Presence and Distribution of Cholinergic Nerves in Rat Mediastinal Brown Adipose Tissue
Institute of Normal Human Morphology, Marche Polytechnic University, Ancona, Italy
Correspondence to: Saverio Cinti, MD, Institute of Normal Human Morphology, Faculty of Medicine, Via Tronto, 10/A, 60020 Ancona, Italy. E-mail: cinti{at}mta01.univpm.it
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Summary |
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Key Words: cholinergic nerves parasympathetic system brown adipose tissue noradrenergic nerves fasting cold exposure thermoneutrality rat
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Introduction |
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Expression of the vesicular acetylcholine transporter (VAChT) has been demonstrated by in situ hybridization (ISH) in a variety of cholinergic neurons in the central and the peripheral nervous systems (Schafer et al. 1994). Subsequently, IHC studies have proved that VAChT is a reliable marker for the terminal fields of central and peripheral cholinergic nervous systems mainly by virtue of its high concentration in nerve terminals, which contain a number of small synaptic vesicles bearing this antigen (Schafer et al. 1995
; Gilmor et al. 1996
; Arvidsson et al. 1997
; Schafer et al. 1998
). Peripheral postganglionic cholinergic nerves are held to belong to the parasympathetic nervous system. The aim of the present work was therefore to establish whether rat subcutaneous and visceral BAT depots are provided with cholinergic, putatively parasympathetic, nerves. To address this question, we investigated by IHC the occurrence and distribution of VAChT-positive nerves in interscapular, cervical, mediastinal, and perirenal BAT depots of rats kept at different environmental temperatures and of fasted rats. The results showed that only mediastinal BAT is provided with cholinergic nerves and that they are modulated by both cold and fasting.
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Materials and Methods |
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All animals were anesthetized [100 mg/kg ketamine (Ketavet; Farm. Gellini, Aprilia, Italy) in combination with 10 mg/kg xylazine (Rompum; Bayer AG, Leverkusen, Germany)] and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). Mediastinal, cervical, interscapular, and perirenal BAT and control tissues (see below) were dissected and postfixed by overnight immersion in the same fixative at 4C. After a brief wash in PB, tissues were cryoprotected in a solution of 30% sucrose in PB for 24 hr at 4C. Then a glass beaker containing 2-methylbutane (isopentane) was cooled in liquid nitrogen and the specimens, embedded in OCT medium, were frozen. Finally, tissues were stored at 80C.
Immunohistochemistry
Four-µm-thick cryosections obtained with a Leica CM1900 cryostat (Leica Microsystems; Vienna, Austria) were collected and air-dried overnight at room temperature (RT). Immunoreactivity was assessed according to the avidinbiotinperoxidase (ABC) method. After two washes in 0.05 M PBS, pH 7.4, for 15 min each, sections were incubated with 0.3% H2O2 in methanol for 30 min at RT to block endogenous peroxidase, washed twice in PBS (15 min each), and incubated in 1:75 v/v normal goat serum [Vector Laboratories, Burlingame, CA; VAChT and vasoactive intestinal peptide (VIP) schedule] or in 1:75 v/v normal rabbit serum [Vector; tyrosine hydroxylase (TH) schedule] in PBS for 20 min at RT to block nonspecific sites. Sections were incubated overnight at 4C with the primary antibody against VAChT (polyclonal rabbit; SigmaAldrich, St Louis, MO) at a concentration of 1 µg/ml, with the primary antibody against VIP (polyclonal guinea pig; Peninsula Laboratories, San Carlos, CA) at a concentration of 2.5 µg/ml, or with the primary antibody against TH (polyclonal sheep; Chemicon International, Temecula, CA) at a concentration of 0.2 µg/ml; washed twice with PBS (15 min each), incubated in 1:200 v/v biotinylated IgG anti-rabbit goat serum (Vector; VAChT schedule), 1:200 v/v biotinylated IgG anti-guinea pig goat serum (Vector; VIP schedule), or 1:200 v/v biotinylated IgG anti-sheep rabbit serum (Vector; TH schedule) in PBS for 30 min at RT, washed twice with PBS (15 min each), incubated in ABC reagent (Vector) in PBS for 1 hr at RT, washed twice with PBS (15 min each) incubated in 0.02% H2O2 and 0.075% diaminobenzidine (Sigma) in 0.05 M Tris buffer (pH 7.6), kept for 5 min in a dark room, rinsed in distilled water, and counterstained with hematoxylin. Sections were finally dehydrated and mounted in Entellan. The ability of the antibodies to specifically detect the antigens was evaluated in sections (prepared as described above) of tissues known to contain the antigens (heart, intestine, and skeletal muscle for VAChT; central nervous system and intestine for VIP; central nervous system and heart for noradrenergic nerves). Negative controls were obtained in each instance by omitting the primary antibody and using preimmune instead of primary antiserum. The images were stored as TIFF files. Brightness and contrast of the final images were adjusted using the PHOTOSHOP 6 software (Adobe Systems; Mountain View, CA).
Morphometry and Statistical Analysis
For semiquantitative evaluations, VAChT and TH IHC reactions were performed under standardized conditions for all samples. The number of spots indicating specific immunostaining in parenchymal cholinergic and noradrenergic nerves and the area of these nerves were evaluated on three representative sections collected every 200 µm for each animal. Five rats were studied for each experimental condition, and an amount of mediastinal BAT containing 500 brown adipocytes was measured for each experimental condition. The number of nerve spots and the area (µm2) occupied by the specific brownish precipitate, indicating both presence and amount of VACht or TH, were measured at high power using a morphological imaging system (LUCIA, Version 4.5; Nikon Instruments, Rome, Italy). To compensate for the relative increase in nerve density due to cell shrinkage during fasting and, to a lesser extent, during cold exposure, results are presented as mean nerve spot number/50 brown adipocytes ± SE and as mean nerve area/50 brown adipocytes ± SE for each group. Group means were compared by two-way ANOVA. Significance was defined as 95% confidence levels.
Immunofluorescence and Confocal Microscopy
Immunofluorescence was performed on cryosections obtained according to the procedure described above. After two washes in PB for 15 min each, sections were incubated in 1:75 v/v normal donkey serum (Jackson ImmunoResearch; West Grove, PA) in PB for 20 min at RT to block nonspecific sites. Sections were incubated overnight at 4C with the mixture containing the primary antibody against VAChT (polyclonal rabbit; Sigma) at a concentration of 1.3 µg/ml and the primary antibody against TH (polyclonal sheep; Chemicon) at a concentration of 0.3 µg/ml, washed twice with PB (15 min each), and incubated in 1:100 v/v FITC donkey anti-rabbit (Jackson) and TRITC donkey anti-sheep (Jackson) antibody in PB for 30 min at RT. Sections were subsequently washed twice with PB, air-dried, and coverslipped using the Vectashield mounting medium (Vector). Fluorescence was detected with a BioRad (Hercules, CA) Microradiance confocal laser scanning microscope equipped with an argon and He/Ne mixed gas laser. FITC and TRITC were excited with the 488- and 543-nm lines, respectively, imaged separately (emissions were separated using 515/30- and 570-nm filters), and then merged using the LaserSharp Processing Bio-Rad software (version 3.2). Sections were viewed in an Eclipse E600 Nikon microscope with a x60 plan apochromat objective and 1.4 numerical aperture. The images (512 x 512 pixels) were then obtained sequentially from two channels using a confocal pinhole of 23. The images were stored as TIFF files. Brightness and contrast of the final images were adjusted using the Photoshop 6 software (Adobe Systems).
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Results |
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Cholinergic Nerve Density Increases After Cold Exposure in Mediastinal BAT
To verify whether cold, the physiological stimulus for BAT heat production and recruitment (Trayhurn and Nicholls 1986), modulates the cholinergic nerve supply to mediastinal fat, we compared by IHC and morphological analysis the density of TH- and VAChT-positive nerves in mediastinal BAT in rats kept at temperatures close to thermoneutrality (28C) and in animals exposed to low temperatures (4C) for 2 days. As expected, TH-positive nerves were sparse in the former animals, whereas their density significantly increased after cold exposure (Figures 4A and 4B)
. Notably, very few vascular and parenchymal VAChT-positive nerves were found in mediastinal BAT in rats kept in a thermoneutral condition. After cold exposure, VAChT immunoreactivity was more evident at perivascular sites and the density of VAChT-positive nerves among brown adipocytes increased significantly (Figures 4A and 4B).
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Discussion |
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A possible cholinergic control over mammalian thermogenesis has been hypothesized. In rats, injection of the muscarinic receptor antagonist atropine sulfate enhances the acute thermogenic response to a meal, an effect that is particularly evident in genetically obese Zucker rats, which normally show defective diet-induced thermogenesis (Rothwell et al. 1981). Furthermore, injection of atropine or surgical vagotomy also prevents metabolic rate decline after glucopenia induced by central or peripheral injections of 2-deoxy-D-glucose (Shiraishi and Mager 1980
; Rothwell et al. 1981
). These findings suggest that, by acting through muscarinic receptors, acetylcholine exerts an inhibitory influence on thermogenesis, particularly in obese rodents. Considering these data, Bryant et al. (1983)
attempted to determine whether rat BAT was provided with cholinergic nerves by measuring tissue acetylcholine levels and acetylcholinesterase activity. Since the study was conducted on interscapular BAT which, in line with our data, was completely devoid of cholinergic innervation, the authors concluded that there was no evidence of parasympathetic innervation in rat BAT. On the contrary, our data suggest that a putative inhibitory influence on thermogenesis exerted by acetylcholine may take place in mediastinal BAT, where acetylcholine released by parenchymal cholinergic axons may act directly on brown adipocytes or, possibly, may exert prejunctional inhibition of norepinephrine release from adjacent sympathetic nerves. Of course, additional central and/or hormonal effects on BAT thermogenesis after experimental manipulation of the cholinergic system cannot be excluded.
Cold exposure and fasting led to increased density of VAChT-positive fibers in addition to noradrenergic sympathetic nerves. Therefore, any influence of cholinergic nerves on brown adipocyte thermogenesis and metabolism increases during cold-dependent and fast-dependent heat production and/or lipid secretion.
Acetylcholine is usually found in the parasympathetic system throughout the body. Therefore, the cholinergic nerves found in mediastinal BAT are probably postganglionic parasympathetic nerves pertaining to the vagal nerve and arising from the many parasympathetic ganglia located close to the mediastinal viscera. Nevertheless, sympathetic postganglionic cholinergic nerves have been described in sweat glands (Landis and Keefe 1983; Leblanc and Landis 1986
), arterial microvasculature of skeletal muscle (Bolme and Fuxe 1970
), and periosteum (Asmus et al. 2000
). Therefore, it cannot be excluded that the cholinergic axons supplying mediastinal brown fat lobules could derive from the cholinergic neurons of the sympathetic thoracic chain (Schafer et al. 1998
).
We did not find cholinergic innervation in interscapular, cervical, and perirenal BAT depots. However, it should be noted that non-cholinergic nitric oxide-, VIP-, and/or calcitonin gene-related peptide-containing parasympathetic nerves have recently been described in human airways (van der Velden and Hulsmann 1999) and in rat heart (Onuoha et al. 1999
). Therefore, our results do not exclude a non-cholinergic parasympathetic nerve supply to other BAT depots. Of note is that a dual sympathetic and parasympathetic innervation has recently been hypothesized for rat white adipose tissue (Kreier et al. 2002
).
On the other hand, if further studies were to exclude the presence of a parasympathetic nerve supply to the other BAT depots, mediastinal BAT would be the sole BAT depot provided with both a sympathetic and a parasympathetic nerve supply. Such double control may be connected with a closer and more subtle modulation of brown adipocyte metabolism and heat production in the mediastinum, possibly in a still unknown relationship to cardiac activity.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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