyrylcholinesterase (BChE, light blue circles). A minority of retrograde-labeled cardiac neurons (green circles) did not stain for Calbindin or BChE. f, Quantification of absolute numbers of ACP neurons (dark blue), ACV neurons (light blue), and double negative cardiac neurons (green) per Amb (mean ± s.d., n = 3 mice).