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LETTERS TO THE EDITOR
1Institute of Normal Human Morphology, Marche Polytechnic University, Ancona, Italy; and 2Department of Biology, Neurobiology and Behavior Program, Center for Behavioral Neuroscience, Georgia State University, Atlanta, Georgia
TO THE EDITOR: Kreier and Buijs (16) have questioned our work (14) demonstrating the lack of parasympathetic nervous system (PSNS) innervation of white adipose tissue (WAT) in laboratory rats and mice, as well as in Siberian hamsters. We appreciate the opportunity to clarify and extend our position and observations on this issue at this time.
The crux of their argument for PSNS innervation of WAT (17) rests largely on one point. Specifically, Kreier et al. (17) suggest that there are two anatomically separate autonomic innervations of the retroperitoneal WAT (RWAT). Given that, Kreier et al. were able to selectively surgically denervate the sympathetic nervous system (SNS) innervation of RWAT, sparing its putative PSNS innervation. Next, Kreier et al. injected the sympathetically denervated RWAT with a transneuronal viral tract tracer, the pseudorabies virus (PRV), to selectively label the remaining PSNS outflow from brain to RWAT. The authors concluded that the PSNS innervation of WAT was demonstrated because the dorsal motor nucleus (DMV) of the brain stem, a prominent component of the PSNS innervation of many peripheral tissues, showed bilateral extensive infections. Biochemical or histochemical evidence that these nerve bundles are indeed sympathetic or parasympathetic does not exist, which questions the interpretation of results when either is severed.
Kreier and Buijs (16) in their rebuttal note that in our paper, which described the sympathetic outflow from brain to WAT for the first time (4), we observed PRV-infected cells in the Siberian hamster DMV after inoculation of the virus into neurally intact WAT. Indeed, we saw unilateral labeling in the peripheral edges of the DMV in that paper (4), as well as in subsequent studies where PRV was injected into WAT (6, 27, 30), including the one being addressed here (14). At no time, however, have we seen such dense, internally located and, most importantly, bilateral labeling of the DMV as that reported by Kreier et al. (17). As we have noted (14), bilateral labeling of the DMV is at odds with the demonstrated unilateral vagal innervation of peripheral tissues in rodents (12, 21–23). One possibility for their observed bilateral infections of the DMV could be a species difference, as we used Siberian hamsters in our work (4, 6, 14, 27, 30) and Kreier et al. (17) used laboratory rats. Alternatively, it could be that of all the peripheral tissues examined to date, the DMV only bilaterally innervates rat RWAT, although this seems unlikely. Interestingly, using PRV to define the SNS innervation of the spleen, a tissue having only SNS innervation and no PSNS efferent or afferent innervation (8), PRV-infected neurons also were localized unilaterally along the boundaries of the DMV (8). Although dismissed by Kreier and Buijs (16), leakage into other sites seems the most parsimonious explanation for their findings.
We tested for evidence of three established parasympathetic nerve markers in other tissues [vesicular acetylcholine transporter (VAChT), vasoactive intestinal peptide (VIP), and neuronal nitric oxide synthase (nNOS)] in three different WAT pads [epididymal WAT, inguinal WAT (IWAT), and RWAT] in three types of animals (ob/obmice, C57BL mice, and Sprague-Dawley rats) with one result: no labeling of any marker in any WAT pad for any species (14). Not addressed in their rebuttal (16) of our paper (14), however, was our finding that when we labeled all nerves in WAT with the protein gene product 9.5, a pan-nerve marker (32), there only were rare cases of a very few fibers that also were not immunopositive for tyrosine hydroxylase, the SNS nerve marker. This is powerful evidence that the vast preponderance of innervation of these three WAT pads is sympathetic in these three species, with the remaining nonnoradrenergic fibers likely being sensory (10, 11, 28, 29).
Kreier and Buijs (16) suggest that our inability to see parasympathetic-associated nerve markers was due to the histological paraffin-embedding procedure we used. However, as stated in our paper (14), "We estimate the loss of immunoreactivity on the basis of comparisons between paraffin-embedded and standard fixation to be 
15–20% and therefore do not feel this loss could explain the lack of VAChT- VIP-, or nNOS-immunoreactivity (ir) in WAT." Therefore, this is not a valid criticism.
Kreier and Buijs (16) next note that the mediastinum shows BAT VAChT-ir (25), a finding we know well from one of our previous publications (13). Consideration should be made, however, that the other BAT depots of laboratory rats are devoid of cholinergic nerves (13) corroborating the lack of biochemical evidence of acetylcholine or acetylcholine esterase in BAT (7). These results parallel our finding that WAT is devoid of PSNS markers (14) and similarly lacks biochemical evidence of cholinergic factors (3). Furthermore, cholinergic sympathetic innervation has been shown for sweat glands (18), arterial microvasculature of skeletal muscle (5), and periosteum (2), and this also could be the case for mediastinal BAT.
Kreier and Buijs (16) suggest possible alternative vagal neurochemicals other than VAChT, VIP, or nNOS that we tested by immunohistochemistry in our various WAT samples, such as pituitary adenylate cyclase-activating polypeptide (PACAP), leucine-enkephalin (L-ENK), and peptide histidine methionine (PHM). These peptides occur in vagal nerves, but there is no evidence that they have parasympathetic-like effects on white adipocyte metabolism (i.e., effects opposite to sympathetically triggered lipolysis). Akesson et al. (1) report that PACAP triggers lipolysis, rather than antilipolytis, whereas L-ENK, as well as other endogenous opiates, have no effect on lipid or glucose metabolism in isolated rat adipocytes (15), and finally no effects of PHM on adipocyte metabolism have been reported. Thus, the alternative neurochemicals proposed by Kreier and Buijs (16) do not seem to be viable parasympathetic neurochemical candidates affecting white adipocytes and have not been demonstrated in WAT nerves.
In our study (14), we injected the specific catecholamine neurotoxin 6-hydroxy-dopamine (6OHDA) locally in IWAT to selectively destroy the sympathetic innervation and thereby spare the putative parasympathetic innervation to WAT. PRV was injected 7 days later into the 6OHDA- or saline vehicle-injected IWAT and the animals were killed 6 days later (13 days post-6OHDA or saline intra-tissue injection). We found no infections in the sympathetic chain, spinal cord, and brain in PRV-injected IWAT that previously was sympathetically denervated by 6OHDA, but normal patterns of infection across the neuroaxis when PRV was subsequently injected into IWAT that was previously injected with the saline vehicle (14). Kreier and Buijs (16) question these results because, at the time of death, IWAT norepinephrine (NE) content of the 6OHDA-injected pads was only depleted by 60% compared with the saline vehicle-injected control IWAT pads (14). First, they state we did not discuss this finding (16). However, as we noted, "in our experience (24) and the experience of others (9, 26, 33), although the immediate effect of 6-OHDA is to produce substantial NE depletions, there is a relatively quick return of NE content likely due to sprouting of nondamaged neurons into areas previously innervated." Second, Kreier and Buijs (16) suggest that the 40% remaining NE content represents "40% of the noradrenergic innervation is still present" (16). The absence or near absence of noradrenergic terminals after 6OHDA is only seen 1–3 days postinjection (e.g., see Refs. 19, 20, 33, 34). After 10–14 days post-6OHDA injection, however, a time similar to our post-6OHDA period (13 days; Ref. 14), the disparities between anatomy and neurochemistry are apparent. For example, BAT from 6OHDA-treated rats has NE content of 50% of control values at this post-6OHDA injection interval, but "this was still not sufficient to induce fluorescent varicosities among the adipocytes" (33), implying no parenchymal sympathetic innervation; instead there was an increase in the number of small, dense core vesicles that store NE in the preterminal portions of the axons, suggesting NE accumulation in axons proximal to their termination (33). A similar conclusion was made for effects of systemic 6OHDA on spleen sympathetic innervation (20). In addition, there is significant contribution to the NE content of the mesentery after 6OHDA treatment by the large nonterminal NE fascicles found along the blood vessels that exhibit exaggerated catecholamine histofluorescence (19). Thus, despite incomplete depletion of NE content after intra-IWAT 6OHDA at the time of death in our study (14), the denervation appears complete.
Regarding the possibility that the putative remaining parasympathetic nerves did not become infected in our study because 6OHDA slows PRV infection in these nerves as Kreier and Buijs suggest (16), we are unsure about this contention based on the evidence they used to make this point (31). They cite Takahashi et al. (31) who injected the wild-type strain of PRV that travels both anterogradely and retrogradely (making interpretation complicated at best), whereas we (14) and Kreier et al. (17) used the Bartha's K strain that only travels retrogradely. Takahashi et al. (31) injected 6OHDA systemically rather than directly into the tissue of interest as we did in our study (WAT, Ref. 14). The suggestion by Kreier and Buijs (16) seems to echo a possibility brought to our attention by Brian Oldfield (personal communication) when we were conducting our study (14) that 6OHDA may stimulate the immune system so as to slow down or block the virus. We therefore examined the sympathetic ganglia (in addition to the spinal cord and brain that most researchers only assay) to test whether 6OHDA caused the PRV to stall there, and it did not (14). The only site examined by Takahashi et al. (31) that is parasympathetic was the Edinger-Westphal nucleus, an accessory parasympathetic cranial nerve nucleus in the midbrain. In that study, 6OHDA did not block PRV infections; instead all of the 6OHDA-treated animals became infected, but at a "markedly decreased level in half of the treated mice" (31). Therefore, all of their animals became infected with diminished PRV infections only occurring in half of them, whereas we found no infection in any of our animals. Therefore, we do not believe this is compelling evidence of a 6OHDA-induced reduction in PRV infection in parasympathetic or sympathetic nerves as Kreier and Buijs (16) contend.
Kreier and Buijs (16) state that our findings showing that surgical and chemical denervation triggers WAT growth (6, 11, 29, 35) is evidence for the PSNS innervation of WAT; we interpret this oppositely: that it instead supports SNS innervation underlying this response. That is, with surgical denervation (6, 11, 29, 35), where both sensory and sympathetic nerve supplies to WAT are destroyed, fat cell number (6, 29, 35) and fat cell proliferation (11) increases. With intra-WAT injection of 6OHDA, however, where only the SNS innervation is destroyed (as evidenced by significantly decreased tyrosine hydroxylase-ir and no effect on calcitonin gene-related peptide-ir, a sensory nerve marker) the surgical denervation-induced increase in fat cell number is duplicated, indicating the underlying mechanism is sympathetic not parasympathetic (11).
Finally, we await the demonstration of parasympathetic ganglia associated with WAT that always accompany parasympathetically-innervated tissue/glands. The Cinti and Bartness laboratories have never observed such ganglia in all of our work with WAT of laboratory rats and mice and Siberian hamsters.
Therefore, the absence of: 1) parasympathetic ganglia in or near WAT, 2) markers clearly shown to be associated with parasympathetic nerves, 3) histological identification of the two neural provisions to RWAT as belonging to separate arms of the autonomic nervous system, and 4) evidence that other possible parasympathetic neurotransmitters participate in antisympathetic lipid or glucose metabolism responses in white adipocytes, fortifies our stance that the PSNS innervation of WAT is either nonexistent or meager at best. It would be extremely exciting and important, however, if WAT PSNS innervation existed, as it would afford WAT the fine control of its physiology that other tissues, such as the heart, possess.
GRANT
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-35254 (to T. J. Bartness).
ACKNOWLEDGMENTS
The authors thank Drs. Gary Picard, Linda Rinaman, Brian Oldfield, and Pat Card for discussions of the issues dealt with herein.
FOOTNOTES
Address for reprint requests and other correspondence: T. J. Bartness, Dept. of Biology, Georgia State Univ., 24 Peachtree Center Ave. NE, Atlanta, GA 30302-4010 (e-mail: bartness{at}gsu.edu)
REFERENCES
This article has been cited by other articles:
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  C. K. Song, C. H. Vaughan, E. Keen-Rhinehart, R. B. S. Harris, D. Richard, and T. J. Bartness Melanocortin-4 receptor mRNA expressed in sympathetic outflow neurons to brown adipose tissue: neuroanatomical and functional evidence Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R417 - R428. [Abstract] [Full Text] [PDF]
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