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American Journal of Physiology - Regulatory, Integrative and Comparative Physiology logoLink to American Journal of Physiology - Regulatory, Integrative and Comparative Physiology
. 2021 Jul 7;321(3):R328–R337. doi: 10.1152/ajpregu.00079.2021

Sympathetic innervation of the mouse kidney and liver arising from prevertebral ganglia

Hayden Torres 1, Clara Huesing 1, David H Burk 1, Adrien JR Molinas 2, Winfried L Neuhuber 3, Hans-Rudolf Berthoud 1, Heike Münzberg 1, Andrei V Derbenev 2,4,✉, Andrea Zsombok 2,4,✉
PMCID: PMC8530761  PMID: 34231420

Abstract

The sympathetic nervous system (SNS) plays a crucial role in the regulation of renal and hepatic functions. Although sympathetic nerves to the kidney and liver have been identified in many species, specific details are lacking in the mouse. In the absence of detailed information of sympathetic prevertebral innervation of specific organs, selective manipulation of a specific function will remain challenging. Despite providing major postganglionic inputs to abdominal organs, limited data are available about the mouse celiac-superior mesenteric complex. We used tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DbH) reporter mice to visualize abdominal prevertebral ganglia. We found that both the TH and DbH reporter mice are useful models for identification of ganglia and nerve bundles. We further tested if the celiac-superior mesenteric complex provides differential inputs to the mouse kidney and liver. The retrograde viral tracer, pseudorabies virus (PRV)-152 was injected into the cortex of the left kidney or the main lobe of the liver to identify kidney-projecting and liver-projecting neurons in the celiac-superior mesenteric complex. iDISCO immunostaining and tissue clearing were used to visualize unprecedented anatomical detail of kidney-related and liver-related postganglionic neurons in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia compared with TH-positive neurons. Kidney-projecting neurons were restricted to the suprarenal and aorticorenal ganglia, whereas only sparse labeling was observed in the celiac-superior mesenteric complex. In contrast, liver-projecting postganglionic neurons were observed in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia, suggesting spatial separation between the sympathetic innervation of the mouse kidney and liver.

Keywords: iDISCO, kidney, liver, retrograde tracing, sympathetic nervous system

INTRODUCTION

Differential regulation of tissue-specific output pathways controls specific physiological functions (1–5); therefore, the use of medical devices that modulate nerve activity to a particular organ is a potentially powerful way to treat disorders. To achieve the desired results with neuromodulation and understand the underlying mechanisms, an accurate mapping and understanding of spatial separation of neurons based on their innervation of organs is necessary. Recently, mouse models became the most widely used animal models due to the availability of genetic manipulation; however, we lack detailed information about the peripheral nervous system and innervation of internal organs in the mouse.

A large number of studies identified the importance of the sympathetic nervous system (SNS) in the proper functioning of organs and in the development of diseases (3, 6–9). Activation of renal sympathetic nerves leads to renin secretion, decrease of sodium excretion, and increase of vascular tone (8), as well as plays a role in inflammatory responses (10), whereas overactivity contributes to the development and persistence of hypertension (7). It has been described that the sympathetic innervation of the kidney arises from the celiac and superior mesenteric ganglia, the aorticorenal ganglia, and partially from the paravertebral chain at the level of spinal segment T11–T13 depending on the species (11–15). Despite the fact that mouse models are widely used to study kidney function and hypertension (16–19), limited information is available about the sympathetic innervation of the kidneys.

Similarly, the importance of the SNS in the regulation of hepatic carbohydrate and lipid metabolism and inflammation has been recognized (20–25). Stimulation of the hepatic sympathetic nerves increases glucose production and glycogenolysis (26–28), and thus systemic glucose levels. Increased sympathetic activity is associated with elevated glucose production, dyslipidemia, and type 2 diabetes mellitus; therefore, manipulation of hepatic sympathetic nerves is potentially beneficial for the treatment of metabolic syndrome (6, 9, 25). Recently, the central nuclei involved in the regulation of hepatic functions were identified (21, 29–34). Neurons in supraspinal areas including the hypothalamus and brainstem transfer signals to preganglionic neurons located in the intermediolateral nucleus (IML) and in the intermediate zone of the spinal cord (T7–T12). In turn, these neurons project to sympathetic postganglionic neurons primarily located in the celiac and superior mesenteric ganglia (21, 31).

Although the consensus from studies in various species is that the celiac, superior mesenteric, and paravertebral ganglia provide the main innervation to the kidney and liver, a detailed neuroanatomical mapping of kidney and liver innervation of the mouse is not available. Consequently, without understanding the details of the sympathetic innervation of the kidney and liver, the development of neuromodulatory approaches to target these tissues to improve cardiometabolic health will be challenging. Here, we provide an overview of the sympathetic innervation of the mouse kidney and liver arising from the prevertebral ganglia. Our study demonstrates the usefulness of different reporter mice for visualization of peripheral sympathetic innervation of the kidney and liver, and it establishes the use of tissue clearing for large abdominal samples combined with immunofluorescence staining to reveal the location of postganglionic sympathetic neurons in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia after pseudorabies inoculation of the mouse kidney and liver.

MATERIALS AND METHODS

Animals

Male and female, young adult (7–10-wk-old) C57BL/6J mice (Stock No. 000664, Jackson Laboratories) were used for viral inoculations. Transgenic dopamine β-hydroxylase (tgDbH)-cre mice [MMRRC No. 032081-UCD, Tg(DbH-cre)KH212Gsat/Mmucd, Mutant Mouse Resources & Research Centers] were crossed with Rosa-tomatofl/fl mice (Stock No. 007914, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, Jackson Laboratories) to generate DbH:tomato reporter mice. Tyrosine hydroxylase (TH-IRES)-cre mice (EMMA No. 00254; B6.129X1-Thtm1(cre)Te/Kieg, European Mouse Mutant Archive) were crossed with Rosa-tomatofl/fl mice to generate TH:tomato mice, respectively. The reporter mice were used to demonstrate differences in visibility of postganglionic neurons without processing of tissues. Mice were grouped housed at a 12-h light-dark cycle with ad libitum access to food and water. Experiments were performed according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of Tulane University and Pennington Biomedical Research Center (Louisiana State University Systems).

Inoculation of the Mouse Kidney and Liver with Pseudorabies Virus

The retrogradely transported pseudorabies virus [PRV-152, reports enhanced green fluorescence protein (EGFP), supplied by National Center for Research Resources (NCRR), Center for Neuroanatomy with Neurotropic Viruses (CNNV) Virus Center, Pittsburgh, PA] was used to identify neurons innervating the left kidney (35–38) and the median lobe of the liver. Under isoflurane anesthesia, a small dorsolateral incision was made to expose the left kidney for injection under direct vision. The injections of PRV-152 (1–2 × 2 µL, 1 × 109 pfu/mL) were made into the left renal parenchyma with a pulled glass pipette (∼50 µm diameter). The injection site was immediately sealed with “liquid skin.” Kidney inoculation was performed in 16 mice, and PRV labeling was confirmed in 13 mice with a fluorescent stereomicroscope (∼80% success rate). Six successfully stained and cleared samples (5 males and 1 female) were imaged with light-sheet microscopy. To ensure that labeling was not due to overall leakage, control mice received surface applications of the same total volume of PRV-152 (2 × 2 µL, 1 × 109 pfu/mL) (n = 2 mice). Animals were maintained in a biosafety level 2 laboratory for up to 72 h after inoculation.

In case of the liver, PRV-152 was injected into the parenchyma of the main (median) lobe of the liver. Seven mice were inoculated, labeling was observed in four (∼60% success rate), and three successfully stained and cleared samples were imaged with light-sheet microscopy (2 males and 1 female). A drop of adhesive “liquid skin” was used to seal each injection sites to prevent backflow. The animals were kept in a biosafety level 2 laboratory for up to 72 h after inoculation.

Fluorescence-Guided Dissection of Sympathetic Ganglia and Postganglionic Neurons

Mice were deeply anesthetized with an overdose of isoflurane followed by exsanguination. Subdiaphragmatic organs including the liver and gastrointestinal tract were placed aside using cotton swabs or removed to identify ganglia and postganglionic neurons in reporter mice and in PRV-inoculated mice with a fluorescent stereomicroscope (Nikon SMZ25, Melville, NY). The whole body was postfixed in 4% paraformaldehyde overnight and further processed for iDISCO staining.

Immunostaining and Tissue Clearing

Immunostaining was performed following the iDISCO method with modifications from Renier et al. (39) and https://idisco.info/idisco-protocol/update-history/, similar to previously described (40, 41). In addition, our adapted protocol is publicly available at protocols.io (https://doi.org/10.17504/protocols.io.bujanuie). The whole body sample was trimmed by carefully removing the entire gastrointestinal (GI) tract, taking care to preserve the celiac-superior mesenteric complex. The limbs, skin, and excessive muscle tissue were removed, and the spinal cord was cut anterior between ∼T7/T8 and posterior right above the level of the thighs. This preserved all abdominal prevertebral ganglia and kidneys in their natural location. The tissue block was transferred to phosphate buffered saline (PBS), then dehydrated in methanol (MeOH; 20%, 40%, 60%, 80%, and 100%, 1 h each) and incubated in 3:1 mix of dichloromethane (DCM) and MeOH (overnight, room temperature). Following two additional washes in methanol (100%, 2 × 1 h), tissues were treated with 5% H2O2 in methanol overnight at 4°C, followed by washes in 100% MeOH for 1 h and rehydration (MeOH/H2O 80%, 60%, 40%, 20% and PBS, 1 h each). Then, samples were incubated in PBS, 0.2% Tween-20 (PTx.2) solution for 2 × 1 h and permeabilized in PTx.2, 11.5 g glycine, and 20% DMSO at 37°C for 2 days. Tissues were blocked in PTx.2, 6% donkey serum (Jackson ImmunoResearch), and 10% DMSO for 2 days at 37°C. Tissues were incubated with primary antibodies [chicken anti-GFP (1:400), Abcam; rabbit anti-tyrosine hydroxylase (1:500), Millipore] in blocking solution for 2 wk. Following staining with primary antibodies, the tissue samples were washed in PBS, 0.2% Tween-20, 1 mg/ml heparin (PTwH) for 4 × 1 h and incubated in PTwH overnight. Samples were incubated with secondary antibodies (Alexa Fluor 555 donkey anti-rabbit and Alexa Fluor 647 donkey anti-chicken, 1:300 for 1.5 wk), then washed four to five times or until next day with PTwH. Following staining, samples were dehydrated in MeOH/H2O series, incubated with 3:1 mix of DCM (Sigma Aldrich) and MeOH at room temperature, then for 2 × 15 min in 100% DCM with agitating on a rocker to rinse out any remaining MeOH. Tissues were stored in dibenzyl ether (Sigma Aldrich) until imaging.

Microscopy and Image Processing

Ganglia in reporter mice and the PRV-152 labeling in abdominal prevertebral ganglia was confirmed with a fluorescent stereomicroscope (Nikon SMZ25, Melville, NY). LaVision light-sheet microscopy (LaVision BioTec, Germany) was used for imaging of processed tissues (https://www.protocols.io/view/light-sheet-microscopy-wz3ff8n). Specimen imaging was performed in organic solvent, which is a capability of the light-sheet microscopy. Overview three-dimensional (3-D) image stacks with a ventral view were generated to reveal PRV-152 labeling in the prevertebral ganglia. Images of tyrosine hydroxylase and PRV-152 were collected for all samples.

RESULTS

Sympathetic Prevertebral Ganglia and Postganglionic Neurons in Reporter Mice

Sympathetic postganglionic neurons express TH and DbH; therefore, TH:tomato and DbH:tomato reporter mice may be useful models for in situ identification of ganglia and postganglionic neurons. After euthanasia, fluorescence-guided stereomicroscopic dissection was used to identify sympathetic postganglionic neurons in the celiac-superior mesenteric complex and nerve bundles without tissue processing. After replacement or removal of the liver and GI tract the postganglionic neurons in the celiac-superior mesenteric complex were visible in the reporter mouse lines. Ganglia, perikarya, and nerve fibers were easily detected in TH:tomato and DbH:tomato reporter mice (Fig. 1). Although difference exists between the reporter mice, our observations suggest that both the TH:tomato and DbH:tomato reporter mice allow the identification of ganglia and fine nerves with a fluorescent dissecting microscope which are not easily seen in bright-field dissection. On the other hand, we have to note that during development TH is expressed in cholinergic parasympathetic neurons (42), which may result in labeling of parasympathetic neurons in TH reporter mice; therefore, TH immunostaining is likely a more reliable marker of the SNS than TH reporter mouse lines with TH-cre-induced reporter expression throughout development (41).

Figure 1.

Figure 1.

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Overview of sympathetic postganglionic neurons in reporter mice under the fluorescent dissecting microscope. A: bright-field overview image showing the location of the celiac-superior mesenteric complex (dotted outline) above the inferior vena cava (IVC) and the renal vein in a TH:tomato reporter mouse. A1: fluorescent view in the same TH:tomato reporter mouse visualizes the celiac-superior mesenteric complex between the spleen and left kidney. The inset shows in greater detail the nerve fibers. Note non-neuronal labeling in the adrenal gland. B: fluorescent view of the entire abdominal area illustrates the location of the celiac-superior mesenteric complex (upper boxed area), the inferior mesenteric ganglion (ggl) (middle boxed area) along the IVC, and the lumbar ggl (bottom boxed area) located near to the rectum and uterus. C and C1: higher-magnification bright-field (C) and fluorescent (C1) views in a DbH:tomato mouse demonstrate that the reporter gene expression greatly improves the visibility of the celiac-superior mesenteric complex. D and D1: bright-field (D) and fluorescent (D1) views of the liver hilum with the portal vein and accompanying portal arteries in a TH:tomato mouse. Sympathetic nerves travel along the portal arteries toward the liver (D1). DbH, dopamine β-hydroxylase; TH, tyrosine hydroxylase.

Mapping of Kidney-Innervating Postganglionic Neurons in Sympathetic Prevertebral Ganglia

Viral tracing with pseudorabies viruses is a common approach to label organ-specific neurons in the peripheral and central nervous system (29, 33, 34, 43–45). PRV-152 was injected into the left kidney and the survival time was kept to ∼72 h to label postganglionic neurons in prevertebral ganglia. It should be noted, that preganglionic neurons may have been labeled at this time point as shown previously (43), but were not investigated in this study. Successful PRV-152 labeling of the left kidney was confirmed with a fluorescent dissecting stereomicroscope in wild-type mice (Fig. 2A). We also verified that PRV labeling is easily detectable in DbH:tomato mice (Fig. 2, C1 and C2). Tissues only with verified labeling in prevertebral ganglia were processed further. In addition, we verified that the labeling was not due to leakage of the virus. Surface application of PRV-152 did not result in labeling in the suprarenal and aorticorenal ganglia or the celiac-superior mesenteric complex (data not shown).

Figure 2.

Figure 2.

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Distinct anatomical location of kidney- and liver-innervating sympathetic postganglionic neurons in the suprarenal and aorticorenal ganglia and celiac-superior mesenteric complex. A: postganglionic neurons were identified under the fluorescent dissecting microscope in the suprarenal and aorticorenal ganglia after inoculation of the left kidney with pseudorabies (PRV). B: PRV inoculation of the liver resulted in labeling of postganglionic neurons mainly in the celiac-superior mesenteric complex. B1: enlarged image of the boxed area shown in B. C, C1, and C2: higher magnification of bright-field and fluorescent views of the location of the celiac-superior mesenteric complex and aorticorenal ganglia after inoculation of the left kidney in a DbH:tomato reporter mouse. C: merged bright-field and fluorescent images illustrate the location of the celiac-superior mesenteric complex (red). C1: merged bright-field and fluorescent views demonstrate the location of postganglionic neurons following PRV inoculation of the left kidney. C2: merged fluorescent view of the location of kidney-innervating neurons compared with the celiac-superior mesenteric complex in a DbH:tomato mouse. DbH, dopamine β-hydroxylase.

Following inoculation of the left kidney, PRV-labeled neurons in the suprarenal ganglia and aorticorenal ganglia on the ipsilateral injection side were seen in whole animal preparation with a fluorescent dissecting microscope following removal of the viscera (Fig. 2). After iDISCO clearance and immunostaining for EGFP and TH, the location of kidney-innervating neurons became visible with the use of light-sheet microscopy (Fig. 3).

Figure 3.

Figure 3.

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Ventral views of kidney-innervating sympathetic postganglionic neurons in the mouse. A: low-magnification light-sheet microscopic extended-focus view of cleared tissue block (iDISCO) with tyrosine hydroxylase (TH) immunohistochemistry from a male mouse showing the location of the celiac-superior mesenteric complex, splanchnic nerve, and intermesenteric plexus. TH staining (red) labels sympathetic neurons and fibers. Note the left suprarenal and aorticorenal ganglia with kidney-innervating neurons (green). Also, note several nonspecifically labeled structures (green) such as connective tissue. B: higher magnification of light-sheet microscopic image of the celiac-superior mesenteric complex, the left suprarenal ganglion, and the aorticorenal ganglia. Note pseudorabies-labeled kidney-innervating postganglionic neurons are located in the suprarenal and aorticorenal ganglia.

Strong, abundant labeling was observed in the suprarenal and aorticorenal ganglia in the ipsilateral side (Fig. 3). Intriguingly, only sparse labeling was identified in the celiac-superior mesenteric complex. The higher-magnification images clearly revealed PRV-labeled neurons (green), TH-positive neurons (red), and PRV-labeled TH-positive postganglionic neurons (yellow) in the suprarenal and aorticorenal ganglia (Fig. 4). Furthermore, PRV-labeled nerve fibers (green) were identifiable among TH-positive nerves (red) (Figs. 3 and 4).

Figure 4.

Figure 4.

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Postganglionic sympathetic neurons innervating the mouse kidney. Light-sheet microscopic images of two examples of PRV-labeled postganglionic sympathetic neurons after unilateral PRV injections into the left kidney. A: retrogradely labeled sympathetic postganglionic neurons (green) in the suprarenal and aorticorenal ganglia following inoculation of the left kidney. Note nonspecifically labeled structures in addition to the neural labeling. A1: tyrosine hydroxylase (TH) immunohistochemistry (red) identified sympathetic postganglionic neurons and nerve bundles in the same mouse. A2: merged image of A and A1. B: enlarged image of boxed area in A2 illustrates kidney-innervating postganglionic neurons (green), TH-positive neurons (red), and colocalization of kidney-innervating neurons with TH (yellow) in the suprarenal ganglion. C, C1, and C2: another example of kidney-innervating sympathetic neurons in the mouse suprarenal and aorticorenal ganglia. Kidney-innervating neurons (C), sympathetic postganglionic neurons identified with TH (C1), and merged image (C2).

Mapping of Liver-Innervating Postganglionic Neurons in Sympathetic Prevertebral Ganglia

PRV inoculation of the liver resulted in widespread labeling in the celiac-superior mesenteric complex (Fig. 5). Successful inoculation of the liver was verified with a fluorescent stereomicroscope (Figs. 2B and 5A) showing a distinct pattern of PRV-labeling compared with kidney-injected samples. Imaging with light-sheet microscopy revealed abundant expression of PRV-labeled neurons in the celiac-superior mesenteric complex, in addition to labeling ipsilaterally and contralaterally in the aorticorenal ganglia and suprarenal ganglia (Fig. 5). Similar to the kidney, individual PRV-labeled neurons (green), TH-positive neurons (red), and PRV-labeled TH-positive neurons (yellow) were identified (Fig. 5C) as well as PRV-labeled nerve fibers.

Figure 5.

Figure 5.

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Ventral overview of sympathetic postganglionic innervation of the liver in the mouse. A: following pseudorabies inoculation of the main lobe of the liver, liver-projecting postganglionic neurons were identified in prevertebral ganglia (green) including the celiac-superior mesenteric complex under the fluorescent dissecting microscope. B: low-magnification light-sheet microscopic image of cleared tissue block with tyrosine hydroxylase (TH) immunostaining (red) illustrates the location of the celiac-superior mesenteric complex, aorticorenal ganglia, and intermesenteric plexus. Postganglionic liver-projecting neurons (green) were identified in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia. C: higher magnification of light-sheet microscopic image of the celiac-superior mesenteric complex shows liver-projecting (green), TH-positive (red), and liver-innervating neurons with TH immunolabeling (yellow).

DISCUSSION

The autonomic nervous system plays a crucial role in the maintenance of organs’ functions including the kidney and liver. Since increased activity of the SNS is a contributing factor in a variety of pathophysiological conditions including hypertension, obesity, and diabetes mellitus, decreasing organ-specific sympathetic activity would allow better management of these diseases (6). Here, we examined whether the celiac-superior mesenteric complex provides differential inputs to the mouse kidney and liver by combining retrograde viral tracing with transgenic mouse models, tissue clearing, immunostaining, and light-sheet microscopy. Our findings revealed the distribution of sympathetic postganglionic neurons in the prevertebral ganglia innervating the kidney and liver in the mouse and demonstrate a clear separation between the sympathetic innervation of these organs (Fig. 6).

Figure 6.

Figure 6.

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Schematic illustration of spatially distinct location of kidney and liver-innervating postganglionic neurons in the mouse prevertebral ganglia. Kidney-related neurons (open circles) were identified in the prevertebral ganglia including the suprarenal and aorticorenal ganglia, whereas liver-related postganglionic neurons (closed circles) were mainly located in the celiac-superior mesenteric complex. In addition, labeling was observed in the aorticorenal and suprarenal ganglia.

The renal nerves contain neuropeptide Y, TH, and DbH (13, 46), whereas the presence of neurotensin, somatostatin, and vasoactive intestinal peptide also has been reported (47–50). The availability of reporter mouse models allows the identification of the SNS including postganglionic neurons and nerve bundles. We found, that both the TH:tomato and DbH:tomato mouse models show strong red fluorescence in the celiac-superior mesenteric complex and suprarenal and aorticorenal ganglia and allow easy identification of TH-expressing or DbH-expressing neurons in prevertebral ganglia and nerve bundles with a fluorescent dissecting microscope. These findings demonstrate that TH and DbH reporter mice are excellent facilitators to visualize specifically the celiac-superior mesenteric complex and select its fine structures and fine nerves. On the other hand, for sites with mixed sympathetic/parasympathetic innervation, it should be recognized that TH-reporter expression is not suitable to distinguish these structures due to TH expression in parasympathetic neurons during development (42), but the use of inducible cre-expression lines could solve this problem. Based on our observations we can further speculate that TH and DbH reporter mice will be also useful models for in vivo identification of ganglia and nerves and thereby for neuromodulation based on their neurochemical phenotype; however, this must be proven.

Stimulation or blockade of nerve activity is a feasible approach to alter or restore the normal function of an organ and thus modulate pathophysiological conditions. Renal sympathetic denervation has been performed throughout the years. In the beginning of the past century, surgical thoracolumbar splanchnicectomy was the procedure of choice for severe hypertension. As of today, catheter-based sympathectomy is used to interrupt neural pathways to reduce blood pressure in drug-resistant patients (7, 9); however, the overall usefulness of renal sympathectomy and the reinnervation of the kidneys after denervation are subjects of ongoing debates among experts (51–53). Although these procedures are performed in the absence of detailed information of sympathetic innervation of the mouse kidney, selective manipulation of the kidney to alter secretion or decrease blood pressure will likely remain challenging in the mouse, which is currently the most widely used model system.

Here, we combined iDISCO methods with immunostaining and performed tissue clearance in the adult mouse. This approach allowed visualization of the SNS including the postganglionic neurons and nerve bundles undisturbed in situ and facilitated the evaluation of prevertebral postganglionic neurons contributing to the innervation of the kidneys. Our data demonstrate that the mouse kidney receives sympathetic innervation from the aorticorenal and suprarenal ganglia, although we must note that in this study, we did not assess the paravertebral ganglia. This largely confirms data from previous studies in other species. In humans, the renal nerves are derived from the celiac plexus, the thoracic and lumbar splanchnic nerves, and the upper parts of the intermesenteric plexus (54). Earlier studies identified ganglia of varying size in the renal plexus and the number of the ganglia decreased with age (54, 55). In primates, the sympathetic innervation originates equally from paravertebral and prevertebral renal, aorticorenal, and superior mesenteric ganglia, but not from the celiac ganglion (56). In dogs, cells projecting to the left kidney were found in the renal ganglia, superior mesenteric ganglion and paravertebral ganglia from T11 to L2, and to a lesser extent in the celiac ganglion (11). In cats, the sympathetic innervation of the kidneys mainly originates from the prevertebral superior mesenteric ganglia and along the renal nerve, and partly from the paravertebral chain (T12 to L3) (12). In contrast to cats and dogs, horseradish peroxidase injection into the hilar region of the rat kidney revealed that the kidney receives their main sympathetic innervation from the paravertebral sympathetic ganglia from T11 to L3 (∼80%) and partially from the prevertebral ganglia (∼20%), especially from the superior mesenteric ganglion and to a smaller extent in the celiac ganglion (15). This is consistent with a more recent study, where kidney-innervating neurons were observed in the celiac ganglion following PRV labeling of the left kidney in rats (43). In the adult mouse, we observed postganglionic neurons in the aorticorenal and suprarenal ganglia and sparse labeling in the celiac-superior mesenteric complex. Although species difference exists regarding sympathetic innervation of the kidney and location of postganglionic neurons, our study demonstrates that in the mouse, the prevertebral ganglia provide innervation to the kidneys. On the other hand, we have to note that our study did not assess the paravertebral ganglia, and we cannot exclude the existence of kidney-innervating postganglionic neurons located in the sympathetic chain ganglia. This is a limitation, and further investigations are required to compare innervation from prevertebral and paravertebral ganglia in the mouse.

Distribution of nerve fibers in the liver is highly species dependent, with considerable variability noted within the individual species (24, 57, 58). In rats and mice, the nerves are mainly restricted into the portal space, hilus, and portal venules, and the parenchyma is sparsely innervated; therefore, gap junctions likely play a role in information transmission (24, 57, 59). In contrast, in human, guinea pig, and cat, dense innervation of the parenchyma was described (60, 61). Sympathetic fibers were found in the periportal fields and interlobular spaces in mouse and rat (62–64). TH and DbH immunoreactivity are markers for sympathetic postganglionic fibers, and recently, dynorphin immunoreactivity was shown in TH-positive nerve fibers (22). It has been shown that stimulation of the splanchnic nerves increases glucose production and decreases glycogenesis (26–28), and there is consensus that sympathetic postganglionic neurons innervating the liver are located in the celiac and superior mesenteric ganglia. In this study, we observed dense PRV labeling in the aorticorenal and suprarenal ganglia and celiac-superior mesenteric complex of the mouse, which is consistent with previous reports in a variety of species. Despite the fact that detailed immunohistochemical studies of the guinea pig celiac ganglion have been available for a long time (65, 66), immunohistochemical analysis of the mouse celiac ganglion just became available (67). This recent study demonstrated that celiac ganglion cells contain noradrenergic neuronal markers including TH; vesicular monoamine transporter 2, a protein required for vesicular storage of catecholamines; and norepinephrine transporter, a protein required for reuptake of catecholamines. Although the majority of mouse celiac ganglion neurons contained both TH and NPY, somatostatin was only found in TH-negative interneurons (67). Our study extends these findings by revealing that TH is colocalized with liver-related neurons in the mouse celiac-superior mesenteric complex. In addition, using iDISCO tissue staining and clearing provides an excellent view of the anatomy of the celiac-superior mesenteric complex and allows a better understanding of the spatial dissociation of kidney-related and liver-related postganglionic neurons and nerve bundles.

In conclusion, the combination of retrograde viral labeling and immunostaining demonstrates that the mouse kidney receives sympathetic innervation from the aorticorenal and suprarenal ganglia, whereas the liver is heavily innervated by neurons located in the celiac-superior mesenteric complex.

GRANTS

This work was supported by research grants from the National Institutes of Health [SPARC OT2 OD-023864 (to H.M.) and DK-122842 (to A.Z. and A.V.D.)].

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

W.L.N., H.-R.B., H.M., A.V.D., and A.Z. conceived and designed research; H.T., C.H., D.H.B., A.J.R.M., W.L.N., H.M., A.V.D., and A.Z. performed experiments; W.L.N., H.-R.B., H.M., A.V.D., and A.Z. interpreted results of experiments; C.H., H.M., A.V.D., and A.Z. prepared figures; A.V.D. and A.Z. drafted manuscript; H.T., C.H., W.L.N., H.-R.B., H.M., A.V.D., and A.Z. edited and revised manuscript; H.T., C.H., D.H.B., A.J.R.M., W.L.N., H.-R.B., H.M., A.V.D., and A.Z. approved final version of manuscript.

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