TNF-alpha activates solitary nucleus neurons responsive to gastric distension

Gregory S. Emch, Gerlinda E. Hermann, and Richard C. Rogers

Department of Neuroscience, College of Medicine, Ohio Sate University, Columbus, Ohio 43210


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor-alpha (TNF-alpha ) 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-alpha may be within the medullary dorsal vagal complex. In this study, we describe the role of TNF-alpha 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-alpha 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-alpha


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYTOKINES ARE RELEASED BY activated macrophages and lymphocytes as part of the immune response to antigenic challenge. Elevation of tumor necrosis factor-alpha (TNF-alpha ) 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-alpha 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-alpha -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-alpha action as well.

Our previous studies (13) have demonstrated that endogenous production of TNF-alpha 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-alpha 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-alpha effect on gastric motility, i.e., within 30 s of application, suggested that TNF-alpha 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-alpha 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-alpha 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-alpha .


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (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-alpha were further diluted with PBS such that the microinjection electrode contained 10-8, 10-9, or 10-10 M TNF-alpha . 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-alpha 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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Fig. 1.   Effects of gastric distension, PBS, and tumor necrosis factor-alpha (TNF-alpha ) microinjection on a single neuron from the nucleus of the solitary tract (NST; A-G are the same neuron). A: raw oscillograph record of the identification of a gastric distension-related neuron in the NST. Antral distension (bottom) produces a brisk increase in firing rate (FR) that is phase locked to the stimulus. Time scale bar = 5 s. B: integrated rate-meter record of the same event as A. Inset: 20 superimposed NST spikes. At lower time scale, bar = 10 s; inset scale bars = 200 µV/2.5 ms. C: control injection of PBS (3 nl at arrow) has no effect on FR; same neuron as in B. Inset: 20 superimposed NST spikes showing that the counted event is unitary; same waveform as the spike in B can be seen. At lower time scale, bar = 1 min; inset scale bars = 200 µV/2.5 ms. D: effect of TNF-alpha (0.03 fmol in 3 nl) injected on the NST neuron. At lower scale, bar = 1 min; inset scale bars = 200 µV/2.5 ms. E: effect of gastric distension on the NST unit 30 min after exposure to and recovery from the TNF-alpha injection in D. Note greatly potentiated response that significantly outlasts the stimulus. At lower time scale, bar = 10 s; inset scale bars = 200 µV/2.5 ms. F: drawing depicting summary of Neurobiotin-labeled recording sites. Marked area represents the spread of all NST neurons recorded in the study. Scale bar = 0.3 mm. AP, area postrema; mNST, medial solitary nucleus; dmn, dorsal motor nucleus of the vagus; st, solitary tract. G: micrograph of the Neurobiotin injection site verifying the neuron's location in the medial NST (arrow). Scale bar = 0.3 mm. cc, Central canal.



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Fig. 2.   Dose-dependent effect of TNF-alpha on NST FR and potentiation of neuronal response to gastric distension. Each bar represents the FR in pulses per second (pps) compiled in 1-min epochs. A: dose-dependent NST response to TNF-alpha was very steep. NST neurons exposed to 0.3 fmol TNF-alpha did not recover from excitation (data not shown). Decreasing the TNF-alpha dose to 0.0003 fmol had no effect on NST neuronal firing (data not shown). Top: application of 0.003 fmol TNF-alpha does not induce a significant increase in NST neuronal FR at 1, 2, 3, and 4 min postinjection. Bottom: application of 0.03 fmol TNF-alpha induces a significant increase in NST FR at all times sampled after TNF-alpha microinjection compared with PBS. * P < 0.01 from Dunnett's test. B: TNF-alpha potentiation of neuronal response to gastric distension. FR sampled at 1, 2, and 3 min after gastric distension is not different from 1 min predistension in neurons exposed to PBS. After exposure to TNF-alpha , gastric distension triggered prolonged activation after inflation was terminated. FR at 1 min postdistension in TNF-alpha neurons was significantly greater than FR at 1 min postdistension in PBS neurons. * P < 0.05, paired t-test.

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-alpha was then injected (e.g., 3 nl of 10-8 M TNF-alpha was delivered for a total dose of 0.03 fmol). This dose of TNF-alpha delivered to the NST is also the threshold dose for TNF-alpha inhibition of gastric motility when applied to the DVC (12). With the use of this dose as a reference point, additional doses of TNF-alpha were tested on other cells by increasing or decreasing the concentration 10-fold.

Cells considered responsive to TNF-alpha must demonstrate a change in FR of a minimum of 50% relative to basal levels of activity (5, 18). At the end of the recording session, iontophoretic current was applied to the recording pipette (100-1,000 nA positive direct current) to eject Neurobiotin to mark the recording site. The amount of TNF-alpha -PBS applied was verified by inspecting the movement of the meniscus with a small microscope (5). Animals were transcardially perfused with saline and 4% paraformaldehyde. The brain stems were removed, postfixed overnight, sectioned at 50 µm, and reacted with Vector ABC and SG reagents for the demonstration of Neurobiotin-labeled NST neurons (23).

Statistical analysis. The effect of TNF-alpha on NST firing was examined by comparing the peak FR after TNF-alpha 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-alpha 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-alpha of NST responsiveness to gastric distension was evaluated by comparing the peak FR after inflation in neurons previously exposed to TNF-alpha with the peak FR after inflation following PBS. Comparisons were made (i.e., pre-TNF-alpha vs. post-TNF-alpha of a given neuron) at 1-min epochs after inflation by using paired t-tests. Statistical significance required P values <0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha ; a representative injection site is included in Fig. 1G. Injection of TNF-alpha from the pipette array typically increased neuronal firing in the NST within 30 s of administration (Fig. 1D). Duration of activation varied with TNF-alpha dose (0.03 fmol = 8.8 ± 2.1 min; 0.003 fmol = 2.4 ± 1.3 min).

Dose dependency of NST firing on TNF-alpha . A dose of 0.03 fmol of TNF-alpha provided the ideal stimulus in that all identified neurons (N = 14 from 11 animals) subjected to this dose responded to TNF-alpha within 30 s of application, all were activated by TNF-alpha , and all recovered from TNF-alpha -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-alpha 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-alpha to elicit changes in NST FR. Decreasing the TNF-alpha 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-alpha altered this relationship in 6 of 14 NST neurons so tested at 0.03 fmol. After these neurons recovered from initial activation after TNF-alpha 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-alpha .


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our previous study (12) demonstrated that injecting subfemtomolar amounts of TNF-alpha into the DVC suppressed centrally stimulated gastric motility. The present study showed that identified gastric NST neurons are excited by TNF-alpha . The observation that TNF-alpha 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-alpha would be expected, given the potent central nervous system (CNS) effect of TNF-alpha 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-alpha , perhaps as a consequence of inhibitory input from the NST (6).

It is likely that TNF-alpha 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-alpha increases vascular-brain permeability (17, 20), and TNF-alpha may gain access to the brain through a specific transport system (9).

The results from the present study support the proposition that TNF-alpha alters autonomic function as a consequence of direct peptide action on regulatory circuits in the medulla. Other routes for TNF-alpha 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-alpha rapidly changes NST excitability are not known. Two types of TNF-alpha 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-alpha -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-alpha . 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-alpha probably produces gastroinhibition by activating NST neurons. Thus in states of elevated circulating levels of TNF-alpha , sensory activation of NST neurons may more readily evoke gastroinhibition.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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