Fos expression in rat celiac ganglion: an index of
the activation of postganglionic sympathetic nerves
Qi
Mei,
Thomas O.
Mundinger,
David
Kung,
Denis G.
Baskin, and
Gerald J.
Taborsky Jr.
Division of Endocrinology and Metabolism, Department of Medicine,
Veterans Affairs Puget Sound Health Care System, Seattle 98108; and
University of Washington, Seattle, Washington 98159
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ABSTRACT |
To develop an index of the
activation of abdominal sympathetic nerves, we used Fos immunostaining
of the celiac ganglion (CG) taken from rats receiving nicotine,
preganglionic nerve stimulation, or glucopenic agents. Subcutaneous
nicotine injection moderately increased Fos expression in the principal
ganglionic cells of the CG (17 ± 4 Fos+ per mm2,
~12% of all principal CG cells), whereas subcutaneous saline had no
effect (0 ± 0 Fos+ per mm2; n = 7;
P < 0.01). Greater Fos expression was obtained by
applying nicotine topically to the CG (71 ± 8 Fos+ per
mm2; 52% of all principal CG cells, n = 5;
P < 0.01 vs. topical saline, n = 4)
and by preganglionic nerve stimulation (126 ± 9 Fos+ per mm2; 94% of all principal CG cells, n = 11; P < 0.01 vs. nerve isolation, n = 7). Moderate Fos expression was also observed in the CG after intraperitoneal 2-deoxy-D-glucose (2DG) injection (21 ± 2 Fos+ per mm2; 16% of all principal CG cells,
n = 5; P < 0.01 vs. saline ip) or
insulin injection (16 ± 2 Fos+ per mm2; 12% of all
principal CG cells, n = 6; P < 0.01 vs. saline ip). Furthermore, Fos expression induced by 2DG was dose and
time dependent. These data demonstrate significant Fos expression in
the CG in response to chemical, electrical, and reflexive stimulation.
Thus Fos expression in the CG may be a useful index to describe various levels of activation of its postganglionic sympathetic neurons.
hypoglycemia; nicotine; nerve stimulation; deoxyglucose; insulin
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INTRODUCTION |
THE CELIAC GANGLION
(CG) is a sympathetic, prevertebral ganglion that receives its
preganglionic input from the spinal cord and projects its
postganglionic fibers to several abdominal organs, including the
pancreas and liver. Activation of these nerve fibers increases hepatic
glucose production indirectly and directly. For example, electrical
stimulation of pancreatic sympathetic nerves increases glucagon
secretion (2, 3, 18, 22), which in turn increases hepatic
glucose production (4). Likewise, electrical stimulation
of the sympathetic nerves innervating the liver also increases hepatic
glucose production (8, 10, 12, 20, 29). These neurally
induced increases of hepatic glucose production either contribute to
the hyperglycemia associated with severe stress (35) or
counterregulate the hypoglycemia induced by insulin administration
(34). Thus it is important to understand when and to what
extent these specific sympathetic pathways are activated.
To evaluate the activities of the sympathetic nerves innervating
pancreas and liver, we have measured pancreatic (15) and hepatic (28) norepinephrine spillover in large animals.
Using this index, we (15) and others (7) have
established in the dog that pancreatic sympathetic nerves are activated
during glucopenia (13-15) or insulin-induced
hypoglycemia (7, 13, 14) and that hepatic sympathetic
nerves are activated by these and other stresses (28).
Because this technique requires large blood samples from the major
veins of the pancreas and liver, as well as measurements of organ blood
flow and norepinephrine extraction, it has not been routinely used in
experiments with the most common laboratory animal, the rat, or the
species commonly used in transgenic studies, the mouse. There are,
however, alternative methods for assessing the activity of peripheral
sympathetic neural pathways in these smaller laboratory animals. One
such method, involving the measurement of tissue norepinephrine
turnover (41), has been used to infer changes of
pancreatic sympathetic activity (42), but only in response
to relatively long-term manipulations such as changes in diet or
environment (42). Another method, recording the firing rates of sympathetic nerves, has also been used to assess changes in
local sympathetic tone (6, 16, 40), but this method cannot, by itself, distinguish the activity of efferent from afferent nerves, nor parasympathetic from sympathetic nerves. Furthermore, such
neural recordings are usually done under anesthesia, which may decrease
the neural tone being measured (25).
A third alternative index of the activity of peripheral nerves in
unanesthetized small animals is Fos expression in their neuronal
nuclei. The expression of the early immediate gene, Fos, in the nuclei
of the neuronal cells of the brain has been widely used as an index of
central neural activity in response to various stimuli
(5), although the functional significance of this
expression has yet to be determined. However, this technique has been
less frequently used to assess the activity of the peripheral autonomic nervous system, including its sympathetic (19,
39), parasympathetic (37), and enteric
branches (26, 27, 31, 43). Regarding Fos expression in
principal ganglia neurons of peripheral sympathetic ganglia, Koistinaho
and Yang (19, 39) showed that subcutaneous nicotine
injection induced a high level of Fos expression in the principal
ganglionic cells of the superior cervical ganglia. On the basis of
these studies, we hypothesized that nuclear Fos expression in certain
principal ganglionic cells of the CG might be an index of the activity
of hepatic and pancreatic sympathetic nerves, because postganglionic
neurons of the CG project to both the liver and pancreas (30,
38).
Before evaluating Fos expression in the CG, we sought to confirm, in
our laboratory, the expression of Fos in the superior cervical ganglia
after nicotine treatment. Thereafter, we determined whether by use of
the same stimulus, the principal ganglionic cells of the CG also
express Fos. We also produced a greater level of chemical stimulation
by injecting nicotine directly into the fascia of CG (topical
application). To induce an even higher level of activation of nicotine
receptors, we released endogenous acetylcholine from the preganglionic
nerves of the CG by electrically stimulating them. Finally, to
determine whether the more physiological activation of pancreatic and
hepatic sympathetic nerves, known to occur reflexively in response to
central glucopenia (13 -15, 28), increases Fos expression
in the CG, we injected either the neuroglucopenic agent 2-deoxy-D-glucose (2DG) or the hypoglycemic agent insulin.
To determine whether CG Fos expression was a quantitative index of physiological activation of the peripheral sympathetic neurons, we
studied the effect of various doses of 2DG on Fos expression. We also
examined the time course of Fos expression. For all stimuli, we
measured the expression of Fos in the nuclei of the principal ganglionic cells by immunohistochemical methods and quantitated that
activation by counting the number of Fos-positive nuclei per square
millimeter of ganglion.
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METHODS |
Animals.
Male Wistar rats (Simonsen Labs, Gilroy, CA) weighing 250-350 g
were used for all studies. Animals were housed under a 12:12-h light-dark cycle and were fed with normal rat chow (Purina, St Louis,
MO) and tap water ad libitum until studies were performed. All animals
included in these studies were certified as healthy by the Veterinary
Medical Officer of the Veterans Affairs Puget Sound Health Care System
(VAPSHCS) and exhibited normal grooming and feeding behavior. All
research involving animals was conducted in an American Association for
Accreditation of Laboratory Animal Care-accredited facility. All
protocols were designed to ensure appropriate ethical treatment of the
animals and were approved by the Institutional Animal Care and Use
Committee of the VAPSHCS.
Experimental protocol.
Nicotine (2 mg/kg sc; Sigma, St. Louis, MO), insulin (2 U/kg ip), 2DG
(100, 200, 400, and 800 mg/kg ip), or saline (1 ml/kg sc or ip) was
administered separately to conscious rats from different groups. After
treatment, rats were returned to their cages for 2 h before being
deeply anesthetized with pentobarbital sodium (60 mg/kg ip) and fixed
in vivo with 4% paraformaldehyde before tissue harvest. In contrast,
in the study of the time course of Fos expression, rats were
anesthetized and fixed at different times, i.e., 0, 1, 2, 4, and 8 h after 2DG (800 mg/kg ip) injection. Topical nicotine application (2 mg/ml × 0.04 ml) and electrical nerve stimulation (8 Hz, 1 ms, 10 mA, 10 min) were performed on pentobarbital-anesthetized (45 mg/kg ip)
rats. After anesthesia induction, rats received a midline laparotomy,
and peritoneal tissue was retracted to expose the CG. For animals
receiving topical nicotine or saline, a 26-gauge needle was gently
inserted under the connective tissue sheath covering the ganglion, with
care taken to avoid impaling the ganglion itself. Approximately 40 µl
of nicotine solution or saline vehicle were injected under the sheath,
producing a localized, visible reservoir surrounding the ganglion. For
animals receiving electrical stimulation, the preganglionic nerve
trunks of the CG were identified (11) and isolated, and a
bipolar electrode (Harvard Apparatus, South Natick, MA) was placed
around the nerve bundle. The electrode was connected to an
oscilloscope, the nerve trunk was stimulated, and the anesthetized rats
remained on a heating pad for 2 h to allow maximum Fos expression (19). The rats were fixed without regaining
consciousness, and the ganglia were harvested for Fos immunostaining.
Fixation.
Two hours after stimulation (19), most of the anesthetized
rats received a thoracotomy, exposing the heart. Reservoirs of isotonic
saline and 4% paraformaldehyde were connected to a common line by a
three-way stopcock, and the common line was fitted with a 16-gauge
needle at the terminus. The needle was inserted into the left ventricle
of the deeply anesthetized rat, and the right atrium was transected to
allow the perfusate to exit from the vascular system. Rats were first
perfused with saline (300 ml/rat) to clear blood from tissue.
Subsequently, the rats were perfused with 4% paraformaldehyde (300 ml/rat) to fix all tissue. The superior cervical and/or celiac ganglia
were harvested and placed in 25% sucrose in phosphate buffer
overnight to dehydrate the ganglia. The next morning, ganglia were
embedded in mounting medium (Tissue-Tek, Miles, Elkhart, IN), frozen,
and stored in a
80°C freezer until immunohistochemical assay. The
alternative fixation procedure, fixing the ganglia ex vivo after
harvest, resulted in greater nonspecific cytoplasmic staining and
false-positive nuclear staining.
Immunohistochemistry.
Sixteen-micrometer sections of ganglia were cut with a cryostat,
mounted on slides coated with chrome alum, and dried in air for 1 h. The dried slides were then permeabilized by immersing them in 0.25%
Triton X-100 in PBS for 1 h and then treated with 0.3%
H2O2 in methanol for 10 min to quench
endogenous peroxidase. After two rinses in 0.25% Triton-PBS within 30 min, the tissues were incubated with 3% normal goat serum to block
nonspecific staining. After a 1-h incubation, the normal goat serum was
removed, and the slides were incubated in a solution containing rabbit Fos primary antibody (AB-5, Oncogene Sciences, Cambridge, MA; 1:10,000)
overnight. Thereafter, sections were washed in Triton-PBS twice within
30 min and incubated for 1 h at room temperature with a solution
containing biotinylated secondary antibody (goat anti-rabbit IgG,
Jackson ImmunoResearch Labs, West Grove, PA; 1:200). After two rinses,
the sections were incubated for 30 min in an avidin-biotin complex
(Vectastain ABC Elite kit, Vector Labs; 1:100). The staining was
visualized by using a Ni-enhanced DAB substrate kit (Pierce Chemical,
Rockford, IL). To determine the extent of nonspecific staining, two
controls were used. In the first, the Fos antibody was replaced by
1:10,000 normal goat serum in PBS containing 0.25% Triton X-100. In
the second, the antigenic segment of the Fos protein (Oncogene
Sciences, Cambridge, MA; final Fos peptide concentration = 1 ng/ml) was preincubated with the primary Fos antibody (1:10,000) before
being applied to the ganglion.
The procedure for PGP 9.5 staining of principal ganglionic cells was
identical to that of Fos, with the exception of the first antibody. The
PGP 9.5 antibody was a kind gift from Dr. Frank Sundler and was used at
a final dilution of 1:20,000.
Quantitation of Fos-positive cells.
Nuclei of cells specifically stained for Fos were identified by
comparing the staining in the presence of the Fos antibody to that
obtained with the two nonspecific staining controls (see Immunohistochemistry). Principal ganglionic cells were
identified by shape and size. The specific black or dark gray staining
in the nuclei of these cells was not observed in the two nonspecific controls.
Quantification of Fos expression in principal ganglionic cells was
performed using a method similar to that previously employed for other
peptides (24). To count the number of Fos-positive principal ganglionic cells, a 10 × 10-mm square grid was inserted into one of the oculars of the microscope. At a set magnification (×250), the square covered an area of 420 × 420 µm. The grid
was placed randomly over each ganglionic section, avoiding areas
containing large nerve bundles where principal ganglionic cells are
less dense. Fos-positive cells within the grid were counted manually, and three such readings were performed on each ganglionic section. The
average of these three readings was multiplied by 5.67, and the data
were expressed as Fos-positive cells per square millimeter. To
determine the average number of principal ganglionic cells in a
420 × 420-µm area, the section was stained with antibodies against PGP 9.5. A mean value was obtained by counting slides from six
normal rats. The mean percentage of principal ganglionic cells
expressing Fos was then calculated by dividing the mean number of
Fos-positive-staining cells in each group by the average number of PGP
9.5-positive cells per area and multiplying by 100%.
Statistical analysis.
To determine whether there was a significant increase in the number of
principal ganglionic cells expressing nuclear Fos, an unpaired
two-tailed Student's t-test was employed to compare the
mean values for control and stimulated groups. The significance of the
dose and time course responses was determined by a one-way ANOVA. All
data are expressed as means ± SE.
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RESULTS |
Effect of subcutaneous nicotine on Fos expression in superior
cervical ganglia.
To confirm our ability to detect and quantify Fos expression in the
nuclei of principal ganglionic cells and to use Fos expression as an
index of the activity of sympathetic postganglionic neurons, we first
reproduced a previously published report (19) of increased nuclear Fos expression in the superior cervical ganglion (SCG) in
response to chemical activation. Two hours after administration of
saline, the number of principal ganglionic cells with detectable Fos
expression in the SCG was negligible (1 ± 1 Fos+ per
mm2; n = 6; Figs.
1A and
2). In nicotine-treated rats (2 mg/kg
sc), Fos expression in principal ganglionic cells was moderate (34 ± 6 Fos+ per mm2; n = 10;
P < 0.01 vs. saline; Figs. 1B and 2) and
was found in neurons randomly distributed throughout the SCG (Fig.
1B). Only principal ganglionic neurons were Fos positive;
satellite and Schwann cells were Fos negative.

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Fig. 1.
Fos expression in the nuclei (black arrows) of principal ganglion
cells of the rat superior cervical ganglion (SCG, A and
B) and celiac ganglion (CG, C and D)
in response to subcutaneous injection of either saline (A
and C) or nicotine (B and D). Scale
bar, 72 µm.
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Fig. 2.
Fos expression in the nuclei of principal ganglion cells
of the SCG in response to subcutaneous injection of either saline
(n = 6) or nicotine (n = 10). Data are
expressed as means ± SE. *P < 0.01.
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Additionally, Fos-positive nuclear staining was not detected when Fos
antibody was replaced with normal goat serum. Furthermore, the Fos
staining in SCG was completely blocked by preincubation of the first
antibody with Fos peptide (data not shown), demonstrating that the
nuclear staining was specific for Fos protein.
Nuclear Fos expression in CG in response to subcutaneous nicotine.
Fos expression in the CG obtained from the same rats was undetectable
in saline-treated rats (0 ± 0 Fos+ per mm2;
n = 4, Figs. 1C and
3). In nicotine-treated rats, Fos
expression was modest but significant (17 ± 4 Fos+ per
mm2; n = 7; P < 0.01 vs.
saline; Figs. 1D and 3). Because PGP 9.5 staining
demonstrated that the rat CG is comprised of 134 ± 5 principal
ganglionic neurons/mm2 (n = 6, Fig.
4), subcutaneous nicotine activated 12%
of principal CG neurons. The Fos-positive neurons were randomly
distributed throughout the CG (see Fig. 1D).

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Fig. 3.
Fos expression in the nuclei of principal ganglion cells
of the CG in response to subcutaneous injection of either saline
(n = 4) or nicotine (n = 7). Data are
expressed as means ± SE. *P < 0.01.
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Nuclear Fos expression in CG in response to topical nicotine.
Because subcutaneous nicotine administration produced Fos expression in
only a small percentage of CG neurons, and because theoretically all
principal ganglionic neurons have nicotinic receptors, we hypothesized
that the maximum dose of nicotine that could be given systemically (2 mg/kg sc) had produced a submaximal exposure of the ganglion to
nicotine. Therefore, we attempted to expose the CG to a higher
concentration of nicotine by applying nicotine directly to the CG of
anesthetized, laparotomized rats (see METHODS) to determine
whether all, rather than a small subset of, principal ganglion cells
were capable of expressing Fos. This topical application of nicotine
induced a higher level of Fos expression in the principal ganglionic
neurons (71 ± 8 Fos+ per mm2 , or ~52% of
PGP-labeled principal ganglionic neurons; n = 5; Figs.
5B and
6). Although topical application of
saline also induced Fos expression in principal ganglionic neurons
(24 ± 11 Fos+ per mm2; n = 4, Figs.
5A and 6), the Fos expression was significantly lower than
that induced by topical nicotine (P < 0.01). Nuclei labeled for Fos were randomly distributed throughout the CG (Fig. 5B). Replacement of the Fos antibody with normal goat serum
(data not shown) or preincubation of the Fos antibody with Fos peptide (Fig. 5C) completely blocked the staining.

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Fig. 5.
Fos expression in the nuclei of principal ganglion cells
(arrows 1 and 2) and satellite cells (arrow
3) of the rat CG in response to topical (top) injection
of either saline (A) or nicotine (B). Fos
staining induced by topical nicotine can be blocked by preincubation of
the first antibody with Fos peptide (C) but not with
phosphate buffer (D). Scale bar, 72 µm.
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Fig. 6.
Fos expression in the nuclei of principal ganglion cells
of the CG in response to topical injection of either saline
(n = 4) or nicotine (n = 5). Data are
expressed as means ± SE. *P < 0.01.
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Topical application of either nicotine or saline also induced Fos
expression in smaller cells, possibly satellite or Schwann cells (see
Fig. 5, A and B). Our quantification of
ganglionic Fos expression specifically excluded these nonprincipal
ganglionic cells.
Nuclear Fos expression in CG in response to nerve stimulation.
Electrical stimulation of the preganglionic nerve trunk of the CG
induced a high level of Fos expression in the principal ganglionic
cells of the CG (126 ± 9 Fos+ per mm2, or ~94% of
PGP-labeled principal ganglionic cells; n = 11; Figs. 7B and
8). Surgical isolation of the nerve
trunk also induced moderate Fos expression in principal ganglionic
cells (62 ± 17 Fos+ per mm2; n = 7;
Figs. 7A and 8). However, the number of Fos-positive nuclei
in the nerve stimulation group was significantly higher than that in
the nerve isolation group (P < 0.01, Fig. 8).

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Fig. 7.
Fos expression in the nuclei of principal ganglion cells of the rat
CG in response to either preganglionic nerve isolation (arrows
1 and 2, A) or stimulation (B).
Scale bar, 72 µm.
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Fig. 8.
Fos expression in the nuclei of principal ganglion cells
of the CG in response to either preganglionic nerve isolation
(n = 7) or electrical stimulation (n = 11). Data are expressed as means ± SE. *P < 0.01.
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Like topical application of nicotine and saline, both nerve stimulation
and isolation also induced Fos expression in nonprincipal ganglionic cells (Fig. 7, A and B).
Nuclear Fos expression in CG in response to surgical stress.
To investigate the effect of surgical stress on Fos expression, we
observed CG Fos in response to induction of general anesthesia and
performance of a midline laparotomy. Animals undergoing this surgical
procedure had less CG Fos expression (13 ± 5 Fos+ per mm2, n = 4) than those with topical saline
and nerve isolation.
Nuclear Fos expression in CG in response to 2DG or insulin.
To determine whether reflex activation of pancreatic and hepatic
sympathetic nerves, caused by central glucopenia, would induce Fos
expression in the CG, we injected either the neuroglucopenic agent 2DG
(800 mg/kg ip) or the hypoglycemic agent insulin (2 U/kg ip). Fos
expression was induced by either 2DG (21 ± 2 Fos+ per
mm2, or ~16% of PGP-labeled principal ganglionic cells,
n = 5; Fig. 9B) or insulin (16 ± 2 Fos+ per mm2; or ~12% of PGP-labeled principal
ganglionic cells, n = 6, Fig. 9D). The
injection of saline (ip) did not induce Fos expression in CG (Fig. 9,
A and C). CG Fos expression induced by 2DG was both dose (P < 0.0001) and time (P < 0.01) dependent (Figs. 10 and
11).

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Fig. 9.
Fos expression in the nuclei of principal ganglion cells of the CG
in response to the ip injection of 2-deoxy-D-glucose (2DG;
arrow in B), insulin (arrow in
D), or saline (A and C). Scale bar, 72 µm.
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Fig. 10.
Fos expression in the nuclei of principal ganglion cells
of the CG in response to the ip injection of saline (n = 3) and 2DG given at doses of 100 (n = 7), 200 (n = 6), 400 (n = 8), and 800 (n = 7) mg/kg.
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Fig. 11.
Fos expression in the nuclei of principal ganglion cells
of the CG immediately and 1-8 h after 2DG (800 mg/kg ip).
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DISCUSSION |
We sought to establish a new method for quantifying sympathetic
neuronal activation that was applicable to small, conscious animals.
Therefore, we investigated the use of nuclear Fos expression as an
index reflecting the activation of sympathetic ganglionic neurons. To
this end, we activated principal ganglionic neurons by stimulating
their nicotinic receptors with either exogenous nicotine or endogenous
acetylcholine, the latter released by either electrical or reflexive
activation of preganglionic neurons. The degree of neuronal activation
was estimated by the number of Fos-positive neurons per square
millimeter of ganglion. The percentage of principal ganglionic cells
expressing Fos was also estimated by dividing by the number of PGP
9.5-positive cells per square millimeter. Our results demonstrate Fos
expression in the CG in response to either nicotine or preganglionic
nerve stimulation. Finally, to judge whether such Fos expression could
be induced by reflexively increasing central sympathetic outflow, we
injected either the neuroglucopenic agent 2DG or the hypoglycemic agent
insulin. Either agent induced a similar degree of Fos expression in a
subset of CG neurons. We also documented the dose-response
characteristics of 2DG-induced Fos expression, demonstrating the
quantitative nature of this index of sympathetic activity and its
sensitivity to detect sympathetic activity in response to more
physiological glucopenia. We found a wide range of Fos expression in
response to these various stimuli. We assume that this wide range of
Fos expression is due to activation of either different populations of
CG neurons or different numbers of nicotine receptors on the same
population of CG neurons.
Nicotine induces Fos expression in SCG.
To confirm our ability to detect Fos expression in the nuclei of
principal ganglionic cells and to confirm that its expression increases
when sympathetic postganglionic neurons are activated, we quantified
Fos expression in the SCG in rats treated with either subcutaneous
nicotine or saline, a model used previously to induce Fos expression in
rat peripheral sympathetic ganglia (19). We found
significant expression of Fos in nuclei of certain large principal
ganglionic cells of the rat SCG 2 h after treating them with a
single, subcutaneous injection of nicotine. In contrast, negligible Fos
staining was detected in the SCG of rats treated with subcutaneous
saline. The specificity of the Fos staining seen in nicotine-treated
rats was confirmed by preincubating the first antibody with the peptide
fragment of Fos used as the immunogen for antibody production; this
preincubation completely blocked the Fos staining (data not shown).
Although subcutaneous nicotine administration did induce Fos expression
in the SCG, the amount of Fos expression observed in our study appeared
to be less than that in previous reports (19, 39).
Potential limitations of our staining method are unlikely to be
responsible for this lower Fos expression, because our method detects
much higher Fos expression in another sympathetic ganglion, the celiac,
in response to more potent stimuli (see below).
Nicotine induces Fos expression in the CG.
Neuronal activation of the CG mediates sympathetic outflow to the liver
and pancreas, thereby stimulating hepatic glucose production and
pancreatic glucagon secretion. To investigate whether Fos expression
also occurs in the CG neurons in response to nicotine stimulation, we
analyzed Fos responses to subcutaneous nicotine in the CG. As in the
SCG, Fos was expressed in CG neurons in response to subcutaneous
nicotine. The immunoreactive Fos was located in large, round nuclei in
the center of ganglionic cells. These cells stained positive for PGP
9.5, a neuronal marker, confirming that they were postganglionic
sympathetic neurons, and not modulating neurons such as small intensely
fluorescent cells (17) or supporting cells such as
satellite or Schwann cells.
Although subcutaneous nicotine administration produced some Fos
expression in CG nuclei, only 12% were Fos positive. Presumably, all
principal ganglionic neurons express nicotinic receptors
(36) and are therefore capable of being activated by
nicotine, if the concentration of nicotine is high enough. To expose
the CG to higher concentrations of nicotine while avoiding the toxicity that occurs with higher systemic doses, we applied nicotine topically to the ganglion by injecting it underneath the fascia surrounding the
CG. The number of cells expressing Fos in response to this topical
nicotine application was higher (52%) than that to subcutaneous nicotine administration. We assume that this greater expression is due
to activation of more nicotinic receptors in response to the higher
local nicotine concentration. Thus it is probable that subcutaneous
administration of nicotine achieved local nicotine concentrations that
were not sufficient to activate all of the nicotinic receptors on the
principal ganglionic cells of the CG. Furthermore, these data suggest
that principal ganglionic cells may have different thresholds of
activation if, in fact, Fos expression, like neuron firing, is an
all-or-none phenomenon.
Nerve stimulation induces Fos expression in the CG.
Although the studies above showed that nicotine administration induces
CG Fos expression, they did not address the question of whether
endogenous acetylcholine, released during the activation of
preganglionic nerve fibers, is also capable of stimulating the
postganglionic neurons to the degree necessary to express detectable
Fos. To answer this question, we electrically stimulated the
preganglionic nerve fibers of the CG. Nerve stimulation did induce CG
Fos expression. In addition, the percentage of Fos-positive staining
cells was very high (94%), suggesting that the amount of endogenous
acetylcholine released by electrical nerve stimulation is sufficient to
activate nearly all principal ganglionic cells. Furthermore, comparison
of the percentage of Fos-positive staining cells in animals treated
with subcutaneous nicotine, topical nicotine, and nerve stimulation
suggests a graded response. The studies with various doses of 2DG (see
below) confirm a graded response of Fos expression.
Fos expression in response to 2DG or insulin injection.
Although Fos expression in rat CG could be induced chemically and
electrically, further studies were needed to demonstrate that Fos
expression occurred in the CG in response to an increase of central
sympathetic outflow. Therefore, to determine whether a reflexively
induced increase of the central sympathetic outflow to the pancreas
(15) and liver (28) can induce the CG Fos expression, we injected either the neuroglucopenic agent 2DG or the
hypoglycemic agent insulin. We found a similar increase of Fos
expression in the CG 2 h after either injection. Further studies with 2DG indicated that this Fos expression was both dose and time
dependent (see below). Together, these data suggest that Fos expression
in the CG may be a useful index of reflex activation of the sympathetic
neurons projecting to the pancreas and liver, although retrograde
tracing experiments to define the target organ of these activated CG
neurons would be necessary to prove this hypothesis.
Time course and dose-response characteristics of 2DG-induced Fos
expression.
We found that the number of neurons expressing Fos after 2DG
administration was maximal 2 h after the injection. This finding is consistent with the time course of Fos expression in the SCG in the
response to nicotine (19). Interestingly, the number of
neurons expressing Fos in response to 2DG did not wane as quickly as
that in the SCG in response to nicotine (19). This
difference between the two studies in the rate of decline of the number
of neurons expressing Fos may be due to the sustained intracellular neuroglucopenia induced by 2DG (28) vs. the transient
neuronal stimulation produced by nicotine. The faster decrease of Fos
expression with time in the studies of Koistinaho (19) and
Yang and Koistinaho (39) using nicotine may more closely
reflect the intracellular half-time of the Fos protein.
The number of neurons expressing Fos was also found to be dependent on
the dose of 2DG. These data suggest that Fos may be a useful index for
documenting the degree of sympathetic neural activation induced by
varying levels of glucopenia. Furthermore, the data suggest that more
moderate and therefore more physiological levels of glucopenia can
activate these sympathetic neurons.
Physical manipulation induces Fos expression in principal and
nonprincipal ganglionic cells.
Two control experiments, the injection of saline underneath the fascia
surrounding the CG (i.e., topical saline) and the simple isolation of
preganglionic nerve trunks (i.e., nerve isolation), also produced
moderate Fos expression in principal CG neurons. This moderate Fos
expression contrasts with the virtual absence of CG Fos expression in
control rats receiving either subcutaneous or intraperitoneal saline.
Because both topical saline injection and preganglionic nerve isolation
require general anesthesia, a laparotomy, and physical manipulation of
the CG or its attached nerve trunks, we speculated that these
potentially stressful factors could account for the moderate Fos
expression observed in these two groups. Because surgical stress
(anesthesia plus laparotomy) induced only minor Fos expression, the
moderate Fos expression in topical saline and nerve isolation was due
mainly to direct physical manipulation of the CG or its nerve trunks.
Nonetheless, the number of CG neurons expressing Fos in response to
topical nicotine and nerve stimulation was significantly greater than the moderate level seen in these respective controls.
In addition to the Fos expression in principal ganglionic cells induced
by nerve isolation or topical saline, we found Fos expression in the
much smaller cells. These small, irregularly shaped cells did not stain
for PGP 9.5, suggesting that they are nonneuronal and therefore may be
satellite and/or Schwann cells (33). Because the Fos
staining in these nonneuronal cells was also blocked by preincubation
of the first antibody with Fos peptide, it appeared to be specific Fos
staining rather than a nonspecific staining artifact. Because the Fos
expression in these nonneuronal cells was not observed in rats injected
with either subcutaneous or intraperitoneal saline, we suspect that
their Fos expression was due mainly to the mechanical manipulation of
the CG or its preganglionic nerves. The functional implication of Fos
expression in nonprincipal ganglionic cells is not clear, but others
have suggested that it is related to nerve damage (32).
Comparison of indexes of sympathetic neural activity.
In the present study, we have introduced Fos expression in the
principal ganglionic cells of the CG as an index of abdominal postganglionic, sympathetic neural activation. This new index circumvents some limitations of the other, older indexes. For example,
measurements of tissue norepinephrine turnover are limited to use in
situations of chronic sympathetic activation or suppression (41,
42) of a few days or weeks in duration. In contrast, as
illustrated by the data we present here, Fos expression can reflect
acute, transient sympathetic activation such as that produced by
nicotine or nerve stimulation. Norepinephrine spillover, another index
(9, 15), can also reflect acute changes in sympathetic activity, yet it requires access to the organ's venous drainage and
estimates of organ blood flow and norepinephrine extraction (1), which effectively limits its use to large species.
Fos expression, on the other hand, can be employed in both large and small species. The recording of neural firing rate, which is a useful
index of both acute and chronic changes of sympathetic activity
(6, 16, 40), has usually required anesthesia, which may
itself suppress the neural activity being measured. Furthermore,
additional experimental techniques are required to distinguish afferent
from efferent neural firing rate. In contrast, Fos expression is
induced in conscious animals and requires anesthesia only for the
fixation and harvest of the ganglia. Furthermore, the anatomic site and
the cell morphology define the neurons as postganglionic and sympathetic.
Although the use of Fos expression as an index of sympathetic
activation circumvents some limitations of the older indexes, it has
its own limitations. Most importantly, this index alone cannot define
the target tissue innervated by the Fos-positive neurons, because
sympathetic ganglia project postganglionic neurons to a variety of
organs. Therefore, retrograde labeling of tissue-specific neurons
(21) must be employed before the Fos staining observed can
be ascribed to activation of the sympathetic neurons of a particular
organ. However, because this dual staining is possible (23), Fos expression in ganglionic neurons is likely to be
a useful tool in describing acute, tissue-specific changes in
sympathetic activity in small laboratory animals, including the rat and
perhaps the mouse. A second limitation is the time delay between
sympathetic stimulation and peak nuclear Fos expression: as we have
demonstrated here, one must wait 2 h for the processes of
transcription, translation, and nuclear translocation of the Fos
protein to be maximal. Furthermore, as also demonstrated here, nuclear
Fos is cleared slowly (5), making it difficult to resolve
repeated episodes of sympathetic stimulation. A third limitation is
that mechanical manipulation of sympathetic ganglia can apparently
induce some Fos expression; therefore, experimental procedures must be
designed to minimize manipulation of the ganglion itself. Although this
"mechanical" stimulation of the neurons may interfere with
detection of a more physiological activation of these neurons, it
likely reflects a real increase in the local sympathetic activity.
Finally, basal sympathetic tone may not be reflected in Fos expression,
as illustrated by the virtual lack of Fos staining in rats treated with
saline. Therefore, this index will probably not be useful for
documenting acute decreases in sympathetic tone. Despite these
limitations, we think that Fos expression in the CG has potential as an
index of acute increases in sympathetic outflow to a variety of
specific tissues, including our two organs of interest, the pancreas
(15) and liver (28).
Summary.
We describe the application of an immunohistochemical technique to
detect Fos expression in the nuclei of principal ganglionic neurons of
the CG as an index of the activation of postganglionic sympathetic
neurons in response to nicotine administration, preganglionic nerve
stimulation, and 2DG or insulin. The data demonstrated a significant
increase of nuclear Fos expression in principal ganglionic cells of the
CG in response to all of the stimuli. The graded Fos response to 2DG
suggests that Fos expression in the CG may be a useful index of the
degree of physiological activation of CG neurons, including those
sympathetic neurons that project to the pancreas and modulate islet
hormone secretion (2) and those that project to the liver
and modulate hepatic glucose production (29). The present
study, along with previous reports (20), suggests that
this method may be applicable to any peripheral sympathetic ganglia and
that it can be a quantitative index of the sympathetic input to their
target tissues.
 |
ACKNOWLEDGEMENTS |
We thank Dr. F. Sundler for the kind gift of PGP 9.5 antibody. We
also thank Drs. J. Kostinaho, G. Hoffman, and S. Ritter for their help
and encouragement early in this project. We thank C. Vathanaprida, H. Tran, and D. Winch for immunohistochemical staining, figure processing,
and preliminary Fos antibody testing.
 |
FOOTNOTES |
This research was supported by the Juvenile Diabetes Foundation
(3-2000-711), the Medical Research Service of the Department of Veterans Affairs, and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-12829, DK-12047, and DK-50154.
Address for reprint requests and other correspondence: Q. Mei,
Division of Endocrinology and Metabolism (151), Veterans Affairs Puget
Sound Health Care System, 1660 S. Columbian Way, Seattle, WA 98108 (E-mail: qmei{at}u.washington.edu).
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. Section 1734 solely to indicate this fact.
Received 5 October 2000; accepted in final form 22 May 2001.
 |
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