Department of Neuroscience, College of Medicine, Ohio Sate University, Columbus, Ohio 43210
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ABSTRACT |
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Tumor necrosis factor- (TNF-
) is liberated as
part of the immune response to antigenic challenge, carcinogenesis, and
radiation therapy. Previous studies have implicated elevated
circulating levels of this cytokine in the gastric hypomotility
associated with these disease states. Our earlier studies suggest that
a site of action of TNF-
may be within the medullary dorsal vagal complex. In this study, we describe the role of TNF-
as a
neuromodulator affecting neurons in the nucleus of the solitary tract
that are involved in vago-vagal reflex control of gastric motility. The results presented herein suggest that TNF-
may induce a persistent gastric stasis by functioning as a hormone that modulates intrinsic vago-vagal reflex pathways during illness.
cytokines; brain; gastric motility; tumor necrosis factor-
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INTRODUCTION |
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CYTOKINES ARE
RELEASED BY activated macrophages and lymphocytes as part of the
immune response to antigenic challenge. Elevation of tumor necrosis
factor- (TNF-
) in the systemic circulation has been correlated
with anorexia, nausea, vomiting, and gastrointestinal stasis (14,
15), which could imply an immune to nervous system communication
during illness. Recent evidence suggests that the nucleus of the
solitary tract (NST) in the medulla oblongata may be one locus for
TNF-
action to control gastrointestinal function (12,
13).
The solitary nucleus receives a host of gastrointestinal mechano- and chemosensory information via the vagus nerve (1, 25). General visceral afferent signals in the vagus uniformly excite second-order NST neurons, which, in the main, inhibit dorsal motor nucleus (DMN) neurons that provide tonic excitatory vagal input to the stomach (25, 30). This vago-vagal reflex (i.e., the accommodation reflex) control of the stomach involves a negative feedback signal produced by the NST. Additionally, the dorsal vagal complex (DVC) possesses the characteristics of a circumventricular organ and is devoid of the blood-brain barrier (3, 8, 29). Previous anatomical work (24, 28) has also shown that dendrites of the NST and DMN enter the area postrema, a well-established chemosensory structure. Thus the NST may be in a position to monitor blood-borne and cerebrospinal fluid-borne factors (25). Indeed, previous work in our laboratory has shown that neurons of the NST and the DMN are subject to modulation by the peptide hormones peptide YY and pancreatic polypeptide. These peptides are produced peripherally yet have specific sites of action within the DVC (5, 19).
The brain stem has been shown to have a high density of TNF--binding
sites (16). Given the anatomical characteristics of the
DVC and the precedence of peptide hormones modulating centrally mediated gastrointestinal function, it was hypothesized that the NST
may be the site of TNF-
action as well.
Our previous studies (13) have demonstrated that
endogenous production of TNF- in response to intravenous
administration of the bacterial cell wall component lipopolysaccharide
(LPS) is sufficient to suppress gastric motility. Our earlier study (12) demonstrated that TNF-
injected unilaterally in
the DVC abolished a thyrotropin-releasing hormone-stimulated vagally
dependent increase in gastric motility in a dose-dependent
manner. The rapid onset of the centrally injected TNF-
effect on gastric motility, i.e., within 30 s of application,
suggested that TNF-
could be directly modulating the firing rate of
neurons in the NST and/or the DMN. Preliminary studies by our
laboratory show that expression of the immediate-early gene product
c-Fos in the NST in response to LPS challenge is significantly elevated
90 min after either intravenous or intraperitoneal injection. This
increase in Fos production is independent of an intact vagal pathway
(6).
Because TNF- powerfully inhibits gastric motility when placed in the
DVC, we hypothesized that this peptide would activate NST neurons
involved in the accommodation reflex. In this way, TNF-
would
suppress gastric motility and tone by mimicking the effects of the
activation of gastrointestinal afferents. Therefore, in the present
study, NST neurons that form the sensory portion of the gastric
accommodation reflex were identified using neurophysiological methods
described by McCann and Rogers (18) and Zhang et al. (30) and were exposed to microinjection of TNF-
.
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MATERIALS AND METHODS |
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Chemicals.
Animals were anesthetized with thiobutabarbitol (Inactin; Sigma
Chemicals, St. Louis, MO) dissolved to a concentration of 100 mg/ml in
saline solution (0.9% NaCl) and administered at a dose of 100 mg/kg
body wt ip. A 2 M NaCl solution was used to record extracellular
neuronal potentials. Neurobiotin (2%; Vector, Burlingame, CA) was
included in the recording pipette to iontophoretically mark the
recording site. PBS (124 mM NaCl, 26 mM NaHCO3, and
2 mM KH2PO4; 304 mosmol; pH = 7.4) was
used as a vehicle injection control. Recombinant rat TNF- (R&D
Systems, Minneapolis, MN) was dissolved in PBS to a concentration of
10
6 M, divided into 25-µl aliquots, and stored at
70°C until use. Stored aliquots of TNF-
were further diluted
with PBS such that the microinjection electrode contained
10
8, 10
9, or 10
10 M TNF-
.
Neurobiotin-injected brain stem slices were reacted with Vector ABC and
SG reagents (Vector) to visualize the marked recording site.
Electrode construction.
Triple-barrel glass micropipettes were constructed such that one
pipette was used for recording (tip diameter of ~1 µm), whereas the
other two were available for drug delivery (combined tip diameter of
10-20 µm). The recording pipette was filled with 2 M NaCl and 2% Neurobiotin. The other pipettes were filled with either PBS or
TNF- in PBS. The electrode array was placed in a stereotaxic electrode carrier, which was oriented at an ~20° rostral angle. The
injection pipettes were connected to a micropressure injection apparatus as described in Ref. 5.
Data collection.
Extracellular neuronal activity was recorded via a silver-silver
chloride wire placed in the recording pipette. Extracellular neuronal
potentials from the recording electrode were amplified (×10,000) and
band-pass filtered (300-10,000 Hz). Signals were then displayed on
an oscilloscope and recorded on magnetic tape. Extracellular spike
potentials were also processed on-line by a window discriminator-rate
meter circuit. The resulting neuronal firing rate (FR) along with the
gastric distension stimulation signal were displayed on a chart
recorder. Figures 1 and 2 were generated by processing the recorded
data with an IBM-PC-based RC Electronics (Goleta, CA)
waveform analysis system.
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Gastric stimulation. A small gastric stimulation balloon was constructed as described previously (18). The balloon catheter was connected to one port of the dome of a Statham P23 pressure transducer. The balloon was fully inflated by injecting 3 ml of air into the other port with a 5-ml syringe (18). The gastric distension signal was transmitted from the pressure transducer to the polygraph and magnetic tape.
Surgical preparations. Male Long-Evans rats (Charles River) weighing 200-600 g were provided with food and water ad libitum and were kept on an ~12:12-h day-night cycle. Before each experiment, the animal was food deprived for ~18 h. The animal was deeply anesthetized with Inactin, and the trachea was cannulated to maintain an open airway. The gastric balloon was secured in the antrum of the stomach according to a previous protocol (18). The animal was placed in a stereotaxic frame, and the dorsal surface of the brain stem was exposed as described previously (18). All experimental protocols were performed according to guidelines set forth by the National Institutes of Health and were approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee.
Experimental design. The pipette array was advanced into the DVC in search of NST neurons responsive to gastric distension. Initial stereotaxic coordinates were 0.3 mm anterior to the calamus scriptorum and 0.3-0.5 mm lateral to the midline of the area postrema (18). A hydraulic microdrive (David Kopf Instruments, Tujunga, CA) was used to advance the array at 10-µm increments. At each electrode advancement through the medullary brain stem, the gastric balloon was momentarily inflated and deflated. Neurons briskly activated by gastric distension were located between 250 and 600 µm below the brain stem surface.
Once a cell was identified as a gastric inflation-related NST neuron, PBS (3 nl) was micropressure injected from the attached pipette, and the spontaneous activity was monitored for at least 5 min. If neuronal activity was altered by PBS microinjection (as a consequence of nonspecific volume effects), it was rejected from further consideration. If PBS injection had no effects on the FR of the neuron, the same volume of TNF-Statistical analysis.
The effect of TNF- on NST firing was examined by comparing the peak
FR after TNF-
injection with basal FR (i.e., post-PBS). FR
comparisons were made at 1-min epochs for 1 min before and 1, 2, 3, and
4 min post-PBS or post-TNF-
injection using a repeated-measures one-way ANOVA followed by Dunnett's posttest. Time of activation, i.e., the duration that the neuron produced action potentials at a rate
>50% above basal FR, was analyzed by a one-way ANOVA and a post hoc
Dunnett's test. Potentiation by TNF-
of NST responsiveness to
gastric distension was evaluated by comparing the peak FR after inflation in neurons previously exposed to TNF-
with the peak FR
after inflation following PBS. Comparisons were made (i.e., pre-TNF-
vs. post-TNF-
of a given neuron) at 1-min epochs after inflation by
using paired t-tests. Statistical significance required P values <0.05.
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RESULTS |
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NST neurons located between 250 and 600 µm below the brain stem
surface were neurophysiologically identified and exposed to PBS and one
of three concentrations of TNF-; a representative injection site is
included in Fig. 1G. Injection
of TNF-
from the pipette array typically increased neuronal firing
in the NST within 30 s of administration (Fig. 1D).
Duration of activation varied with TNF-
dose (0.03 fmol = 8.8 ± 2.1 min; 0.003 fmol = 2.4 ± 1.3 min).
Dose dependency of NST firing on TNF-.
A dose of 0.03 fmol of TNF-
provided the ideal stimulus in that all
identified neurons (N = 14 from 11 animals) subjected to this dose responded to TNF-
within 30 s of application, all were activated by TNF-
, and all recovered from TNF-
-induced activation (Fig. 1). If the dose was increased 10-fold (0.3 fmol), NST
neurons did not recover from excitation (data not shown). If the dose
was decreased 10-fold (0.003 fmol), a more subtle activation by TNF-
was documented. At this dose, approximately one-half (4:10 from 7 animals) of the neurons elicited a response, implying that the lower
dose of 0.003 fmol is close to the ED50 for TNF-
to
elicit changes in NST FR. Decreasing the TNF-
dose to 0.0003 fmol
had no effect on NST neuronal firing (data not shown). In contrast, a
dose of 0.03 fmol elicited a significant increase in neuronal FR at 1, 2, 3, and 4 min after microinjection (Fig.
2A) as well as an activation
time that was significantly greater than in controls in all neurons tested.
Potentiation of NST response to gastric stimulation.
NST cells exhibit a stereotypic response to gastric distension
stimulation (Fig. 1, A and B). That is, NST
neurons demonstrate an increased FR tightly locked to gastric balloon
inflation. Once mechanoreceptor activation is terminated, NST neurons
quickly return to basal FRs (18, 30). Activation by
TNF- altered this relationship in 6 of 14 NST neurons so tested at
0.03 fmol. After these neurons recovered from initial activation after
TNF-
microinjection, i.e., return to basal FR, subsequent balloon
inflation triggered prolonged activation (Figs. 1E and
2B). The potentiation response ranged from 1 to 7 min in
duration and could be elicited for up to 90 min after recovery from
exposure to TNF-
.
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DISCUSSION |
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Our previous study (12) demonstrated that injecting
subfemtomolar amounts of TNF- into the DVC suppressed centrally
stimulated gastric motility. The present study showed that identified
gastric NST neurons are excited by TNF-
. The observation that
TNF-
can affect the sensory component of this vago-vagal reflex
circuit may account for the rapid and large reductions in gastric
motility observed in our previous studies (12). The
principal action of NST neurons activated by gastrointestinal
distension appears to be the inhibition of DMN neurons providing
excitation to the stomach (18, 23, 30). Therefore, the
activation of gastric-related neurons by TNF-
would be expected,
given the potent central nervous system (CNS) effect of TNF-
to
suppress gastric motility via a vagal efferent route (12).
Preliminary results from our laboratory show that DMN neurons appear to
be inhibited by TNF-
, perhaps as a consequence of inhibitory input
from the NST (6).
It is likely that TNF- generated as a consequence of infection or
other proinflammatory processes inhibits gastric motility by directly
affecting the sensitivity of gastric vagal control circuitry in the
medulla. This vago-vagal reflex circuit is outside the blood-brain
barrier and therefore is readily accessible to large circulating
peptides (3, 8, 29). Additionally, TNF-
increases
vascular-brain permeability (17, 20), and TNF-
may gain
access to the brain through a specific transport system (9).
The results from the present study support the proposition that TNF-
alters autonomic function as a consequence of direct peptide action on
regulatory circuits in the medulla. Other routes for TNF-
modulation
of brain stem autonomic control are possible. Studies by Sehic and
Blatteis (27) indicate a role of the afferent vagus in the
transduction of information about systemic cytokine production, which
results in the onset of fever. However, this pathway seems to be
specific for the detection of cytokines in the abdominal cavity and is
not involved in the detection of cytokines in the circulation
(27). Results that suggest that the afferent vagus is
involved in the initiation of cytokine-evoked visceral aversion
behavior (2) are controversial. Schwartz et al.
(26) have shown that cytokine-induced visceral aversion
behavior is not altered by specific afferent vagotomy. These authors
concluded that cytokine-induced behavioral aversions must be produced
as a consequence of direct action in the CNS.
The cellular mechanisms by which TNF- rapidly changes NST
excitability are not known. Two types of TNF-
receptors (TNFR) have
been identified, the P75 TNFR and the P55 TNFR (4, 11). Although the neuronal distributions of these receptors have not yet
been characterized (21), the highest density of
TNF-
-binding sites is in the medullary brain stem (16).
Studies of P55 transduction mechanisms in other cells show that this
receptor can rapidly activate (within 1-2 min) a sphingomyelinase
pathway that produces ceramide (10, 22). Intracellular
ceramides can, in turn, increase intracellular calcium levels and
regulate protein kinase cascades, which can modulate properties of
membrane ion channels (7), and may be involved in the
augmented response to afferent stimulation. However, very little is
presently known about the specifics of how cytokines can rapidly alter
the excitability of neurons.
In summary, this study shows that NST neurons are strongly activated by
TNF-. Sensory afferent activation of NST neurons produces
gastroinhibition as a consequence of their action on vagal motor
neurons. Therefore, our study suggests that centrally acting TNF-
probably produces gastroinhibition by activating NST neurons. Thus in
states of elevated circulating levels of TNF-
, sensory activation of
NST neurons may more readily evoke gastroinhibition.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52142 to R. C. Rogers and G. E. Hermann.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. C. Rogers, Ohio State Univ., 4197 Graves Hall, 333 W. Tenth Ave., Columbus, OH 43210 (E-mail: Rogers.25{at}osu.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. §1734 solely to indicate this fact.
Received 24 January 2000; accepted in final form 5 April 2000.
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