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EDITORIAL FOCUS
APPETITE, OBESITY, DIGESTION, AND METABOLISM
1Neurobiology of Nutrition Laboratory, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana; 2Department of Psychology, Purdue University, West Lafayette, Indiana; and 3Anatomy Institute, University of Erlangen-Nürnberg, Erlangen, Germany
ADIPOSE TISSUE DOES NOT SEEM to be innervated in any significant manner by the vagus nerve after all. As reported by Giordano et al. (9) in this issue, retrograde tracing with pseudorabies virus from at least one fat pad in the Siberian hamster reveals only the occasional neuron in the vagal motor nucleus and nerve fibers in adipose tissue of mouse, rat, and hamster do not exhibit any of the markers typically associated with vagal motor innervation.
Adipose tissue has recently attained high prominence as a key player in obesity and its secondary health problems such as type-2 diabetes and cardiovascular disease. Originally considered as a passive storage organ, it has been found to secrete a number of important hormones and cytokines (17). It is now apparent that in obesity the expanding adipose tissue is chronically inflamed (10). The combination of increased secretion of inflammatory cytokines and reduced secretion of adiponectin leads to dyslipidemia and insulin resistance (11). Hence, there is renewed interest in the biology of adipose tissue including its innervation by the autonomic nervous system.
White adipose tissue is distributed in characteristic pads in the abdominal and thoracic cavities, under the skin, and, under certain circumstances, within specific organs such as muscle and liver (7). Adipose tissue is clearly innervated by the sympathetic nervous system. Noradrenergic nerve fibers found mainly along the vasculature and occasionally contacting adipocytes are present in all fat depots of most mammals, including humans (16). They originate from postganglionic neurons located in the pre- and paravertebral ganglia. The postganglionic neurons are driven by cholinergic sympathetic preganglionic neurons located in the intermediolateral column of the spinal cord that receive input from a number of brain areas. The generally lipolytic action of this sympathetic innervation has been demonstrated in numerous studies using a variety of techniques.
Until recently, there was little or no evidence for a similar innervation of white adipose tissue by the parasympathetic nervous system through outflow from either the vagal system or the lower spinal cord. While more or less extensive vagal efferent innervation of most thoracic and abdominal organs was demonstrated using a variety of techniques, adipose tissue together with the spleen, adrenal gland, and gonads was not among them. According to classic autonomic theory, vagal parasympathetic innervation consists of cholinergic preganglionic neurons located in the caudal brain stem and postganglionic neurons expressing either acetylcholine or other transmitters such as vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS), the so called noncholinergic, nonadrenergic nerves. Because markers for the cholinergic phenotype thus do not unequivocally point to vagal origin, retrograde (from the periphery to the brain) and anterograde (from the brain to the periphery) tracing of vagal fibers is a better approach.
In contrast to the sympathetic innervation where the ganglia are located in the paravertebral or prevertebral chain (celiac ganglion, etc.), vagal ganglia are typically located within or very close to the innervated organ. The implication is that while conventional retrograde tracers injected into a given organ readily label vagal preganglionic neurons, they only label sympathetic postganglionic but not preganglionic neurons. Thus, retrograde tracers were important for providing a first qualitative assessment of vagal innervation of various organs. Initial enthusiasm for this convenient new method was, however, rapidly curtailed when serious pitfalls became apparent. It turned out that in some studies, retrograde labeling of vagal motor neurons to organs receiving relatively little innervation was vastly exaggerated by tracer leaking out of the injected target site and contaminating adjacent organs with a much heavier vagal innervation (8). Thus, unless very stringent controls are employed, false positives are often the result.
Better results can be obtained with modern anterograde tracing techniques introduced and adapted to the vagal motor system in the 1980s and 1990s (1, 4, 15). Because vagal preganglionic neurons are conveniently located in two motor nuclei, a large percentage of them can be impregnated with the anterograde tracer in a single or a few injections. Also, labeling of anterogradely traced axons in the innervated organs does not compete with any other immunohistochemical marker. Therefore, anterograde tracing of vagal innervation is better suited to identify the extent of, and specific compartments/cells targeted by the innervation. This approach was instrumental in demonstrating the dense innervation by vagal preganglionic fibers of the entire alimentary canal all the way down to the colon, and the more moderate innervation of the associated pancreas, liver, and portal vein (3, 5). Double-labeling strategies also clearly demonstrated that the overwhelming majority of vagal preganglionic axons terminate on intrinsic ganglion cells located in the myenteric plexus of the gastrointestinal tract and in interlobular pancreatic ganglia (2). Most important to this discussion is that in none of these numerous studies was any significant innervation of adipose tissue noted, although white adipose tissue was not systematically searched.
Transneuronal retrograde tracing with pseudorabies virus is the most recent, very powerful tool to identify neural pathways, and is ideally suited to visualize the multisynaptic autonomic outflow to peripheral organs. Giordano et al. (9) now show that virus injected into the left or right subcutaneous inguinal fat pad of the Siberian hamster infects only a handful of neurons in the area of the dorsal motor nucleus of the vagus. In addition, this scarce labeling was completely absent when the fat pad was previously sympathetically denervated by local treatment with the noradrenergic toxin 6-hydroxydopamine. This clearly demonstrates that at least in the Siberian hamster, vagal efferent innervation of subcutaneous fat is either not present or extremely scarce, and thus confirms the long-held view that white adipose tissue does not receive any significant vagal efferent innervation.
In stark contrast, Kreier et al. (13, 14) recently reported significant vagal innervation of several fat depots in the rat. Using both pseudorabies virus and Fluorogold, a conventional retrograde tracer, combined with selective microsurgical sympathectomy, they reported that retroperitoneal and subcutaneous inguinal fat pads are each innervated by separate pools of vagal motor neurons in both the dorsal motor nucleus and nucleus ambiguus. This claim is reminiscent of the exaggerated maps reported in the vagal motor nuclei when conventional retrograde tracing started some 20 years ago and the strong labeling is likely due to tracer leakage problems. The massive labeling of both sides of the dorsal motor nucleus after unilateral virus injection into the sympathetically denervated left retroperitoneal fat pad in the Kreier et al. study (13) is highly suspicious for two reasons. First, much of the labeling is in the stomach area of the nucleus, strongly suggesting that the virus leaked out from the surgically traumatized fat pad and entered the stomach wall that also may have been traumatized. Simply putting virus on top of the fat pad or injecting it intravenously are not adequate controls for the slower leakage from the injection depot. More appropriate controls would have been to cover the injected fat pad or the stomach with a protective coating, or to monitor the potential transfer of virus to the stomach or adjacent tissues over the period of the experiment. Second, vagal motor outflow is pretty much lateralized, and, therefore, unilateral virus injection should only label one side of the dorsal motor nucleus. Bilateral labeling is another strong argument supporting the leakage hypothesis.
Likewise, retrograde labeling of vagal neurons in the brain stem on Fluorogold injection into the inguinal fat pad can be explained by inadvertent leakage into the peritoneal cavity. Fluorogold readily diffuses through soft tissues and may easily penetrate the delicate fascia covering the spermatic cord in male rats (and the round uterine ligament in females), thus gaining access to the peritoneal cavity through the open inguinal canal (12). Over the relatively long postinjection survival time of 4 days, the tracer can reach vagal nerve endings in intestines and retrogradely label vagal brain stem neurons. An obvious control experiment for this pitfall would be tracer injection into fat pads far remote from the inguinal canal.
As mentioned above, a more straightforward approach to test vagal innervation of any tissue is anterograde tracing of preganglionic fibers from the dorsal motor nuclei. If this results in labeled fibers terminating in adipose tissue, it would be clear proof of innervation, because the likelihood of false positive results is very small with this method. The major problem with the use of this approach for examining parasympathetic innervation of fat is that according to classic autonomic physiology, preganglionic fibers terminate on postganglionic neurons, and there are no known ganglia and neurons in adipose tissue. The only organ known to receive direct preganglionic input is the adrenal medulla receiving direct sympathetic preganglionic fibers.
Although there is a possibility that the vagal postganglionic neurons are located outside the adipose tissue, the wide anatomical distribution of the major fat pads makes it extremely difficult to come up with potential locations. In one anterograde tracing study, a few vagal preganglionic axons were found to terminate on neurons of unknown phenotype in the adrenal, celiac, and superior mesenteric ganglia (6). Because neurons in these ganglia are likely to contribute to the sympathetic innervation of at least intra-abdominal adipose tissue, this may be a potential pathway for vagal innervation too.
Parasympathetic postganglionic neurons are known to express not only the cholinergic, but also the VIP and/or NOS phenotype. Despite very careful and expert examination, Giordano et al. (9) did not find one single fiber with markers for these phenotypes in various fat pads in mouse, rat or hamster. At the same time, many tyrosine hydroxylase-positive nerve fibers were easily demonstrated. Although it is theoretically possible that there is an as yet unknown additional phenotype of postganglionic parasympathetic neurons, these results strongly argue against the presence of any significant parasympathetic innervation of adipose tissue.
Maybe the last straw for explaining at least some of the findings of both groups of investigators is that neurons in the prevertebral ganglia innervated by vagal preganglionics (6) and projecting to abdominal adipose tissue express a noradrenergic phenotype. This pathway could be responsible for the scarce infection of vagal motor neurons in intact animals, and it would be wiped out with the 6-hydroxydopamine treatment of Giordano et al., (9) but not necessarily with the surgical sympathectomy of Kreier et al. (14).
It is unfortunate that some important parameters, such as species, fat pad, and batch of virus were not better matched between the two teams, as these vagaries could account for some of the discrepant findings. Clearly, a definitive study along the lines discussed here is necessary to put the important question of vagal innervation of adipose tissue to rest.
FOOTNOTES
Address for reprint requests and other correspondence: H.-R. Berthoud, Neurobiology of Nutrition Laboratory, Pennington Biomedical Research Center, Louisiana State Univ. System, 6400 Perkins Road, Baton Rouge, LA 70808 (e-mail: berthohr{at}pbrc.edu)
REFERENCES
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