Characterization of a cell bridge variant connecting the nodose and superior cervical ganglia in the mouse: prevalence, anatomical features, and practical implications

Abstract

While autonomic ganglia have been extensively studied in rats instead of mice, there is renewed interest in the anatomy of the mouse autonomic nervous system. This study examined the prevalence and anatomical features of a cell bridge linking two autonomic ganglia of the neck, namely, the nodose ganglion (NG) and the superior cervical ganglion (SCG) in a cohort of C57BL/6J mice. We identified a cell bridge between the NG and the cranial pole of the SCG. This cell bridge was tubular-shaped with an average length and width of 700 and 240 μm, respectively. The cell bridge was frequently unilateral and significantly more prevalent in the ganglionic masses from males (38%) than females (21%). On each of its extremities, it contained a mixed of vagal afferents and postganglionic sympathetic neurons. The two populations of neurons abruptly replaced each other in the middle of the cell bridge. We examined the mRNA expression for selected autonomic markers in samples of the NG with or without cell bridge. Our results indicated that the cell bridge was enriched in both markers of postganglionic sympathetic and vagal afferents neurons. Lastly, using FluoroGold microinjection into the NG, we found that the existence of a cell bridge may occasionally lead to the inadvertent contamination of the SCG. In summary, this study describes the anatomy of a cell bridge variant consisting of the fusion of the mouse NG and SCG. The practical implications of our observations are discussed with respect to studies of the mouse vagal afferents, an area of research of increasing popularity.

Graphical Abstract

This study describes the anatomy of a cell bridge (CB) variant consisting of the fusion of the mouse nodose ganglion (NG) and the superior cervical ganglion (SCG). This anastomosis was tubular-shaped and significantly more prevalent in the ganglionic masses from males (38%) than females (21%). It contained a mixed of vagal afferents (blue, TH-positive and Hand2- and isolectin B4-negative; purple, TH- and Hand2-negative and isolectin B4-positive; grey, TH- and Hand2- and isolectin B4-negative) and postganglionic sympathetic neurons (green, TH- and Hand2-positive). The two populations of neurons abruptly replaced each other in the middle of the cell bridge. The practical implications of our observations are discussed with respect to studies of the mouse vagal afferents, an area of research of increasing popularity.

Introduction

The mammalian autonomic nervous system (ANS) consists of parasympathetic and sympathetic nerves and ganglia connecting the central nervous system and the viscera. The conventional view in most reviews, atlases, and textbooks is that the parasympathetic and sympathetic systems are segregated until they reach their target tissues (Espinosa-Medina, Saha, Boismoreau, & Brunet, 2018; Fox, 2005; Furness, 2006; Netter, 2016; Udit & Gautron, 2013). However, detailed anatomical studies revealed a more complicated picture in which parasympathetic and sympathetic fibers are intimately intermingled by means of small nerve branches (Randall & Armour, 1977). This is particularly evident in the case of the mammalian vagus nerve. For example, many studies have found that adventitial fibers of sympathetic origin travel in the vagus nerve of mammalian species including humans (Agostoni, Chinnock, De Daly, & Murray, 1957; Hoffman & Kuntz, 1957; Kawagishi et al., 2008; Kemp, 1973; Prechtl & Powley, 1990; Verlinden, Rijkers, Hoogland, & Herrler, 2016). Anatomical and electrophysiological studies also demonstrated that vagal efferents directly innervate sympathetic neurons located in the solar plexus (Berthoud & Powley, 1993; Bratton et al., 2012). Pelvic ganglia also contain a mix of parasympathetic and sympathetic neurons (Furness, 2006; Keast, 1999). Another example is that of the ciliary nerves supplying the eyeballs, which contain a mix of postganglionic sympathetic and parasympathetic fibers (Johnson, 1988). The aforementioned observations make it abundantly clear that the mammalian parasympathetic and sympathetic nervous systems are not always anatomically separated.

The superior cervical ganglion (SCG), a large sympathetic ganglion of the neck, is an interesting example of the complicated anatomical relationships between the parasympathetic and sympathetic nervous systems. The SCG is well-known to be in close proximity of the nodose ganglion (NG) in most mammals including humans (Appelgren, Hansson, & Schmiterloew, 1963; Chungcharoen, De Burgh Daly, & Schweitzer, 1952; Fioretto, de Abreu, Castro, Guidi, & Ribeiro, 2007; Jamieson, Smith, & Anson, 1952; Phillips, Randall, & Armour, 1986; Randall, Armour, Randall, & Smith, 1971; Sato et al., 2014). Connections between the SCG and other cervical nerve systems are established by numerous small nerve branches and filaments arising from the NG, vagus nerve and cervical plexus (Fioretto et al., 2007; Janes et al., 1986; Mitsuoka, Kikutani, & Sato, 2017; Nourinezhad, Mazaheri, & Biglari, 2015; Rodrigues, 1930). These connections carry preganglionic and postganglionic sympathetic fibers, as well as a small sensory supply originating from the vagus, glossopharyngeal, and upper cervical nerves (Takaki, Nakamuta, Kusakabe, & Yamamoto, 2015; Tseng, Lue, Lee, Wen, & Shieh, 2001; Zaidi & Matthews, 2013). Hence, there is good evidence that the SCG is anatomically connected by small branches to the parasympathetic nervous system. However, the exact arrangement of these connections is highly variable between animal species and individuals. Intriguingly, Altschuler and colleagues briefly noted the existence of a cell bridge connecting the NG and the cranial pole of the SCG in the rat (Altschuler, Bao, Bieger, Hopkins, & Miselis, 1989). The latter study provided little information as to the anatomical features of this cell bridge other than its occurrence in 40% of their samples and that it contained the perikarya of neurons projecting to the oesophagus. This observation indicated that the SCG and NG may sometimes be connected by means of a cell bridge. We are not aware of any other studies that mentioned the existence of such a cell bridge in any other animal species or autonomic ganglia. Therefore, we sought to investigate the prevalence and anatomical features of a putative cell bridge between the SCG and NG in the C57BL/6J mouse, a laboratory animal increasingly used in the fields of autonomic neurosciences and vagal biology. In particular, while the anatomy of autonomic ganglia has been extensively studied in large animals and rats instead of mice, there is a resurgence of research on the mouse autonomic nervous system. This is exemplified by many recent publications on the molecular make-up, anatomy, and functions of the mouse vagal afferents (Bai et al., 2019; Kaelberer et al., 2018; Kupari, Haring, Agirre, Castelo-Branco, & Ernfors, 2019; Williams et al., 2016). Therefore, a better understanding of the anatomical relationships between autonomic ganglia in the mouse is warranted.

1. Materials and Methods

1.1. Mice and sample collection.

A total of 40 C57BL/6J mice from the ages of 4 weeks to 9 months were used for the studies described below. Of the 40 mice, 21 and 19 were male and female, respectively. All of our mice were grouped-housed in an accredited vivarium with an ambient temperature of 21 ± 2°C and a relative air humidity of 40–60%. Sacrifice typically occurred during the light phase between 10am and 2pm. Mice had ad libitum access to tap water and a commercial rodent chow. Experiments were conducted in accordance with our animal protocols #2017–101994 and #2016–101605 which were approved by our Institutional Animal Care and Use Committee. On the day of sacrifice, mice received an overdose of chloral hydrate (500 mg/kg, ip), followed by intracardiac perfusion with phosphate buffered saline (PBS) and 10% formalin (Sigma-Aldrich). Following decapitation, we dissected formalin-fixed mice using a bench microscope and fine forceps. The vagus nerve and SCG were localized and the NG carefully isolated from adjacent tissue, including adipose and conjunctive tissues. We paid special attention to collect the entire nodose-jugular ganglionic mass and to maintain intact appendages leaving the ganglionic mass. Samples were kept at 4ºC in formalin for 24–48 hrs. Digital images of fixed samples were taken using the 4x objective of an Axioskop 2 microscope (Zeiss). The length and width of the identified cell bridge were measured using the measuring tools available with the Axiovision 4.8 software. Thereafter, samples were switched to a solution of 20% sucrose at 4ºC for an additional 24–48 hrs. Finally, 8 samples with a cell bridge were used for immunohistochemistry. The samples were surrounded by a drop of O.C.T. compound (Tissue-Tek, Sakura), frozen on a bed of dry ice, and stored at −20ºC until further processing.

1.2. Immunohistochemistry and antibodies.

A total of 8 cell bridge were used for immunofluorescence. Briefly, fixed ganglionic masses were cryostat-cut to generate 14 μm tissue sections on SuperFrost Plus slides (Fisher Scientific). Thereafter, slides were stored at − 80ºC. For immunohistofluorescence, slides were brought to room temperature and quickly washed in phosphate buffer saline (PBS). Next, slides were incubated in a solution of PBS with Triton (PBT) containing a mixture of anti-tyrosine hydroxylase (TH) (RRID: AB_10013440; 1/1,500 dilution) and Griffonia simplicifolia lectin (GS-IB4) (2.5μg/ml). Please see Table 1 for details on the reagents used in this study. After an overnight incubation at room temperature, slides were washed and incubated in a secondary anti-chicken antibody (RRID: AB_142924; see also Table 1) for 2 hr at a 1/2,000 dilution. Finally, tissue was rinsed in PBS and mounted with VectaShield Hard set containing DAPI (H-1500 Vector Laboratories, Burlingame, CA). A camera lucida attached to a Zeiss epifluorescence microscope was used to produce drawings of the outlines of the immunostained ganglionic masses. Digital images of representative cases were imported to Adobe Photoshop for annotation.

Table 1.

List of reagents used to perform histology, qPCR, or ISH.

Reagent name	Registry ID	Manufacturer	Cat#	Lot#
Antibodies and lectin
Chicken anti-TH antibody	RRID:AB_10013440	Aves Lab	TYH	6917982
GS-IB4 AlexaFluor 594	n/a	Invitrogen	I21413	2076360
AlexaFluor 488 anti-chicken	RRID:AB_142924	Invitrogen	A11039 AB152	584970
Goat anti-TH antibody	RRID:AB_390204	Millipore	705-065-147	2493925
Anti-goat biotinylated secondary	RRID:AB_2340397	Jackson		118762
Streptavidin AlexaFluor 594	n/a	ImmunoResearch Invitrogen	s32356	1816618
qPCR probes
18S rRNA	n/a	ThermoFisher	Hs99999901_s1
DBH	n/a	ThermoFisher	Mm00460472_m1
Gata2	n/a	ThermoFisher	Mm00492301_m1
Gata3	n/a	ThermoFisher	Mm00484683_m1
Glp1R	n/a	ThermoFisher	Mm00445292_m1
Hand2	n/a	ThermoFisher	Mm00439247_m1
Nav1.8/Scn10a	n/a	ThermoFisher	Mm00501467_m1
NPY	n/a	ThermoFisher	Mm00445771_m1
Phox2b	n/a	ThermoFisher	Mm00435872_m1
Prdm12	n/a	ThermoFisher	Mm01324476_m1
Slc17a6/VGlut2	n/a	ThermoFisher	Mm00499876_m1
TH	n/a	ThermoFisher	Mm00447546_m1
VIP	n/a	ThermoFisher	Mm00660234_m1
ISH probes
DapB-C1	Advanced Cell Diagnostics		310043
Hand2-C1	RRID: SCR_012481		499821
Glp1r-C1			418851
Hand2-C1			499821
Gata3-C1			403321
Npy-C1			313321
Dbh-C1			407851

GS-IB4 has been widely used in the literature to label unmyelinated afferents (La, Feng, Kaji, Schwartz, & Gebhart, 2016). The staining patterns here matched very well with its known distribution. For instance, intense staining was observed in a Golgi-like pattern in a majority, but not all, vagal afferents. Staining did not occur in neurons located in the SCG. Last, pre-incubation of GS-IB4 with 1M D-galactose (Sigma) prevented GS-IB4 binding to vagal afferents (Figure 1A).

Figure 1.

Validation of reagents. (A) Nodose ganglion (NG) incubated with GS-IB4 as described in the main manuscript or in the presence of 1M D-Galactose (Sigma). Labeling of vagal afferents was prevented. (B-E) Superior cervical ganglion (SCG) were stained with the anti-TH antibody described in the main manuscript. Tissue was simultaneously incubated with a goat anti-TH (Millipore AB152 lot 2493925). This antibody is raised against denaturated TH from rat. Both antibodies were diluted at 1/2,000 overnight. This was followed by anti-goat biotinylated secondary (Jackson immunoreasearch 705-065-147) and streptavidin AlexaFluor 594 (Invitrogen s32356). SCG neurons were labeled identically by both antisera. In the absence of primary antibodies, tissue was completely devoid of immunoreactivity.

The anti-TH antibody was raised against synthetic peptides corresponding to different regions of the Tyrosine Hydroxylase gene product that were shared between the human (P07101, NCBI) and mouse (P24529, NCBI) sequences. It was previously used in several publications on the mouse nervous system including in the Journal of Comparative Neurology (Merchan-Sala, Nardini, Waclaw, & Campbell, 2017). The TH immunoreactivity pattern in our hands was consistent with its expected cellular and anatomical distribution. In particular, cells resembling neurons were intensely labeled in the entire SCG but sparsely labeled in the NG. To further ascertain specificity, we performed double immunostaining using this antibody in combination with a goat anti-TH (AB_390204; Millipore AB152; lot 2493925). This antibody was raised against denatured TH from rat. Briefly, both primaries were simultaneously incubated overnight at a dilution of 1/2,000. The other TH antibody was detected using an anti-goat biotinylated secondary (AB_2340397; Jackson ImmunoResearch; cat #705–065-147) and streptavidin AlexaFluor 594 (Invitrogen s32356). The two antibodies labeled exactly the same cells (Figure 1B–E). In the absence of antibodies, tissue was completely devoid of immunoreactivity. More importantly, using in situ hybridization (ISH) combined with TH immunofluorescence (see later), we found that TH immunoreactivity colocalized perfectly with Hand2 mRNA, a marker of sympathetic neurons (Kupari et al., 2019).

1.3. Chromogenic ISH.

Ganglia were prepared on SuperFrost slides as described above to perform chromogenic ISH using the RNAScope 2.5 HD Red Assay (ACD; RRID: SCR_012481). Slides were kept at −80ºC until needed. On the day of experiment, slides were baked at 60ºC for 30 min to prevent the tissue from detaching. Thereafter, pretreatment was performed following the manufacturer’s instructions with slight modifications. The retrieval solution was applied for 2–5 min at 90ºC and protease Plus solution for 15–30 min at 40ºC. The probes listed in Table 1 were applied for 2hrs at 40ºC. Amplification steps were performed exactly as directed by the manufacturer. FastRed accumulated to form red dots (magenta after correction) that could be detected using either bright-field or fluorescence microscopy. Of note, we were highly confident of the specificity of the observed ISH signals. This is because the RNAScope amplification steps rely on the binding in tandem of two Z probes, which greatly limit the likelihood of unspecific hybridization. Additionally, the distribution of the FastRed signals matched well the known distribution of the examined genes. Moreover, a probe for a prokaryotic gene was also used as a negative control and confirmed the absence of background staining. As we have done it in the past (Yuan, Caron, Wu, & Gautron, 2018), ISH was followed by immunofluorescence. Briefly, slides were incubated with a chicken TH primary antibody and the appropriate secondary exactly as described above. Finally, coverslips (1–1/2 thickness) with EcoMount (Biocare medical) were carefully placed on the slides.

1.4. Microscopy and illustrations.

Bright-field images were captured using a Zeiss Axioplan light microscope attached to a digital camera. Images of immunolabeled tissues were captured using a Zeiss LSM880 confocal microscope available at UT Southwestern Live Cell Imaging Facility. Digital images (1024×1024 pixels; 8bit) were captured using either the 20x or 63x (oil) objectives. Z-stacks were collected with 3–7 planes with a step of 0.5–1.5 μm and were saved as czi files. Fiji ImageJ (NIH) was used to generate projection of the Z-stacks to form a single tiff image and to convert DAPI to gray. Images of FluoroGold-labeled samples were taken using Leica DM6 equipped with the adequate filter (BP546/12 Beam Splitter FT 580 Emission LP590) and connected to the LAS X software with Thunder Imager. Adobe Photoshop CS5 was used to adjust digital image size, resolution, and contrast. All modifications were uniformly applied across images in the same plate. Finally, images were converted to green/magenta and annotations and scale bars were added as needed.

We estimated the relative proportion of TH-immunolabeled profiles also positive for either GS-IsB4 (double fluorescence), or Hand2 or Glp1r mRNA (ISH combined with immunofluorescence). Data are represented as percentages of counted profiles positive for TH alone or in combination with the aforementioned markers (mean ± SEM, n = 3 for mRNAs or n=6 for IsB4). We were only interested in the proportion of profiles being positive for the above markers rather than the absolute numbers of positive profiles. Counts were performed manually using the 20x objective of an epifluorescent microscope on ganglionic masses with a clearly identified NG, cell bridge, and SCG.

1.5. Quantitative PCR (qPCR).

A total of 12 C57BL/6J male mice from the ages of 4 weeks to 9 months were used for the qPCR studies described below. On the day of sacrifice, mice received an overdose of choral hydrate (500 mg/kp, ip) and were rapidly decapitated. Using a dissection bench scope, ganglionic masses were rapidly isolated. A total of 24 ganglionic masses were collected consisting of SCG and NG with or without cell bridge. Samples were immediately frozen on a bed of dry ice and subsequently stored at −80ºC. Total RNA was isolated using RNAStat60 drawn through a 21G needle until the tissue was completely homogenized. RNA was precipitated in the presence of GlycoBlue to aid in higher RNA yield and visualization of the RNA pellet. cDNA synthesis and qPCR were performed as described (Bookout et al., 2006) with the TaqMan gene expression probes listed in Table 1. Another 3 mice were used to validate our qPCR findings by chromogenic ISH (see above for methods).

1.6. Microinjection studies.

Microinjections were performed on a total of 9 fixed ganglia using a prepulled micropipette (WPI, TIP20TWI) with 20 μm tip and 750 μm capillary diameter. With the help of a stereotaxic arm and a dissecting scope, we gently inserted the tip of the pipette into the NG itself. Over period of 2 minutes, we administered 440 nl of a solution of 1% FluoroGold (in 0.9% saline; FluoroChrome, CO) by air pressure. Ganglia were rinsed, transferred in 30% sucrose, and frozen on dry ice. Cryostat-cut sections of the injected ganglia were later examined by epifluorescence.

1.7. Data Availability Statement

All the unedited and uncropped original images, the data that support the specificity of the antibodies and histology, and the qPCR data are all available from the corresponding author.

2. Results

Gross anatomy, size and prevalence.

We dissected 80 formalin-fixed vagal ganglionic masses from 40 mice. In a majority of mice, the shape of the NG and the arrangement of its main appendages corresponded well to that described in the literature (Altschuler et al., 1989; Mazzone & Undem, 2016; McGovern et al., 2015) (Figure 2A, B). Briefly, the NG was more or less ovoid and fused with the jugular ganglion (JG). In addition to the vagus nerve, several smaller nerves were seen attached to the ganglionic mass (Figure 2A, B). Notably, a cluster of melanocytes was systematically seen at the surface of the JG (Figure 2A, B). They likely correspond to meningeal melanocytes previously described in the literature (Gudjohnsen et al., 2015). The SCG was close to the NG but not directly connected to it, as they were separated by some conjunctive tissue, adipose, and a few occasional thin filaments. In many of the dissected ganglionic masses (see below for prevalence), a large cell bridge was seen between the caudal NG and the cranial pole of the SCG (Figure 2C, D; Table 2). At higher magnification, the cell bridge in question resembled a tubular-shaped cell bridge (Figure 2D). The half of the cell bridge that is attached to the NG appeared translucent with a slightly fibrous core (Figure 2D). Half that is attached to the SCG appeared entirely translucent. No distinct demarcation was noticed between the two halves of the cell bridge. Most notably, the overall size of the cell bridge varied considerably between ganglionic masses. Its average length and width were 700 and 240 μm, respectively (Figure 3). However, the longest cell bridges were slightly over 1 mm long. The shortest were a little less than 250 μm long (Figure 3). Differences in size did not correlate with gender or anatomical side.

Figure 2.

Photomicrographs showing the anatomical relationships between the SCG and the vagal ganglionic mass in the mouse. (A) In agreement with conventional descriptions (Altschuler et al., 1989), the NG and SCG of most mice were in close proximity, but not connected. (B) At higher magnification, the NG resembled a translucent swelling of the vagus nerve with a fibrous core corresponding to passing vagal fibers. (C) Occasionally, we observed a large cell bridge between the SCG and NG. (D) At higher magnification, the NG appeared to be fused with a tubular-shaped structure extending toward the rostral tip of the SCG. The cell bridge was in continuity with the NG and shared its translucent appearance with a fibrous core. The pole of the cell bridge attached to the SCG was less fibrous with an appearance similar to that of the SCG. Of note, the mouse petrosal ganglion contains a subset of sensory neurons that innervate the tongue and carotid body {Retamal, 2014 #30706}. However, due to its small size in the mouse, we are not entirely confident about its exact location relative to jugular and nodose ganglia. We decided to refer to the jugular/petrosal ganglionic complex as a single structure. Abbreviations: C, carotid artery; m, melanocyte; NG, nodose ganglion; PJG, petrosal-jugular ganglion; PR, pharyngeal ramus; SCG, superior cervical ganglion; SLN, superior laryngeal nerve; IX, glossopharyngeal nerve; X, vagus nerve; XI, accessory nerve. The scales in A and B apply to C and D. The rostro-caudal axis is indicated by a symbol in the right bottom corner of A.

Table 2.

Contingency tables regarding the prevalence of the NG-SCG anastomosis in mice. The gender prevalence was defined by comparing the number of samples in males and females with an identified anastomosis relative to the total number of collected samples (80 ganglia). Note that the prevalence of NG-SCG anastomosis was significantly higher in males than females. The anatomical side prevalence was defined by comparing the number of left and right samples with an identified anastomosis relative to the total number of collected samples (80 ganglia). The laterality prevalence was defined by comparing the number of mice with a unilateral and bilateral anastomosis relative to the total number of mice (40 mice). The anastomosis was unilateral in most mice.

	Gender (80 ganglia)			Side (80 ganglia)			Laterality (40 mice)
	Male	Female	Total	Left	Right	Total	Unilateral	Bilateral	Total
YES	17	8	25	15	10	25	21	5	26
NO	25	30	55	25	30	55	0	14	14
Total	42	38	80	40	40	40	21	19	40
x2	df, 3.503, 1			df, 1.455, 1			df, 23.81, 1
	z, 1.872			z, 1.206			z, 4.879
	P=0.0306			P=0.1139			P<0.0001

Figure 3.

Scatter plot depicting the length and width of 25 individual cell bridges. Each point corresponds to one cell bridge. The 8 numbered points refer to the cases represented later in Figure 5.

The overall prevalence of the aforementioned SCG-NG cell bridge was 30% of all of the dissected ganglionic masses (gender, laterality, and sides combined). The cell bridge was slightly more frequently found on the left side (60%), but not in a statistically significant manner (Table 2). In 78% of the mice, the cell bridge was observed in a unilateral manner (Table 2). A bilateral cell bridge was seen only in 5 out of 26 mice with identified at least one cell bridge (p<0.0001). Interestingly, we estimated the cell bridge prevalence to be 38% and 21% of collected ganglionic masses in males and females, respectively (Table 2). The difference was statistically significant (p=0.0306), thus indicating sexual dimorphism.

2.1. Neuronal composition.

To ascertain the identity of the neurons contained in the cell bridge, we performed additional immunostainings as described below. As shown in Figures 4 and 5, we labeled ganglionic masses with GS-IB4 and TH to identify vagal unmyelinated afferents and sympathetic postganglionic neurons, respectively. The NG contained a large number of cells positive for GS-IB4 staining (Figure 4A, B; Table 3). GS-IB4 staining decorated a majority, but not all vagal afferents. As anticipated (Brumovsky, 2016), a small subset of vagal afferents were also TH-positive (Figure 4B; Table 3). TH and GS-IB4 staining never colocalized (Table 3). All neurons in the SCG appeared to be TH-positive (Figure 4C, D; Table 3). GS-IB4 was not observed in neurons of the SCG. Despite variations in shape and size, the cellular composition of the cell bridge was comparable. A typical cell bridge consisted of an elongated cell bridge connecting the NG and SCG (Figure 4E). It contained a dense population of large cells resembling neurons surrounded by non-neuronal cells (Figure 4E, F). On the side connected to the NG, cellular profiles were GS-IB4-positive and resembled vagal afferents found in the NG itself (Figure 4E; Table 3). A typical cell bridge was in perfect continuity with the rest of the NG, without a clear demarcation or gap. On the side of the cell bridge connected to the SCG, the cellularity resembled that of the rest of the SCG with a continuum of TH-positive neurons (Figure 4E; Table 3). We sometimes noticed a constriction at the point of junction between the cell bridge and the SCG (Figures 4 and 5). Hence, the cell bridge itself was enriched with both vagal afferents and sympathetic neurons. Toward the middle of the cell bridge, vagal afferents were abruptly replaced by sympathetic neurons and vice-versa (Figure 4F). Strikingly, vagal afferents located in that transition area were immediately adjacent to TH sympathetic neurons (Figure 4F). In most cases, regardless of gender or anatomical side, the cell bridge consisted of a thin and elongated cell bridge (Figure 5A–F). However, as previously noticed when examining the whole ganglia, the size and shape of the cell bridge varied considerably between animals. For instance, in 3 cases, the cell bridge was very short, resulting in the SCG and NG appearing to be almost fused with one another (Figure 5G, H).

Figure 4.

Digital images representative of the neuronal composition of the NG-SCG cell bridge. Images were acquired using confocal microscopy (Zeiss LSM880). (A, B) NG consisted mainly of GS-IB4-positive neurons (magenta), a few TH-positive neurons (double arrow, green) and GS-IB4-negative neurons (asterisks). DAPI (gray) was used to help delineate our tissue and identify neurons with a large and lightly stained nucleus. The inset in A is shown as image B. (C, D) In the SCG, we found only TH-positive neurons (green), but not GS-IB4-positive cells. A few capillaries were also observed. The inset in C is shown as image D. (E) Images of the cell bridge were taken at 20x and stitched together using the Zen software. In this example, GS-IB4-positive neurons (vagal afferents) were contained in the part of the cell bridge fused with the NG, whereas TH-positive cells were mostly found in the part fused with the SCG. In the middle of the cell bridge, vagal afferents and sympathetic neurons abruptly replaced each other. Inset in E is shown at higher magnification F. (F) High-magnification view of the transition area between vagal afferents (red) and sympathetic neurons (green). The two populations of neurons were not separated by an obvious gap or demarcation, with the exception of a few non-neuronal cells. A few neurons that were TH- and GS-B4-negative were presumptive vagal afferents. Abbreviations: NG, nodose ganglion; psn, postganglionic sympathetic neuron; SCG, superior cervical ganglion; van, vagal afferent neuron. Scale bars in A and B apply to C and D, respectively.

Figure 5.

Camera lucida-assisted drawings of representative ganglionic masses with a NG-SCG cell bridge. Immunolabeled tissue for GS-IB4 (red) and TH (green) was used to survey the general morphological feature of our samples. Due to its small size and zigzagging shape, our histological sections never traveled perfectly across the entire length of the cell bridge. Outlines of representative ganglionic masses demonstrated that the NG-SCG cell bridge varied in size and shape. (A-F) In most cases, the cell bridge was identified as an elongated bridge fused with the NG and SCG on its extremities. Sometimes, the cell bridge was stubby and short. (G, H) In two cases, the NG and SCG appeared fused without a clear cell bridge per se. Abbreviations: cb, cell bridge; NG, nodose ganglion; SCG, superior cervical ganglion; X, vagus nerve. The scale bar in B applies through I. The rostro-caudal axis is indicated by a symbol in the right bottom corner of A.

Table 3.

Mean percentages of neuronal profiles positive for select markers of primary afferents (GS-IsB4 or Glp1r mRNA) or sympathetic neurons (Hand2 mRNA) in combination with TH-IR. For GS-IsB4, data were obtained by double immunofluorescence and are expressed as mean ±SEM (n = 6 different ganglia). For Hand2 and Glp1r studies, data were obtained by combined in situ hybridization and immunofluorescence and are expressed as mean ±SEM (n = 3 different ganglia). The total number of counted profiles is also indicated in the bottom row (per anatomical structure) and last column (per markers combination). Glp1r, glucagon like receptor 1; GS-IsB4, Griffonia simplicifolia isolectin B4; Hand2, Heart- and neural crest derivatives-expressed protein 2; TH-IR, tyrosine hydroxylase immunoreactivity; NG, nodose ganglion; SCG, superior cervical ganglion.

	SCG	Anastomosis	NG	Total profiles
GS-IsB4 and TH-IR	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	458
TH-IR	99.8 ± 0.2	58.7 ± 12.6	13.1 ± 3.9	1126
GS-IsB4	0.2 ± 0.2	41.3 ± 12.6	86.9 ± 3.9	0
Total profiles	809	459	316	1584
	SCG	Anastomosis	NG	Total profiles
Hand2 mRNA and TH-IR	99.8 ± 0.2	77.3 ± 0.9	0.0 ± 0.0	982
TH-IR	0.0 ± 0.0	20.4 ± 2.4	100.0 ± 0.0	130
Hand2 mRNA	0.2 ± 0.2	0.5 ± 0.5	0.0 ± 0.0	3
Total profiles	843	202	70	1115
	SCG	Anastomosis	NG	Total profiles
Glp1r mRNA and TH-IR	7.3 ± 0.7	3.3 ± 0.9	2.8 ± 0.3	138
TH-IR	92.7 ± 0.7	83.6 ± 8.2	42.0 ± 5.4	1921
Glp1r mRNA	0.0 ± 0.0	13.1 ± 7.5	55.3 ± 5.2	227
Total profiles	1483	553	250	2286

2.2. Gene expression studies.

We used qPCR to assess the expression of markers of autonomic neurons in the SCG and NG with or without cell bridge (Figure 6). As expected, the SCG was highly enriched in “traditional” sympathetic markers including Gata3, Hand2, Dbh, Npy, Th, Gata2 (Figure 6A, B). These genes were also expressed in samples of the NG with cell bridge, reflecting its mixed sympathetic/vagal nature. Several sympathetic markers including Th and Gata2 were also detectable, although at lower levels, in the NG without cell bridge (Figure 6B). Thus, the latter genes cannot be considered strictly specific to the sympathetic nervous system. In contrast, Gata3, Hand2, Dbh, and Npy were undetectable in the NG alone (Figure 6A). Therefore, the latter genes can be considered selective sympathetic markers. Markers of vagal afferents such as Glp1r, Vip, Phox2b, Prdm12, Nav1.8, and Vglut2 were all enriched in the NG with or without cell bridge (Figure 6B, C). Glp1r, Vip, and Phox2b were also found at low levels in the SCG (Figure 6B), thus indicating their slight enrichment in the sympathetic nervous system.

Figure 6.

QPCR analysis of select autonomic genes in the mouse superior cervical ganglion (SCG) compared to the nodose ganglion (NG) with or without cell bridge (a) compared to the hypothalamus. (A) Genes considered specific for sympathetic postganglionic neurons were enriched in both the SCG and NG with cell bridge, but not significantly expressed in the NG. (B) Genes detectable in both the SCG and NG with or without cell bridge. (C) Genes considered specific for vagal afferents were significantly expressed in the NG with or without cell bridge, but not detectable in the SCG. Averaged Ct values were 31 (Gata3), 34 (Dbh), 33 (Hand2), 35 (Npy), 34 (Th), 36 (Gata2), 32 (Glp1r), 34 (Vip), 31 (Phox2b), 29 (Prdm12), 30 (Nav1.8), 30 (Vglut2).

Chromogenic ISH for a few select genes was performed to confirm our qPCR data. In particular, we used ISH to ascertain the distribution of above sympathetic-enriched genes. As expected, ISH signals for Gata3, Hand2, Dbh were found in most neurons across the entire SCG (Figure 7A,C, G, I). By comparison, Npy was only expressed in a subset of sympathetic neurons (Figure 7E), in agreement with the literature {Landry, 2000 #30703}. Vagal afferents in the NG were always negative for Hand2 and Npy (Figure 7B, F). In contrast, Gata3 and Dbh were occasionally detectable in a small subset of NG neurons (Figure 7D, H). Likewise, in the cell bridge linking SCG and NG, Dbh expression marked sympathetic neurons and a small number of vagal afferents (Figure 7I). Thus, among the examined genes, Hand2 seems to be most selectively enriched in sympathetic neurons. In further agreement with this view, robust chromogenic signals for Hand2 (red) were seen across the entire SCG and infiltrating the part of the cell bridge fused with the SCG (Figure 8A). In the NG and the part of the cell bridge fused to it, Hand2 signals were undetectable, including in TH-positive vagal afferents (Figure 8B; Table 3). In the SCG, Hand2 mRNA colocalized perfectly with TH immunoreactivity, thus confirming its expression in sympathetic neurons (Figure 8C; Table 3). In parallel, we assessed Glp1r. In agreement with our qPCR, moderate signals were seen in a subset of vagal afferents across the NG and the cell bridge until it reached the SCG (Figure 8D). Glp1r and TH immunoreactivity did not colocalize in vagal afferents (Figure 8E; Table 3). In agreement with our qPCR, Glp1r mRNA was also seen in TH-positive neurons of the SCG itself, although at very low levels (Figure 8F; Table 3). Dapb, a prokaryotic gene, was undetectable (Figure 8G–I). Together, our findings indicate that the NG does not normally contain positive sympathetic neurons. However, NG samples can be easily contaminated by positive neurons originating from the SCG if the sympathetic pole of the previously described cell bridge is inadvertently included during dissection.

Figure 7.

Chromogenic in situ hybridization (ISH) for genes enriched in sympathetic neurons (FastRed dots) in the C57Bl/6J mouse. Hand2 was expressed in the entire superior cervical ganglion (SCG) (A), but not in neurons of the nodose ganglion (NG) (B). Gata3 was detected in most sympathetic neurons, although at moderate levels (C). In the NG, only a few vagal afferents were positive for Gata3 (black arrows) (D). In comparison, Npy signals were only seen in a subset of SCG neurons (black arrows) (E). Vagal afferents were devoid of ISH signals for Npy (white asterisk) (F). Robust ISH signals for Dbh were observed in most sympathetic neurons across the SCG (G) and within the cell bridge (I). In the case of Dbh, a few vagal afferents were positive for its transcript in the NG with or without cell bridge (H, I). Tissue was counterstained with hematoxylin. Scale bar in (A) applies though (H). Abbreviations: X, vagus nerve.

Figure 8.

Ganglionic masses were labeled using chromogenic ISH (magenta) following by TH immunofluorescence (green). Digital images were captured using confocal microscopy (Zeiss LSM880). (A-C) Robust Hand2 signals were observed across the SCG, but not the NG and the part of the cell bridge connected to it. TH-positive neurons located in the NG and cell bridge were not positive for Hand2 mRNA (double arrowheads). In contrast, TH-positive neurons in the SCG expressed high levels of Hand2 mRNA (empty arrows). (D-F) Moderate signals for Glp1r were seen in the NG and cell bridge (asterisks). Typically, Glp1r mRNA rarely colocalized with TH immunoreactivity in the NG and cell bridge (double arrowheads). However, low levels of Glp1r mRNA were detectable in TH-positive neurons of the SCG (empty arrows). (G-I) As anticipated, the mRNA for DapB was not detected in the examined ganglia. TH-positive neurons in the NG, cell bridge, and SCG were devoid of hybridization signals. Abbreviations: NG, nodose ganglion; SCG, superior cervical ganglion; X, vagus nerve.

2.3. Microinjection studies.

We wondered whether a solution injected into the NG could potentially “contaminate” sympathetic neurons by diffusion through the cell bridge. We investigated this potential issue by assessing the diffusion of 1% FluoroGold directly injected into the NG (Figure 9A, B). Out of 9 cases, one injection was missed with little FluoroGold observed in the ganglionic mass, but some diffuse fluorescence in the epineurium (Figure 9C). In 3 cases, FluoGold was seen filling the NG, entirely or partially, without significantly leaking toward the cell bridge and SCG (Figure 9D). This result implies that vagal afferents located within the cell bridge may often be missed during microinjections studies. In 4 other cases, however, FluoroGold was clearly seen filling the cell bridge with or without contaminating the SCG (Figure 9E–G). In one case, FluoroGold was more clearly seen in the SCG itself, mostly confined to the rostral pole connected to the cell bridge (Figure 9E). This demonstrated that the microinjection of a small volume of solution into the NG can occasionally diffuse toward the cell bridge and SCG, thereby resulting in the contamination of sympathetic neurons. As a remark, signs of tissue damage were sometimes noticeable close to the site of injection in the NG (Figure 9D).

Figure 9.

A posteriori validation of tracer microinjections into the fixed NG with cell bridge. (A,B) Representative example of a microinjected ganglionic mass with a solution of 1% FluoroGold. The site of injection was clearly visualized using epifluorescence of the whole ganglionic mass. (C-G) Injected ganglia were processed for histology and FluoroGold diffusion was assessed by epifluorescence. Faint yellow Fluorescent was seen in the epineurial layer of the ganglia, as well as intense fluorescence in the parenchyma of injected ganglia. However, the extent to which the tracer diffused across each ganglionic mass varied considerably between samples. In C, the injection was considered to be “missed” due to limited fluorescence inside the ganglia. In D, FluoroGold only partially filled the NG and did not diffuse far from the injection site. Of note, tissue damage was seen at the site of injection itself (small white arrow). In E, F, and G, we could see FluoroGold within the cell bridge and, to a varying extent, into the SCG parenchyma. Abbreviations: cb, cell bridge; NG, nodose ganglion; SCG, superior cervical ganglion; X, vagus nerve.

3. Discussion

Centuries of dissection of human cadavers have shown that anatomical variants of the ANS are a common finding. For instance, the human vagus nerve has been described to display a high degree of inter-individual variability (Boyd, 1949; Tubbs et al., 2007). Thus, the anatomical organization of the ANS that is depicted in textbooks is oversimplified and only represents average anatomical models. To complicate the matter, the categorization of the ANS divisions is still a matter of dispute among experts (Ernsberger & Rohrer, 2018; Espinosa-Medina et al., 2016; Janig, Keast, McLachlan, Neuhuber, & Southard-Smith, 2017). Moreover, critical components of the ANS, such as autonomic neurons that establish inter-organs connections, are often ignored in textbooks (Furness, 2006). Last, the scientific literature often underappreciates that the subdivisions of the ANS are not fully separated. Here, we provided another example of the complexity of the ANS. Our data show that the NG and SCG, two autonomic ganglia that belong to distinct ANS divisions, are not always completely separated ganglia in the mouse. A normal anatomical variant exists consisting of a cell bridge linking the SCG and NG. This cell bridge consists of a cell bridge made of both sympathetic neurons and vagal afferents.

A survey of the literature indicates that the dog NG has been described as more or less fused with the SCG (Chungcharoen et al., 1952). Additionally, Randall and Armour (Randall & Armour, 1977) described a thin filament connecting the NG and the SCG in one human cadaver. Lieberman also mentioned the existence of small clusters of ectopic autonomic neurons in the rabbit NG in its superior pole (Lieberman, 1976). Solely based on morphological criteria, Lieberman deduced that these neurons were likely sympathetic neurons resembling those found in the SCG. Nonetheless, the Lieberman study does not describe a connection between the SCG and NG. Likewise, Kupari and colleagues in a recent single cell-sequencing analysis of the NG reported finding a small cluster of neurons that displayed “a clear profile of sympathetic neurons” and likely originated from the superior cervical ganglion (Kupari et al., 2019). They did not explain how sympathetic neurons from the SCG could possibly be found in a preparation of vagal afferents. Therefore, to the best of our knowledge, Altschuler and colleagues are the only investigators to have explicitly described the existence of a cellular bridge between the NG and SCG (Altschuler et al., 1989). However, their study was not aimed at assessing the anatomical features of such anastomoses. Here, we described without ambiguity the existence of a cell bridge made of a mix of sympathetic postganglionic neurons and vagal afferents. Remarkably, at the junction area between the two poles of the cell bridge, vagal afferents are lying immediately next to postganglionic sympathetic neurons. Considering that the main divisions of lower vertebrates ANS are even less separated than in mammals (Burnstock, 1969; Nilsson, 2011), the cell bridge described in this study may represent a vestigial feature. In particular, the NG-SCG cell bridge may be formed during embryonic life due to the accidental fusion of the two ganglia. Due to its small size and variable shape, we may have occasionally missed a cell bridge. That said, the reported 38% prevalence in males is almost identical that reported by Altschuler and colleagues in the rat (Altschuler et al., 1989).

Very little is known about the vagal afferents contained in the cell bridge described in this study. The distribution of vagal afferents in the NG doesn’t follow a precise topography (Altschuler et al., 1989). For example, within the NG, one neuron supplying the stomach may lie immediately adjacent to a neuron supplying the lung. To complicate the matter, at least 18 different subtypes of vagal afferents can be differentiated based on their transcriptional profile in the mouse NG (Kupari et al., 2019). Because of this inherent heterogeneity and lack of topography, the cell bridge described in this study is unlikely to be enriched in any particular subsets of vagal afferents. In other words, the vagal composition of the cell bridge is probably comparable to that of the NG per se. In support of this view, we found the cell bridge to contain a mixed population of Isolectin B4-positive and negative neurons, as well as a subset of Glp1r-positive neurons.

The functional implications (if any) of the cell bridge described in this study are unclear. Minor anatomical variants of the peripheral nervous system are common and typically without ill-effects (Willan & Humpherson, 1999). Nonetheless, our observations raise intriguing questions as to the anatomical variability of the vagus nerve. For instance, the cell bridge described here was unilateral in most cases. It has long been appreciated that the vagus nerve is not a symmetrical system (Berthoud & Neuhuber, 2000). The male prevalence of the cell bridge is also an interesting observation. Sexual dimorphism of vagal afferents and related-functions has been reported in the past (Khasar, Green, Gear, Isenberg, & Levine, 2003; Li et al., 2008). That said, how vagal functions differ across sexes remains an understudied area of research. Furthermore, vagus nerve stimulation is an area of research of increasing popularity (Koopman et al., 2016; Shikora et al., 2015; Somann et al., 2018; Spatola et al., 2013). One study reported high inter-individual variability of vagus nerve stimulation on cardiovascular function (Frei & Osorio, 2001). Assuming that the cell bridge described in rats and mice also exists in humans, one wonders whether the outcome of vagus nerve stimulation may be altered by its anatomical variability. Further research is therefore warranted to establish the existence of SCG-NG variants in other animal species including humans.

Our findings have immediately obvious practical implications in experimental studies related to vagal afferents. For instance, in gene expression and cell culture studies, the inclusion (or lack thereof) of the cell bridge during dissection of the NG may be a source of experimental variability and/or contamination by sympathetic neurons. In support of this view, Kupari et al. found a small subset of nonglutamatergic Hand2-positive neurons among vagal afferents that were deduced to be sympathetic neurons (Kupari et al., 2019). The authors suggested that these sympathetic neurons originated from a contamination by the SCG. Our data agree with their proposition and provide a practical explanation for it. Without knowledge of the cell bridge described in our study, it is easy to inadvertently “contaminate” samples supposedly made solely of vagal afferents with sympathetic neurons contained in the cell bridge. Our qPCR and ISH data confirmed that NG samples with a cell bridge were enriched in sympathetic markers including Hand2, Dbh and Npy. This finding was not observed in NG without cell bridge. Whenever experimental design demands, it may therefore be useful to ascertain “contamination” by sympathetic neurons. This is not a straightforward task because many sympathetic markers, such as TH, are also produced by small subsets of non-sympathetic neurons including vagal afferents. However, our study confirmed the observation of Kupari et al. (Kupari et al., 2019) that Hand2 is selectively enriched in postganglionic neurons and thus useful in ascertaining the presence of sympathetic neurons among vagal afferents.

A better knowledge of the molecular make-up of autonomic neurons may help restrict the expression of molecular reagents (for example, Cre) and fluorescent reporters using genetically-guided strategies. In particular, the microinjection of viral vectors directly into NG is an increasingly popular tool which is used as a mean to alter gene expression in vagal afferents in a selective manner (Bai et al., 2019; Chang, Strochlic, Williams, Umans, & Liberles, 2015; Diepenbroek et al., 2017; Han et al., 2018; Krieger et al., 2016; Nonomura et al., 2017). In the mouse, the volume of injected solution typically ranges from 140 nl (Chang et al., 2015; Nonomura et al., 2017) to 200 nl (Bai et al., 2019), up to 500 nl (Diepenbroek et al., 2017; Han et al., 2018). However, the existence of a cell bridge linking the NG and SCG led us to wonder if a solution injected into the NG could diffuse to the SCG, thereby leading to the unwanted “contamination” of sympathetic neurons. This is important considering that SCG neurons influence a wide range of autonomic and metabolic functions including food intake (Mul Fedele, Galiana, Golombek, Munoz, & Plano, 2017) and cardiovascular functions (Witt et al., 2017), among other examples. Moreover, because the cell bridge is only present in a subset of animals, and more frequently in males, the unwarranted contamination of SCG neurons may introduce experimental variability and false cases of gender differences. Here, we found that a moderate volume of FluoroGold (<440nl) injected directly into the NG can occasionally reach the cell bridge and, to a lesser extent, the SCG. Conversely, subsets of vagal afferents contained in the cell bridge may be missed. This is because we also observed that FluoroGold often remained confined to the NG without traveling toward the cell bridge. As a general precaution, it is advisable to systematically verify the presence of any NG-SCG connections in studies related to the molecular biology and functions of the mouse vagal afferents. More importantly, it is advisable to process the ganglia for histology to verify a posteriori the size of the injection site, as well as signs of tissue damage. Taken together, our results warrant the consideration of anatomical variability of autonomic ganglia when designing and interpreting experimental studies on vagal afferents.