Investigation of vagal sensory neurons in mice using optical vagal stimulation and tracheal neuroanatomy

Abstract

In rats and guinea pigs, sensory innervation of the airways is derived largely from the vagus nerve, with the extrapulmonary airways innervated by Wnt1+ jugular neurons and the intrapulmonary airways and lungs by Phox2b+ nodose neurons; however, our knowledge of airway innervation in mice is limited. We used genetically targeted expression of enhanced yellow fluorescent protein-channelrhodopsin-2 (EYFP-ChR2) in Wnt1+ or Phox2b+ tissues to characterize jugular and nodose-mediated physiological responses and airway innervation in mice. With optical stimulation, Phox2b+ vagal fibers modulated cardiorespiratory function in a frequency-dependent manner while right Wnt1+ vagal fibers induced a small increase in respiratory rate. Mouse tracheae contained sparse Phox2b-EYFP fibers but dense networks of Wnt1-EYFP fibers. Retrograde tracing from the airways showed limited tracheal innervation by the jugular sensory neurons, distinct from other species. These differences in physiology and vagal sensory distribution have important implications when using mice for studying airway neurobiology.

Keywords: Sensory neuroscience; Techniques in neuroscience.

Graphical abstract

Figure 1

Effect of left and right electrical vagus nerve stimulation (VNS) on cardiorespiratory function in anesthetized mice (A) Schematic diagram of the breeding strategy of Wnt1-Cre;loxP-ChR2 and Phox2b-Cre;loxP-ChR2 mouse lines. (B) Schematic diagram of physiological recording with electrical VNS. (C) Example tracings of blood pressure (upper) and diaphragm EMG (lower) recordings with right electrical VNS (black bar labeled “Electrical VNS on”) at 20 Hz. Accompanying mean ± SD physiological response data showing right (open symbol and dashed line) and left (filled symbol and solid line) electrical VNS train frequency-dependent changes in (D) heart rate, (E) blood pressure, (F) respiratory rate, and (G) minute-dEMG. D′, D″, E′, E″, F′, F″, G′, and G″ show Emax and EF50 values for heart rate, blood pressure, respiratory rate and minute-dEMG respectively. n = 8 and 1 respectively for Phox2b-Cre;loxP-ChR2 and Wnt1-Cre;loxP-ChR2 mice. n = 5 and 4 respectively for right and left electrical VNS. Abbreviations: BP, blood pressure; dEMG, diaphragmatic electromyography; DMNV, dorsal motor nucleus of vagus; EF50, the stimulation frequency that produced effect in 50% of animals; Emax, the stimulation frequency that produced a maximum effect; eVNS, electrical vagus nerve stimulation; GIT, gastrointestinal tract; NA, nucleus ambiguus; nTS, nucleus of the solitary tract; Pa5, paratrigeminal nucleus.

Figure 2

Differential sensorimotor effects evoked by left and right optical vagus nerve stimulation (VNS) in Phox2b-Cre;loxP-ChR2 and Wnt1-Cre;loxP-ChR2 mice (A) Schematic diagram of physiological recording with optogenetic VNS. (B) Example tracings of blood pressure (upper), diaphragm EMG (middle), and esophageal pressure (lower) recordings with optical VNS (blue bar labeled “Laser on”) at 20 Hz of (i, iii) left and (ii, iv) right vagal fibers in Phox2b-Cre;loxP-ChR2 (green) and Wnt1-Cre;loxP-ChR2 (blue) mice. Accompanying mean ± SD data optical pulse train frequency-dependent changes in (C and G) heart rate, (D and H) blood pressure, (E and I) esophageal pressure and (F and J) respiratory rate are shown in the green and blue stimulus-response curve plots. n = 10 and 6 respectively for left (light green symbol and solid line) and right (dark green symbol and dash line) Phox2b-Cre;loxPChR2 and n = 6 for loxP-ChR2 (open symbol and dotted black line). n = 9 and 7 respectively for left (light blue symbol and solid line) and right (dark blue symbol and dash line) Wnt1-Cre;loxP-ChR2 and 6 for loxP-ChR2 (open symbol and dotted black line). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; #p = 0.05–0.08 for comparison of left and right optical VNS groups against loxP-ChR2 (Dunnett’s post hoc tests). Abbreviations: BP, blood pressure; dEMG, diaphragmatic electromyography; DMNV, dorsal motor nucleus of vagus; EMG, electromyography; EP, esophageal pressure; GIT, gastrointestinal tract; NA, nucleus ambiguus; nTS, nucleus of the solitary tract; Pa5, paratrigeminal nucleus.

Figure 3

Optical recruitment of different fiber types in ex vivo vagus nerve EYFP-ChR2 expression the axons of the vagus nerve from (A) Phox2b-Cre;loxPChR2 and (B) Wnt1-Cre;loxP-ChR2 mice. Representative compound actional potentials induced by ex vivo optical stimulation of vagi harvested from (C) Phox2b-Cre;loxP-ChR2 and (D) Wnt1-Cre;loxP-ChR2 mice. Representative compound actional potential trace during ex vivo optical stimulation of vagi harvested from (E) Phox2b transgene-negative and (F) Wnt1 transgene-negative loxP-ChR2 mice. Representative compound actional potentials induced by electrical stimulation of vagi harvested from (G) Phox2b-Cre;loxP-ChR2 and (H) Wnt1-Cre;loxP-ChR2 mice. G′ and H′ show the magnified compound action potential from the dotted box areas in G and H respectively. Scale bars represent 100 μm in (A and B). ChR2, channelrhodopsin-2; EYFP, enhanced yellow fluorescent protein.

Figure 4

Transgenic expression of EYFP in the vagal ganglia and the brainstem of Phox2b-Cre;loxP-ChR2 and Wnt1-Cre;loxP-ChR2 mice (A–D) Differential EYFP-ChR2 expression in the jugular and nodose portions of the vagal ganglia and colocalization with the pan neuronal marker PGP9.5, the satellite glial cell marker GS, CGRP, and TH in (A and B). (C and D) Representative examples of expression of CGRP and TH in optically cleared vagal ganglia and (E and F) quantitative relative abundance of each. (G and H) Differential EYFP-ChR2 expression at rostrocaudal levels of the medulla. Numbers on the right lower corners indicate the nearest bregma levels. Lower panels in (G and H) show magnified views of the white rectangles delineated in the respective upper panels. Scale bars represent 100 μm in (A, B, and C), 200 μm in (D), and 500 μm in (G and H). Abbreviations: 7N, facial nucleus; AP, area postrema; CGRP, calcitonin gene related peptide; ChR2, channelrhodopsin-2; Cu, cuneate nucleus; DMV, dorsal motor nucleus of vagus; EYFP, enhanced yellow fluorescent protein; GS, glutamine synthetase; NA, nucleus ambiguus; nTS, nucleus of the solitary tract; Pa5, paratrigeminal nucleus; PGP9.5, protein gene-product 9.5; TH, tyrosine hydroxylase.

Figure 5

Innervation pattern of tracheal EYFP fibers in Phox2b-Cre;loxP-ChR2 mice (A) Representative image showing overall distribution of Phox2b-derived nerve fibers and bundles in the mouse trachea. (B) A higher magnification of Phox2b-derived nerve fiber bundle on the adventitial surface. Phox2b-derived nerve fibers were (C) positive for PGP9.5 but negative for (D) CGRP, (E) TH, and (F) SP. White arrows indicate EYFP-positive fibers in close proximity to (E) TH-positive or (F) SP-positive fibers. (G) Some of adventitial Phox2b-derived fibers were positive for ChAT (white arrows). (H) Relationship between Phox2b-derived mucosal nerve fibers and ASMA-stained blood vessels. ASMA, alpha smooth muscle actin; CGRP, calcitonin gene related peptide; ChAT, choline acetyltransferase; EYFP, enhanced yellow fluorescent protein; PGP9.5, protein gene-product 9.5; SP, substance P; TH, tyrosine hydroxylase. Scale bars represent 100 μm in all images except in (A) where it represents 1 mm.

Figure 6

Innervation pattern of tracheal EYFP fibers in Wnt1-Cre;loxP-ChR2 mice (A) Representative image showing overall distribution of Wnt1-derived nerve fibers and bundles in the mouse trachea. (B) Wnt1-derived EYFP fibers at different z planes on a projected image. The color bar on the right indicates superficial-to-deep z planes. (C) The majority of Wnt1-derived EYFP fibers were positive for PGP9.5 (solid filled white arrows) but some were negative for this pan-neuronal marker (open white arrows). (D) The majority of Wnt1-derived mucosal EYFP fibers were positive for CGRP. On the adventitial surface, only select few EYFP (E) fibers and (F) intrinsic neurons (solid filled white arrows) were positive for CGRP. Open white arrow in (F) indicates an artifact in the sample. CGRP, calcitonin gene related peptide; EYFP, enhanced yellow fluorescent protein; PGP9.5, protein gene-product 9.5. Scale bars represent 100 μm for all images except in (A) where it represents 1 mm.

Figure 7

Neurochemical characterization of tracheal EYFP-ChR2 fibers in Wnt1-Cre;loxP-ChR2 mice Mucosal (A) but not adventitial (B) Wnt1-derived EYFP-ChR2 fibers expressed TH. (C) Many mucosal EYFP-ChR2 fibers additionally stained positive for SP. (D) Some Wnt1-derived intrinsic ganglionic neurons on the adventitial surface expressed SP (solid filled white arrows) but some did not (open white arrow). (E) A limited number of Wnt1-derived fibers and intrinsic neurons on the adventitial surface were positive for ChAT (solid filled white arrows). (F) Wnt1-derived EYFP-ChR2 fibers running along ASMA-positive blood vessels. ASMA, alpha smooth muscle actin; ChAT, choline acetyltransferase; EYFP, enhanced yellow fluorescent protein; SP, substance P; TH, tyrosine hydroxylase. Scale bars represent 100 μm in all images.

Figure 8

Identification and characterization of neurons retrogradely traced from the trachea and the lungs in Phox2b-Cre;loxP-ChR2 and Wnt1-Cre;loxP-ChR2 mice (A) Schematic diagram of the experiment where the retrograde viral tracer AAV-CAG-tdTomato was injected into the trachea and lungs along with sectioning of left vagus and superior laryngeal nerves in two mouse lines. Expression of virally induced tdTomato in the trachea of (B) Phox2b-Cre;loxP-ChR2 and (C) Wnt1-Cre;loxP-ChR2 mice. Localization of virally traced tdTomato-positive neurons in the left and right optically cleared whole vagal ganglia and their quantification of (D and F) Phox2b-Cre;loxP-ChR2 and (E and G) Wnt1-Cre;loxP-ChR2 mice. Representative images of virally traced tdTomato-positive neurons in the optically cleared whole (H and I) stellate and (J and K) superior cervical ganglia of (H and J) Phox2b-Cre;loxP-ChR2 and (I and K) Wnt1-Cre;loxP-ChR2 mice. Expression of virally traced tdTomato-positive vagal terminals in the medulla in both (L) Phox2b-Cre;loxP-ChR2 and (M) Wnt1-Cre;loxP-ChR2 mice was localized to the nTS and vagal fiber tract (solid white arrows) with no expression observed in the Pa5. CGRP, calcitonin gene related peptide; ChR2, channelrhodopsin-2; DMNV, dorsal motor nucleus of vagus; EYFP, enhanced yellow fluorescent protein; nTS, nucleus of the solitary tract; Pa5, paratrigeminal nucleus; SLN, superior laryngeal nerve; VG, vagal ganglia. Scale bars represent 100 μm in (B and C), 150 or 200 μm in (D, E, H, and I), and 500 μm in (L and M).

Figure 9

Identification of neurons retrogradely traced in a Cre-dependent manner from the trachea in CGRP-Cre mice (A) Schematic diagram of the experiment where the Cre-dependent retrograde viral tracer AAV-FLEX-tdTomato was injected into the trachea of CGRP-Cre mice. (B) Representative image of optically cleared vagal ganglia showing conditionally traced tdTomato-positive neurons (which were CGRP-positive) in relation to TH-expressing neurons. (C) Quantification of the total number of TH-expressing and conditionally traced neurons from n = 4 separate experiments. (D and E) Representative images showing virally induced tdTomato-positive fibers and local intrinsic neurons (solid white arrow) in the trachea. (F) Representative images showing localization of virally traced tdTomato-positive nerve terminals along with ChAT staining for neuroanatomy at different bregma levels of the medulla. Expression of tdTomato was restricted to the nTS and the vagal fiber tract (solid white arrows). In the overlay images, the tdTomato was expressed in a red pseudo color. ChAT, choline acetyltransferase; ChR2, channelrhodopsin-2; EYFP, enhanced yellow fluorescent protein. Scale bars represent 300 μm in (B), 100 μm in (D and E), and 100 μm in (F).

Figure 10

Identification of neurons retrogradely traced from the trachea of Phox2b-Cre;loxP-ChR2 mice (A) Schematic diagram of the experiment where the retrograde tracer cholera toxin B (CTB) was injected into the trachea of Phox2b-Cre;loxP-ChR2 mice (n = 6). (B) Representative images of vagal ganglia section from a Phox2b-Cre;loxP-ChR2 animal with tracheal CTB injection. (C) Quantification of the total number of EYFP-positive or CTB-positive cells in the vagal ganglia (n = 11 vagal ganglia from n = 6 animals). (C′) Proportion of CTB-positive and EYFP-positive cells and CTB-positive and EYFP-negative cells. (C″) Proportion of CTB-positive cells in the jugular and nodose portions of the vagal ganglia. (D) Representative images showing expression of EYFP and CTB in different bregma levels of the medulla. Numbers on the right upper corners of EYFP images indicate the nearest bregma levels. Lower panels in (D) show magnified views of the white rectangles delineated in the respective upper panels. AP, area postrema; ChR2, channelrhodopsin-2; CTB, cholera toxin B; DMV, dorsal motor nucleus of vagus; EYFP, enhanced yellow fluorescent protein; NA, nucleus ambiguus; nTS, nucleus of the solitary tract; Pa5, paratrigeminal nucleus. Scale bars represent 100 μm in (B) and 500 μm in (D).