Extended Data Fig. 2: PAG innervation of RAm Nts+ and Nts- neurons.
From: A brainstem circuit for phonation and volume control in mice
a, Experimental schematic: AAV-GFP was injected into the caudolateral PAG of male mice to label projections of vocalization-gating neurons, and 3 weeks later male mice were induced to vocalize towards a female for 90 minutes before GFP and c-Fos immunostaining. b, Immunostaining for GFP and c-Fos in coronal brain slice containing caudal PAG following injection of AAV-GFP into left caudolateral PAG and subsequent vocalization induction. Note c-Fos expression across the PAG, and injection site targeting the left caudolateral PAG. Scale bar, 200 µm. c, Quantification of PAG innervation of RAm c-Fos+ neurons (n = 4 mice). d, Representative immunostaining image of RAm showing three c-Fos+ neurons (arrowheads) also labeled with Nissl (neuron-filling stain), tdTomato (NtsCre+ neurons), and GFP (PAG innervation). Note right NtsCre + /c-Fos+ neuron receiving PAG innervation (puncta), and two left NtsCre-/c-Fos+ neurons also receiving PAG innervation. Scale bar, 10 µm. e, Additional representative immunostaining image showing four c-Fos+ neurons (arrowheads). Note three top neurons are NtsCre + /c-Fos+ and receive PAG innervation, whereas bottom-right neuron is NtsCre-/c-Fos+ and does not receive PAG innervation. Scale bar, 10 µm. All data shown as mean ± S.D.
