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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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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Fig. 4.   PGP-stained immunoreactive cells in rat CG (arrow). Scale bar, 72 µm.

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.

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.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 281(4):E655-E664
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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