Article Figures & Data
Figures
- Figure 1.
Characterization of Emx1-tdtomato signals in the brain. The fluorescence signal is inverted and shown in black color. A–C, A midsagittal section shows strong fluorescence in forebrain structures. The overexposed image in B shows minor but significant signals present in hindbrain regions. The box in B is enlarged in C. D–F, Coronal sections of rostral (D) and caudal (E) medulla. Fluorescence signals were present in the tractus solitarius and corticospinal tract. The box in E is shown enlarged in F. Dense fluorescence is present in the tractus solitarius, and labeled single axonal fibers with varicosities are detected in the NTS field.
- Figure 2.
3D imaging of Emx1-tdtomato signals in the brainstem. The formalin-fixed brainstem was optically cleared and scanned on a light-sheet microscope. A, Cleared brainstem preparation. B–D, Coronal serial images of Emx1-tdtomato signals in the left half of the brainstem. At the rostral level, the Emx1-tdtomato signal is detected in the vagus nerve rootlets of the brainstem (B), traveling the dorsal medulla at the caudal level (C), and terminates within the NTS at the caudal end (D). The full scanned image is available as Movie 1. E–G, 3D reconstruction of Emx1-tdtomato signals presented in coronal (E), sagittal (F), and horizontal (G) views. The Emx1-tdtomato+ vagus nerve traverses the medulla. Outlines of brainstem tissue and NTS are depicted by yellow dashed lines. The full image is available as Movie 2. Arrowheads in the scale bar indicate the axis. V: ventral, L: lateral, C: caudal, VN: vagus nerve, CP: choroid plexus, NTS: nucleus tractus solitarius, CST: corticospinal tract.
- Figure 3.
Characterization of Emx1-tdtomato signals in the nodose ganglion. A, Dissected nodose ganglion on the vagus nerve. B, Pseudocolor image merging, DIC (C), and tdtomato fluorescence (D) images of a whole-mount nodose ganglion. E, High-magnification image of a sectioned nodose ganglion. Note fluorescence signals in D, E are inverted and shown in black. F, Emx1-tdtomato is not expressed in the Choline acetyltransferase (Chat)+ premotor neurons within the dorsal motor vagus nucleus (DMV) or Nucleus ambiguus neurons expressed Emx1-tdtomato.
- Figure 4.
Characterization of Emx1-tdtomato signals at the carotid bifurcation. A, Dissected carotid artery and associated neural tissue, as well as the superior cervical ganglion (SCG). B, C, A whole-mount image of dissected tissues in DIC (B) and Emx1-tdtomato fluorescence (C). Note fluorescence is shown in black in C. D, Higher magnification image of Emx1-tdtomato fibers in a cleared carotid tissue and SCG. Fluorescence signals (in white) are detected at the carotid sinus (CS), SCG, and surrounding nerve fibers. The outline of the carotid artery is shown by the dashed line. E, F, Emx1-tdtomato signals in the SCG section. Emx1-tdtomato signals are only sparsely detected as pieces of nerve fibers in the SCG (arrowheads). Sympathetic preganglionic neurons are identified by in situ hybridization of tyrosine hydroxylase (Th) and Neuropeptide Y (Npy). E, Emx1-tdtomato only. F, Color image merged with Th (green) and Npy (yellow). CCA: common carotid, ICA: internal carotid artery, ECA: external carotid artery.
- Figure 5.
Optogenetic stimulation of Emx1-Cre+ nerve fibers in vitro. Vagus afferent nerve (tractus solitarius) was stimulated by a LED located over the brainstem slice (A). The border of NTS is indicated by the dashed line. Axis D: dorsal, V: ventral. C–G, Photostimulation evoked either synchronous (D, F) or asynchronous (F, G) EPSCs in all recorded NTS neurons (n = 36) with little variability in the onset latency (B). C, 28% (10/36) of NTS neurons received synchronous, whereas 72% (26/36) of neurons received asynchronous EPSCs. Representative EPSC traces (D, F) and histograms (F, G) of mean EPSC (50-ms bins). Note the elevation of EPSC frequency following optogenetic afferent stimulation in E. Values are mean ± SE. H, Capsaicin effect on the NTS neurons receiving asynchronous EPSC. Capsaicin depolarizes the TRPV1+ vagus nerve terminals, resulting in increased sEPSC and block of optogenetically evoked EPSC. The inset shows responses to optogenetic stimulation during baseline (a) and after exposure to capsaicin (b).
- Figure 6.
Optogenetic stimulation of Emx1-Cre+ nerve fibers in vivo urethane anesthetized mouse. A, EKG and respiratory signals were converted to heart rate (HR) and respiratory rate (RR). B, Example of respiratory and cardiac depression response to vagus nerve photostimulation. C, D, Cardiorespiratory response to vagus nerve photostimulation. In these recordings, n = 6 responses from 5 mice. E, F, Cardiorespiratory responses to photostimulation of the carotid bifurcation. n = 5 responses from 5 mice. G, H, Cardiorespiratory response to photostimulation of the dorsal medulla. An LED was horizontally positioned over the exposed dorsal medulla surface. A spacer was used to prevent direct contact. Stimulation consistently depressed heart rate while changes in respiration tone were variable. n = 5 responses from 4 mice.
Movies
- Movie 1.
Images of Emx1-tdtomato brainstem. Video shows rostral to caudal direction.
- Movie 2.
3D reconstructed Emx1-tdtomato brainstem. Vagus nerve and emanating nerve fibers are seen at the dorsal level and dense corticospinal tracts are present at the ventral surface.
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