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1 Neuropsychology and
Behavioral Neurosciences Program, Brown adipose
tissue (BAT) plays a critical role in cold- and diet-induced
thermogenesis. Although BAT is densely innervated by the sympathetic
nervous system (SNS), little is known about the central nervous system
(CNS) origins of this innervation. The purpose of the present
experiment was to determine the neuroanatomic chain of functionally
connected neurons from the CNS to BAT. A transneuronal viral tract
tracer, Bartha's K strain of the pseudorabies virus (PRV), was
injected into the interscapular BAT of Siberian hamsters. The animals
were killed 4 and 6 days postinjection, and the infected neurons were
visualized by immunocytochemistry. PRV-infected neurons were found in
the spinal cord, brain stem, midbrain, and forebrain. The intensity of
labeled neurons in the forebrain varied from heavy infections in the
medial preoptic area and paraventricular hypothalamic nucleus to few
infections in the ventromedial hypothalamic nucleus, with moderate
infections in the suprachiasmatic and lateral hypothalamic nuclei.
These results define the SNS outflow from the brain to BAT for the
first time in any species.
pseudorabies virus; thermogenesis; obesity; autonomic nervous
system; tract tracing; central nervous system
BROWN ADIPOSE TISSUE (BAT)
plays a critical role in the increased heat production occurring with
cold exposure (for review see Ref. 19) or overeating (for review see
Ref. 41). BAT receives a dense innervation by the sympathetic nervous
system (SNS; for review see Ref. 20), and this innervation plays an
important role in heat production by BAT. The BAT pad receiving the
most attention is interscapular BAT (IBAT) because of its size,
accessibility (8), and clear SNS innervation [5 bilateral nerve
bundles (i.e., intercostal nerves) going to each pad] (12).
Injections of the retrograde tract tracer horseradish peroxidase into
laboratory rat IBAT pads labeled the proximate neuroanatomic distribution of the postganglionic neurons that innervate this BAT
depot (49). The central nervous system (CNS) sites that innervate the
preganglionic sympathetic neurons in the spinal cord that, in turn,
project to these postganglionic neurons remain to be determined
neuroanatomically, however. The location of these sites has been
inferred using lesions or electrical or chemical stimulation of the CNS
followed by assessment of changes in IBAT morphology, biochemistry, and
electrophysiology (for review see Ref. 20). For example, the
ventromedial hypothalamus (VMH) has been implicated in the control of
BAT thermogenesis, because electrical or chemical stimulation of the
VMH results in morphological and biochemical changes in IBAT as well as
changes in the firing rates of the SNS intercostal nerves innervating
IBAT (e.g., Ref. 4; 2, 5, 18, 23, 35, 40, 46, 54, 56-58).
Complementary lesion studies appear to implicate further the role of
VMH in BAT thermogenesis (e.g., Ref. 14; 21, 22, 32, 38, 43, 45, 48, 60, 61). The interpretation of these VMH stimulation or lesion data and
of stimulation and lesion data of other brain sites (e.g., Ref. 6; 3, 10, 13, 15, 25, 50) on BAT thermogenesis can be difficult because of
the inherent problems of confining the stimulation or lesion to these brain targets of interest. Although several brain regions have been
implicated in the regulation of BAT function as a result of these
stimulation and lesion studies, no neuroanatomic data exist for the
origins of the SNS outflow from brain to BAT.
The present experiment was designed to answer the question: what is the
neuroanatomic chain of functionally connected neurons from the CNS to
BAT? This was accomplished with the use of a transneuronal viral tract
tracer, Bartha's K strain of the pseudorabies virus (PRV). A similar
strategy was successful in defining the CNS origins of the SNS outflow
from brain to white adipose tissue (WAT) recently (7).
Animals. Adult male Siberian hamsters
(Phodopus sungorus sungorus) were
obtained from a breeding colony maintained at Georgia State University.
The hamsters were initially group-housed (10-12/cage) in polyvinyl
cages (48 × 27 × 15 cm) and kept in a light-dark (LD) cycle
that simulated long-day, summerlike conditions (16:8 LD with lights on
at 0300). Purina Rodent Chow (no. 5001) and water were available ad libitum.
Surgical procedures. Eight hamsters
were anesthetized with pentobarbital sodium (50 mg/kg), and the IBAT
was exposed. An attenuated strain of the Bartha's
gCKa PRV was injected into IBAT.
The virus initially was supplied by Dr. Arthur Loewy (Washington Univ.,
St. Louis, MO) and then by Dr. Teryl Frey (Georgia State Univ.). PRV
(108 plaque-forming units/ml) was
injected unilaterally into the right IBAT pad in 120-nl volumes into
five areas across its length (n = 8 hamsters). All injections were made in the morning between 0900 and 1100 under a fume hood. The time course for PRV infection rate
was determined by killing the animals at various intervals after PRV
injections. Our previous PRV study in WAT of Siberian hamsters (7)
showed that infection in brain stem neurons could be detected 96 h
after PRV injection into this tissue. The infection reaches the
hypothalamus and extends into the forebrain areas by 144 h after PRV
injections. Therefore, to induce infections extending into the
forebrain areas, hamsters were killed at intervals of 96 h, or 4 days
(n = 4 hamsters), and 144 h, or 6 days
(n = 4 hamsters). Animals were
perfused intracardially with 4% paraformaldehyde in the morning
between 0900 and 1100.
Immunocytochemical procedures. The
brains were postfixed overnight in 4% paraformaldehyde followed by
overnight incubation in 25% sucrose. Brains were cut coronally into
50-µm-thick sections on a freezing microtome and stored in five vials
of 0.1 M sodium phosphate buffer with 0.1% sodium azide. One of every
five sets of stored sections was incubated in a pig polyclonal antibody to PRV (donated by Dr. Kenneth Platt, Iowa State Univ.) overnight at
room temperature. The PRV antibody was diluted 1:30,000 in a buffer
containing 2% normal rabbit serum and 0.3% Triton X-100. The sections
were then incubated in biotinylated rabbit anti-pig secondary antibody
(Sigma) at a 1:100 dilution for 2 h at room temperature. Next, the
sections were incubated in the avidin-biotin horseradish peroxidase
complex (Vectastain ABC Elite kit; Vector Laboratories, Burlingame, CA)
at a 1:200 dilution for 1 h at room temperature. PRV-infected neurons
were visualized with 3,3'-diaminobenzidine. The sections were
cleared with xylene and placed under coverslips with DPX.
The specificity of the immunocytochemical staining for the pig anti-PRV
antibody used in this study has been validated previously (51).
The animals that had PRV infections in the brain stem and forebrain
neurons were checked for signs of lysis of the infected neurons in the
spinal cord. The spinal cords were removed from the infected animals
killed on day
6, the longest postinjection interval.
The cords were postfixed overnight in 4% paraformaldehyde followed by
overnight incubation in 25% sucrose. The entire length of the spinal
cord, including all sections of the cervical, thoracic, and lumbar
regions, was cut coronally into 50-µm-thick sections on a freezing
microtome. The sections were stored in 0.1 M sodium phosphate buffer
with 0.1% sodium azide. They were processed immunocytochemically following the same procedure as described above for brain sections.
Histological quantification. The
PRV-labeled neurons in brain sections were localized and quantified
using Image Tracer software (Translational Technology) and a
stage-mounted position transducer system (MD3 Microscope Digitizer,
Minnesota Datametrics). Camera lucida pictures of each brain section,
stained with cresyl violet, were drawn and scanned into the computer. A
digital image of each drawing was projected onto the computer screen
with the Image Tracer program. The image of each brain section was
registered with the same section on the microscope stage with the use
of the stage transducers. For all animals, the position of PRV-labeled neurons on each brain section was visualized with the microscope and
marked in the exact position on the computerized image of that section.
The number of marked PRV-labeled neurons was counted in each nucleus
for each animal. The absolute number of infected neurons was combined
for ipsilateral and contralateral sides of the injection site for each
brain region and analyzed statistically using a two-way ANOVA (brain
site × time postinjection). Mean percentages of total infected
cells for the brain stem, midbrain, and forebrain areas were
calculated. Post hoc comparisons were done using Duncan's new multiple
range tests (29).
Of eight hamsters injected with PRV, five became infected. One of the
animals killed on day
4 and two of the animals killed on
day 6 had no infections. There was no sign of illness in the infected animals
with exception of slight weight loss in day
6 animals.
Spinal cord. Quantification of virus
distribution within the spinal cord was not conducted; however, the
entire length of the spinal cords of day
6 animals, those with the longest postinjection survival period, were inspected microscopically to screen for lysis of
the infected cells. In addition, the presence of infected cells in the
ventral horns also was determined, because such infections would
indicate spread of the virus from the IBAT pad to underlying musculature. The spinal cords were sectioned coronally, and the cervical, thoracic, and lumbar regions were not marked before cord
removal; thus it was not possible to determine the exact level of the
cord at which neurons were infected. Lysis did not occur in any of the
infected neurons, nor were there any cases of infected cells in the
ventral horns. Infected neurons were seen only in a well-defined
cluster located in the intermediolateral cell group and the central
autonomic nucleus of the spinal cord ipsilateral to the injection site
(i.e., the SNS preganglionics; Fig. 1).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (116K):
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Fig. 1.
Photomicrograph illustrating distribution of pseudorabies virus
(PRV)-labeled cells in spinal cord of day
6 hamster (longest postinjection period). Bar = 200 µm. CC, central canal; IML, intermediolateral
nucleus.
Data presentation. The distribution of
infected neurons is presented schematically in Fig.
2. These seven levels of the neuroaxis were
chosen because the densest infections were seen at these levels. Figure
3 is composed of
representative microphotographs from infected animals at low and high
magnifications. The letter-number designations correspond as closely as
possible to the letter designations for each level of the neuroaxis in
Fig. 2.
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Brain stem. Figures 2, A-C, and 3, A-C, show the distribution of and examples of, respectively, some of the infected neurons in brain stem at day 6 after injection of the virus into IBAT. At this level of the neural axis, the infection was bilateral and uniform.
In day 4 animals, the virus invaded
most of the ventrally located nuclei in the brain stem. Labeling was
sparse among dorsally located nuclei. For example, among dorsally
located nuclei, only a few infected neurons were found throughout the
rostrocaudal extent of the dorsal aspects of the nucleus of the
solitary tract (Sol) and the medial aspects of the medial vestibular
nucleus (MVe). At the most caudal levels of the brain stem (bregma = 
14.30 to 13.30 mm), infection was found in low levels in
the neurons of the lateral reticular nucleus and medullary reticular
nuclei, dorsal and ventral aspects. At this level, a few infected
neurons were found in the raphe pallidus nucleus (Rpa). At the level of the C1 epinephrine cells (C1) and rostroventrolateral reticular nucleus
(RVL) regions (bregma = 13.30 to 12.72 mm), intense labeling was seen in the intermediate reticular nucleus (IRt), with
moderate labeling of the C1/RVL neurons. Some of the neurons of the
parvocellular reticular nucleus also were labeled. More rostrally, the
infection in the IRt and C1/RVL regions had intensified. At the level
of the MVe, neurons were heavily infected in the C1/RVL region, the
caudal raphe, including the Rpa and raphe obscurus nucleus (ROb), and
all aspects of the gigantocellular reticular nucleus (Gi). At the level
of the facial nucleus, PRV infected the norepinephrine cells (A5)
region. The caudal raphe nuclei, the Gi, the lateral
paragigantocellular nucleus (LPGi), and the raphe magnus remained
heavily infected at this level.
In day 6 animals, the pattern of distribution of infected neurons was quite similar to that of day 4 animals; the infection was contained within the same nuclei. In the majority of nuclei examined, however, intensity of infection was much more severe in day 6 vs. day 4 animals. For example, the virus that had infected the dorsal aspects of the Sol in day 4 animals was transmitted to the lateral and ventral portions of the Sol by day 6. In some regions, such as the caudal raphe (ROb/Rpa), the virus infected neurons in the entire rostrocaudal extent of the nuclei.
Table 1, which shows the mean ± SE of
infected neurons in each brain region across days, has been provided to
illustrate the difference in degree of infection between
day 4 and day
6 animals. The average number of infected neurons
combined for all brain stem regions was significantly higher in
day 6 than in day 4 animals (F = 30.10, P < 0.001). The difference in
average number of infected neurons among brain regions across the two
postinjection intervals approached significance
(P = 0.07). The mean percentage of
total infected cells across all brain stem regions in
day 6 hamsters showed that 20.9% of
total infected cells were found in the IRt. The A5 had 15.2%, the
Rpa/ROb had 12.2%, the LPGi had 11.0%, and the C1 had 10.3% of the
total number of infected cells. The lowest mean percentage of total
infected cells was in the Gi at 4.3%.
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Midbrain. Figures
2D and
3D show the distribution of and
examples of, respectively, some of the infected neurons in brain stem
at day
6 after injection of the virus into
IBAT. The infection also was bilateral and uniform at this level of the
neural axis. The most heavily infected area in the midbrain was the
central gray (CG). Infections throughout the rostrocaudal extent of the CG were found in hamsters killed 4 days after PRV injections. The most
intense labeling was found in the caudal regions, at the level of the
pyramidal tract (Py) in the ventral portions of the CG. More rostrally,
at the level of pontine nuclei (Pn; bregma = 8.00 to 7.64
mm), PRV-infected neurons were seen in lateral and ventral portions of
the central gray (CGLV) and surrounding areas and posterior to the
dorsal raphe nucleus (DR). A few scattered PRV neurons were found in
the lateral dorsal central gray (CGLD), but none were found in the
dorsal central gray (CGD) or medial central gray.
The PRV was transmitted more extensively by the day 6 postinjection interval. As in day 4 animals, hamsters killed on day 6 had the majority of their PRV-infected neurons in the ventral portion of the CG at the level of Py. A few infected neurons had invaded the DR. More rostrally, throughout the Pn sections, intense labeling was seen in the CGLV. In day 6 animals, more infected neurons were found in the CGLD, and a few were seen in the CGD portion. At the level of the ventral tegmental area, a few PRV-infected neurons also were found in the CG and CGD areas.
Forebrain. Figures 2, E-G, and 3, E-G, show the distribution of and examples of, respectively, some of the infected neurons in the forebrain at day 6 after injection of the virus into IBAT. Infections were bilateral at this level of the neural axis, but a greater density of labeling was seen ipsilateral to the injection site. The average number of infected neurons at each site is shown in Table 1. As in the brain stem and midbrain, with an increase in postinjection interval from 4 to 6 days, the PRV infection significantly intensified in each nucleus (F = 15.57, P < 0.001) but remained confined to the same regions.
In day 4 animals, labeled neurons were found in the lateral hypothalamus (LH) and zona incerta (ZI) at the level of the VMH. A few infected neurons also were seen in the dorsal hypothalamic area. In only one animal, two infected neurons were found at the border of the VMH. Rostrally, at the retrochiasmatic area, the majority of infected cells were in the medial parvicellular and ventral aspects of the paraventricular nucleus (PVN); however, the infection also had extended to the borders of, and slightly within, the magnocellular portion. At the level of the suprachiasmatic nucleus (SCN), the anterior parvicellular PVN and medial preoptic area (MPA) were the only regions labeled. A few infected cells were seen at the lateral border of the SCN. No infected neurons were seen in the more rostral sections of the forebrain.
In day 6 animals, the infection intensified within the same nuclei. For example, at the level of the VMH, many more infected neurons appeared across the entire LH and ZI. The infection also penetrated the VMH. A similar situation was observed in the SCN and all areas of the PVN. PRV-infected neurons were seen throughout the lateral SCN and were visible in the medial aspects of this nucleus. Both parvicellular and magnocellular portions of the PVN were heavily labeled with PRV. In addition, the virus was transmitted to more rostral sections of the forebrain and infected the lateral and ventral septum as well as the bed nucleus of the stria terminalis (BNST). There was a significant difference in the total number of infected neurons among forebrain regions (F = 4.37, P < 0.05), with significantly larger numbers of infected neurons in magnocellular and parvicellular portions of the PVN (combined) and in the MPA (P < 0.05). Specifically, 6 days after PRV injections, 38.7% and 32.3% of the total number of infected neurons were found in the PVN and the MPA, respectively. In contrast, only 1.6% of the total number of infected neurons was found in the VMH. Moderate PRV infection was seen in the LH (9.5%) and in the SCN (8.8%). The areas with the lowest percentage of total infected cells were the BNST (4.2%) and the lateral septum (LS; 2.2%).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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These results provide the first neuroanatomic description of the SNS outflow from the brain to BAT in any species. The pattern of labeling observed in the present study after PRV was injected into IBAT resembled the pattern of labeling seen after PRV injections into WAT of Siberian hamsters and laboratory rats (7) and of labeling seen after PRV injections into the adrenal medulla of laboratory rats (53) or Siberian hamsters (unpublished observations). Collectively, these suggest that some of the labeling seen after injections of the virus into IBAT labeled part of the general SNS outflow from the brain to the periphery (52). We found, however, that although it was similar to the labeling after PRV injections into WAT or the adrenal medulla, there were some differences in the degree of labeling of certain brain structures. Specifically, higher percentages of cells were infected in the A5 and caudal raphe (ROb and Rpa collectively, Table 1) regions of the brain stem after BAT (15.2% and 12.2%, respectively) vs. WAT (4% and 5%, respectively) (7) injections of the virus. There also were more infected neurons in the LH and the BNST in animals injected with PRV into IBAT than there were in animals injected with the virus into WAT (7).
Previous attempts to identify the CNS origins of the SNS innervation of IBAT used nonneuroanatomic techniques (i.e., stimulation or lesions) and measured changes in IBAT morphology, biochemistry, or neurophysiology. The VMH has been implicated in most of these studies (for review see Ref. 20), yet we found little or no neural connections between the VMH and IBAT using this transneuronal viral tract tracer (see Table 1 and Fig. 1G). One possible reason for the discrepancy between the results of the nonneuroanatomic studies and those of the present neuroanatomic study is that the targeted stimulation or destruction of the VMH secondarily affected the caudally projecting neurons of the hypothalamic PVN that course near and around the VMH (24, 28, 33). When these PVN neurons are stimulated or destroyed more directly, BAT physiological, biochemical, and/or morphological thermal responses are affected (e.g., Ref. 9; 3, 10, 13). Therefore, previous attempts to affect IBAT thermogenesis through manipulations of the VMH may have altered these caudally projecting PVN neurons known to make direct connections with the spinal preganglionics (for review see Ref. 31). In this manner, BAT thermogenesis may ultimately have been affected.
Concerning the infections seen in the magnocellular region of the PVN, it should be noted that we believe this labeling was not a result of vascular transport of PRV to, for example, the posterior pituitary. This interpretation is based on the inability of PRV injected directly into peripheral venous or arterial blood to induce CNS infections, even at long postinjection survival times (30). Rather, it seems more likely that the scattered spinally projecting parvicellular neurons located within the magnocellular area (36) as well as the merging of the dorsal and ventral parvicellular spinally projecting areas more caudally within the hamster PVN (36) are the origins of the infections seen in the magnocellular PVN in the present study. The magnocellular neuronal infections seen in the present study and the lesser infections seen in other studies wherein PRV was injected into peripheral tissues (52, 53), including our own study after injections of virus into WAT (7), could be explained by transsynaptic spread of PRV within and between the nucleus subdivisions.
Innervation of BAT by the SCN has been suggested by an increase in IBAT thermogenesis after electrical stimulation of the retinohypothalamic tract that innervates the SCN (1) or by glutamate injections directly into the SCN (6). In the present study, a substantial number of infected neurons were visualized in the SCN after injection of PRV into IBAT. It is tempting to speculate that the SCN-BAT connection may be involved in the circadian timing of torpor bouts in Siberian hamsters, because SCN lesions block the expression of the torpor-associated rhythmic daily decreases in body temperature in this species (42).
We also found extensive labeling of PRV-infected neurons in the MPA. These data may help explain why electrical (59) or chemical (11) stimulation of the MPA increases IBAT thermogenesis and the firing rate of the sympathetic nerves that innervate IBAT (11, 59), respectively. The roles of the MPA and the SCN in BAT thermogenesis, however, are not well understood.
Although the pattern of infection in the present study after PRV injections into IBAT was more similar than different compared with the pattern after virus injections into the adrenal medulla of laboratory rats (52, 53), there were some notable differences, especially in the forebrain. These sites included the SCN and MPA as well as the LS and the BNST. These areas also were infected after PRV injections into WAT pads in Siberian hamsters and laboratory rats (7). One possible explanation for these differences may be that we used a longer survival period after the virus was injected into WAT (7) and IBAT than was used in the studies wherein PRV was injected into the adrenal medulla (52, 53) (i.e., 6 vs. 4 days, respectively). We believe that this explanation fails to account for some of the differences in labeling, because the MPA was one of the more rostral forebrain structures labeled, yet it showed the second largest number of infected neurons across the neural axis 6 days post-PRV injection (Table 1).
In a previous study, PRV was injected into WAT of Siberian hamsters and laboratory rats (7). The distribution of infected neurons in the brain after PRV injections into BAT seen in the present study is quite similar to that found in WAT. Some differences do exist between the CNS infections for each type of adipose tissue, however. First, PRV infected the brain much faster after injections into BAT than it did after injections into WAT. Within 4 days after injections into BAT, PRV had spread into several areas in the forebrain, whereas after injections into WAT, it took 6 days before a similar pattern of labeling was seen. This most likely was a result of the shorter distance the virus traveled from the BAT pad than it did from the WAT pads (inguinal and epididymal) (7). Second, PRV infections in all brain regions examined were much heavier after injections into BAT than they were after injection into WAT (7). In addition, there were some differences between BAT- and WAT-injected animals in the number of cells labeled in some brain regions (i.e., greater infections in the A5 and caudal raphe nuclei, and LH and BNST in BAT vs. WAT). The functional significance of these differences remains to be determined (see Perspectives).
In conclusion, the results of the present study suggest that the general SNS outflow to the periphery (52) also is involved in the SNS innervation of BAT (specifically, IBAT). In addition, other CNS sites identified as being connected neuroanatomically to BAT, such as the BNST, SCN, MPA, and LS, also are parts of the SNS innervation of WAT (7).
Perspectives
Our understanding of the central control of peripheral metabolism has been hampered by the inability to trace the chain of neurons originating in the brain and terminating in peripheral glands and organs. The use of transneuronal viral tract tracers, such as the PRV, permits the definition of neural circuits, such as those involved in the central control of peripheral metabolism, within the same animal. The work to date delineating the SNS innervation of a variety of peripheral tissues, including BAT (in the present study), WAT (7), the adrenal medulla (26, 52, 53), the kidney (47), and other peripheral tissues, such as the heart (26, 27, 55), suggests a general SNS outflow from the CNS to the periphery (52). A critical question is raised because of these similarities: how is this general SNS outflow from the brain to the periphery regulated under conditions where there are differential SNS drives on peripheral tissues? Moreover, given the present findings for BAT and our previous findings for WAT (7), a more specific question can be posited: how can there be both separate and simultaneous SNS drives to BAT and WAT? An example of the separate SNS control of these adipose tissues is starvation or severe food restriction. In these conditions of reduced caloric intake, the SNS drive to BAT, and consequently BAT thermogenesis, is decreased (44), and the SNS drive to WAT, and consequently WAT lipolytic activity, is increased (34). An example of the simultaneous SNS control of these adipose tissues is cold exposure. In this condition, the SNS drive to BAT, and consequently BAT thermogenesis, is increased (37, 62); the SNS drive to WAT, and consequently WAT lipolytic activity, is also increased (16, 17). Because of the relatively separate postganglionic SNS innervation of the epididymal and inguinal WAT pads, there may be an analogous separate postganglionic SNS innervation of WAT and BAT. Alternatively, the differences that are seen in the degree of innervation of WAT and BAT by the SNS in some CNS structures should not be overlooked as a means by which this differential control of the SNS drive on adipose tissues or, for that matter, other tissues and organs innervated by the SNS may be controlled.| |
ACKNOWLEDGEMENTS |
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The authors thank Drs. Arthur Loewy, Michael Stock, Patrick Card, and Frank Gordon for helpful discussions of these data. We also thank Dr. Kenneth Platt for providing the PRV antibody, Dr. Arthur Loewy for providing the initial supply of the virus, and Dr. Teryl Frey for providing the current supply of PRV. Finally, we thank Drs. Arthur Loewy and Patrick Card for continued encouragement and suggestions.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-35254 and National Institute of Mental Health Research Scientist Development Award KO2 MH-00841.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. J. Bartness, Dept. of Biology, Georgia State Univ., Atlanta, GA 30303 (E-mail: bartness{at}gsu.edu).
Received 11 May 1998; accepted in final form 10 February 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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