Figure 1. Inhibitory neurons in the hypothalamus and amygdala provide input to the periaqueductal gray (PAG) vocal gating circuit.
(A) (Left) Viral strategy shown for transsynaptic labeling of direct inputs to PAG-USV neurons (performed in N = 4 males). (Right) Confocal images are shown of starter PAG-USV neurons, upstream neurons labeled within the preoptic area of the hypothalamus (POA), and upstream neurons labeled within the AmgC/M and CeA. (B) Same, for transsynaptic labeling of direct inputs to GABAergic PAG neurons (performed in N = 4 males, N = 2 females). (C) Representative confocal image of in situ hybridization performed on transsynaptically labeled POA neurons that provide direct input to GABAergic PAG neurons (labeled with GFP, shown in white), showing overlap with expression of VGAT (green) and VGlut2 (red). DAPI shown in blue. (D) Quantification of overlap of transsynaptically labeled POA and amygdala neurons (CeA and AmgC/M combined) with VGAT and VGlut2 (N = 2 male mice per condition). Total numbers of neurons scored for overlap in each condition are indicated by the numbers over the bars. See also Figure 1—figure supplements 1–4, and Figure 1—source datas 1–2.
Figure 1—source data 1. Source data for Figure 1A–C.
elife-63493-fig1-data1.docx (13.8KB, docx)
Figure 1—source data 2. Source data for Figure 1D.
elife-63493-fig1-data2.xlsx (16.1KB, xlsx)
Figure 1—figure supplement 1. Monosynaptic rabies-based tracing reveals preoptic and amygdala inputs to the midbrain vocalization circuit.
(A) Example confocal images showing transsynaptic labeling of neurons in the preoptic area of the hypothalamus (preoptic area [POA], green) that provide monosynaptic input to GABAergic periaqueductal gray (PAG) neurons (left-most confocal image, VGAT-Cre female; middle confocal image, VGAT-Cre male) or to PAG-USV neurons (right confocal image, FosTVA male; blue, Neurotrace). (B–C) Same, for transsynaptically labeled neurons in (B) the central-medial boundary region of the amygdala (AmgC/M) and (C) the central amygdala (CeA). Coronal brain atlas images show the approximate plane of section of the confocal images to the right. Red rectangles superimposed on the atlas images indicate the approximate location of the brain regions shown in the confocal images. See also Figure 1—figure supplements 2–4. Scale bars, 250 µm. All brain atlas images were obtained from the Allen Mouse Brain Reference Atlas, (https://mouse.brain-map.org/experiment/thumbnails/100048576?Image_type=atlas).
Figure 1—figure supplement 2. Monosynaptic rabies-based tracing reveals cortical inputs to the midbrain vocalization circuit.
(A) Example confocal images showing transsynaptic labeling of neurons in the infralimbic cortex (green) that provide monosynaptic input to GABAergic periaqueductal gray (PAG) neurons (left-most confocal image, VGAT-Cre female; middle confocal image, VGAT-Cre male) or to PAG-USV neurons (right confocal image, FosTVA male; blue, Neurotrace). (B–E) Same, for transsynaptically labeled neurons in (B) insular cortex, (C) motor (M1, M2), and cingulate (Cg) cortices, (D) M1, M2, and somatosensory cortex (S1), and (E) auditory cortex. Coronal brain atlas images show the approximate plane of section of the confocal images to the right. Red rectangles superimposed on the atlas images indicate the approximate location of the brain regions shown in the confocal images. See also Figure 1—figure supplements 1, 3 and 4. Scale bars, 250 µm. All brain atlas images were obtained from the Allen Mouse Brain Reference Atlas, (https://mouse.brain-map.org/experiment/thumbnails/100048576?Image_type=atlas).
Figure 1—figure supplement 3. Monosynaptic rabies-based tracing reveals additional hypothalamic inputs to the midbrain vocalization circuit.
(A) Example confocal images showing transsynaptic labeling of neurons in the anterior hypothalamus (green) that provide monosynaptic input to GABAergic periaqueductal gray (PAG) neurons (left-most confocal image, VGAT-Cre female; middle confocal image, VGAT-Cre male) or to PAG-USV neurons (right confocal image, FosTVA male; blue, Neurotrace). (B–D) Same, for transsynaptically labeled neurons in (B) the paraventricular nucleus (PVN), lateral hypothalamus (LH), and zone incerta (ZI), (C) ventromedial hypothalamus (VMH) and LH, (D) posterior hypothalamus, and (E) premammillary nucleus. Coronal brain atlas images show the approximate plane of section of the confocal images to the right. Red rectangles superimposed on the atlas images indicate the location of the brain regions shown in the confocal images. See also Figure 1—figure supplements 1, 2 and 4. Scale bars, 250 µm. All brain atlas images were obtained from the Allen Mouse Brain Reference Atlas, (https://mouse.brain-map.org/experiment/thumbnails/100048576?Image_type=atlas).
Figure 1—figure supplement 4. Monosynaptic rabies-based tracing reveals additional subcortical inputs to the midbrain vocalization circuit.
(A) Example confocal images showing transsynaptic labeling of neurons in the ventral pallidum (green) that provide monosynaptic input to GABAergic periaqueductal gray (PAG) neurons (left-most confocal image, VGAT-Cre female; middle confocal image, VGAT-Cre male) or to PAG-USV neurons (right confocal image, FosTVA male; blue, Neurotrace). (B–D) Same, for transsynaptically labeling of neurons in (B) lateral septum, (C) bed nucleus of the stria terminals (BNST), (D) lateral habenula, and (E) paraventricular nucleus of the thalamus (PVT). Coronal brain atlas images show the approximate plane of section of the confocal images to the right. Red rectangles superimposed on the atlas images indicates the location of the brain regions shown in the confocal images. See also Figure 1—figure supplements 1–3. Scale bars, 250 µm. All brain atlas images were obtained from the Allen Mouse Brain Reference Atlas, (https://mouse.brain-map.org/experiment/thumbnails/100048576?Image_type=atlas).