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. 2003 Oct 10;553(Pt 3):1005–1018. doi: 10.1113/jphysiol.2003.045906

The central nucleus of the amygdala modulates gut-related neurons in the dorsal vagal complex in rats

Xueguo Zhang 1, Jinjuan Cui 1, Zhenjun Tan 1, Chunhui Jiang 1, Ronald Fogel 1
PMCID: PMC2343616  PMID: 14555729

Abstract

Using retrograde tract-tracing and electrophysiological methods, we characterized the anatomical and functional relationship between the central nucleus of the amygdala and the dorsal vagal complex. Retrograde tract-tracing techniques revealed that the central nucleus of the amygdala projects to the dorsal vagal complex with a topographic distribution. Following injection of retrograde tracer into the vagal complex, retrogradely labelled neurons in the central nucleus of the amygdala were clustered in the central portion at the rostral level and in the medial part at the middle level of the nucleus. Few labelled neurons were seen at the caudal level. Electrical stimulation of the central nucleus of the amygdala altered the basal firing rates of 65 % of gut-related neurons in the nucleus of the solitary tract and in the dorsal motor nucleus of the vagus. Eighty-one percent of the neurons in the nucleus of the solitary tract and 47 % of the neurons in the dorsal motor nucleus were inhibited. Electrical stimulation of the central nucleus of the amygdala also modulated the response of neurons in the dorsal vagal complex to gastrointestinal stimuli. The predominant effect on the neurons of the nucleus of the solitary tract was inhibition. These results suggest that the central nucleus of the amygdala influences gut-related neurons in the dorsal vagal complex and provides a neuronal circuitry that explains the regulation of gastrointestinal activity by the amygdala.

The amygdala, an essential component of the brain's limbic system, plays an important role in a variety of complex behaviours including emotional response, motivation, learning, memory and feeding (Henke et al. 1991; Giraudo et al. 1998; Cardinal et al. 2002). These activities are often associated with changes in gastrointestinal tract function (Giraudo et al. 1998; Monnikes et al. 2001; Mayer & Collins, 2002). Neuronal circuits involving the central nucleus of the amygdala (CNA) and the dorsal vagal complex (DVC) may mediate the changes in gastrointestinal activity.

Anatomical studies have demonstrated a connection between the CNA and the DVC (Hopkins & Holstege, 1978; van der Kooy et al. 1984; Veening et al. 1984; Danielsen et al. 1989; Liubashina et al. 2000; Saha et al. 2000, 2002). Anterograde tract-tracing studies have revealed that efferent fibres from the CNA terminate in both the nucleus of the solitary tract (NST) and the dorsal motor nucleus of the vagus (DMNV), in regions that are involved in the regulation of gastrointestinal activity. The ipsilateral medial NST (mNST) and the subpostremal subnuclei of the NST are the primary targets of CNA axons. These areas are also targets for the primary vagal afferent fibres from the gastrointestinal tract (Shapiro & Miselis, 1985; Altschuler et al. 1989; Zhang et al. 2000). The DMNV is the origin for the majority of parasympathetic neurons that innervate the gut. It has been reported that injection of retrograde tracers into the DVC results in the appearance of labelled neurons in the CNA (Ricardo & Koh, 1978; Schwaber et al. 1980; van der Kooy et al. 1984; Veening et al. 1984; Thompson & Cassell, 1989). At present, a detail topographical distribution of CNA neurons that project to the DVC is lacking.

Physiological studies suggest that the DVC mediates the effect of the CNA on gastrointestinal function. Electrical stimulation of different regions of the amygdala induces vagal-dependent changes in gastrointestinal activities, including increased pancreatic secretion (Hahn et al. 1994), gastric acid secretion (Innes & Tansy, 1980), increased and decreased gastric motility (Henke et al. 1991; Liubashina et al. 2002) and gastric ulceration (Innes & Tansy, 1980). Electrical stimulation of the CNA changes the activity of neurons in the NST and DMNV and induces c-fos expression in NST neurons (Veening et al. 1984; Nishimura, 1987; Petrov et al. 1996; Liubashina et al. 2002). Stimulation of the CNA excites NST neurons in the rabbit (Cox et al. 1986) and inhibits the majority of rat NST neurons that respond to electrical stimulation of the cervical vagus nerve (Liubashina et al. 2002).

Although both anatomical and physiological data suggest that the DVC mediates the influence of the CNA on gastrointestinal function, we do not know whether this control is mediated by the NST alone, the DMNV alone, or both nuclei. Using a retrograde tracing method, we provide new details regarding the topographic distribution of the CNA neurons that project to the DVC. Using extracellular recording and intracellular injection techniques, we characterized the response of individual NST and DMNV neurons to CNA stimulation and gastrointestinal stimulation. These data indicate that gut-related neurons in the DVC are sensitive to stimulation of the CNA. The results provide additional information regarding the central regulation of gastrointestinal function and feeding behaviour.

METHODS

The Institutional Animal Care and Use Committee of the Henry Ford Health Sciences Center approved all of the experimental procedures.

Neuronal tract-tracing study

Five adult male Sprague Dawley rats weighing 270–350 g were used to characterize the neuronal projections from the CNA to the DVC. Animals were anaesthetized with sodium pentobarbital (50 mg (kg body weight)−1, i.p.). The degree of anaesthesia was assessed by the paw pinch reflex and muscle tone. Supplemental doses of sodium pentobarbital (25 mg (kg body weight)−1, i.p.; total volume < 100 mg (kg body weight)−1) were administered upon evidence of either a withdrawal response or increased muscle tone in response to pinching of a paw. Anaesthetized rats were placed in a Kopf stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). While in the frame, body temperature was maintained at 37 °C by a thermostatically controlled heating blanket (custom-made). The rat's head was shaved and cleaned with 70 % ethanol. A glass pipette (tip diameter 20–30 μm) was used to infuse 0.1 μl of the retrograde tracer Alexa-Fluor-555-conjugated cholera toxin subunit B (in 1 % sterile saline; Molecular Probes, Eugene, OR, USA) into the DVC at the level of the obex (0.5 mm lateral to the obex and 0.5 mm ventral to the dorsal surface of the brainstem). The surgical area was cleaned with warm sterile saline solution, the excess solution blotted with sterile cotton tips and the wound closed. The entire surgical procedure was performed under aseptic conditions.

After surgery, the animals received routine postoperative care according to our institutional protocol. Buprenorphine (Reckitt & Colman Pharmaceuticals, Richmond, VA, USA) was administered to reduce postsurgical pain (0.05 mg (kg body weight)−1, i.p.). Additional doses were given as needed (0.05 mg (kg body weight)−1, i.p.; twice daily for 2 days).

After 7–10 days, the rats were again deeply anaesthetized with sodium pentobarbital (100 mg (kg body weight)−1, i.p.) and then perfused through the heart with 500 ml of 0.9 % saline in 0.1 m sodium phosphate buffer (PB; pH 7.4, room temperature). The rinse solution was followed by a 500 ml fixative solution containing 4 % paraformaldehyde in 0.1 m PB (pH 7.4, room temperature). The brain, including the CNA and the DVC areas was removed and serial 200 μm coronal sections were collected using a M720 vibratome (Campden Instruments, Loughborough, UK). Labelled neurons in the CNA were identified and photographed using a Nikon PCM 2000 confocal microscope (Nikon, New York, USA). The injection site in the DVC was inspected. Animals were excluded from analysis if any of the label leaked beyond the borders of the DVC.

Electrophysiology protocol

Surgery

Adult male Sprague Dawley rats weighing 270–350 g were anaesthetized with sodium pentobarbital (50 mg (kg body weight)−1, i.p.). The heart rate was monitored continuously, and the level of anaesthesia was assessed as described above. Supplemental doses of sodium pentobarbital (25 mg (kg body weight)−1, i.p.) were administered following any signs of pain. The electrophysiological recording protocol requires that the animal be deeply anaesthetized with complete muscle relaxation. This level of anaesthesia was obtained using sodium pentobarbital alone. Neuromuscular blocking agents and analgesics were not required. The animal was killed at the conclusion of the experiment by an overdose of sodium pentobarbital (100 mg (kg body weight)−1, i.p.).

The surgical procedure was similar to that described previously (Zhang et al. 1995; Fogel et al. 1996). The trachea was cannulated and the animal ventilated with room air (100 strokes min−1, 2.0-2.4 cm3 tidal volume) using a Harvard 683 rodent ventilator (Harvard Apparatus, Holliston, MA, USA). Briefly, a midline abdominal incision exposed the abdominal vagus nerve, the stomach and the duodenum. A pair of Teflon-coated, pure gold wire stimulating electrodes (A-M Systems, Everett, WA, USA) was placed around the anterior and posterior branches of the subdiaphragmatic vagal nerve. The stomach and rostral intestine were cannulated separately. The abdomen was closed and the animal was placed in the stereotaxic frame.

While the animal was in the stereotaxic frame, a thermostatically controlled heating plate (custom-made) maintained the body temperature at 37 °C and warmed all perfusion fluids to body temperature. A pair of parallel stimulation electrodes was placed into the CNA at coordinates of 2.0 mm caudal to the bregma, 4.5 mm lateral to the midline and 8.5 mm ventral to the brain surface (Paxinos & Watson, 1986). The brainstem was exposed by removing the atlanto-occipital membrane and a portion of the occipital bone. A bevelled glass micropipette (A-M Systems; tip diameter 0.08–0.1 μm, resistance = 50–70 MΩ), filled with 2.0 % neurobiotin (Vector Laboratories, Burlingame, CA, USA) in 1.0 m KCl was lowered into the vagal complex between 100 μm rostral and 400 μm caudal to the obex. The brainstem was covered with 3 % agar in saline to reduce the influence of the ventilation and heart beating.

Electrophysiological recording of DVC neurons

After a 1 h stabilization period, biphasic electrical search stimuli (0.5 ms duration, 0.5–3.0 mA, 1 Hz) were delivered to the abdominal vagus by an isolated pulse stimulator (A-M Systems). The recording micropipette was advanced until a unit driven by the vagal stimulating electrode was encountered. All units driven by the stimulating electrode were tested for a response to duodenal and/orgastric distension, as described previously (Zhang et al. 1992, 1995). Action potentials were amplified by a high-input-impedance preamplifier (A-M Systems), displayed and then stored on an IBM-compatible computer using Axotape software (Axon Instruments, Foster City, CA, USA).

Neurons that were responsive to electrical stimulation of the subdiaphragmatic vagus nerve (gut-related neurons) were tested for responses to stimulation of the CNA and to gastrointestinal stimuli. The CNA was stimulated (0.2–0.4 mA, 0.5 ms, 15 Hz) for 1 min using a separate isolated pulse stimulator. The gastrointestinal distension stimulus was administered by raising the outlet tube of the stomach or intestine to 20 cm H2O above the animal. The HCl perfusion stimulus consisted of perfusing the intestinal loop with 0.1 m HCl with the efflux catheter in the plane of the animal. If the neuron showed electrophysiological stability (i.e. consistent action potential amplitude and stable basal activity) and the responses to the CNA and gastrointestinal stimuli were reproducible, we tested the response of the DVC neuron to simultaneous administration of the CNA and gastrointestinal stimuli.

DMNV neurons were identified by retrograde activation following electrical stimulation of the subdiaphragmatic vagal nerve. Criteria included a constant latency after stimulation following high-frequency electrical stimulation of the vagus nerve (75–150 Hz) and a positive collision test. NST neurons were recognized by an anterograde response that included variable latency, inability to follow high-frequency stimulation, and multiple action potentials in response to a single vagal stimulus. Occasionally, anterograde activation may be seen in a DMNV neuron that receives direct projections from the primary vagal afferents. If a neuron did not show specific properties of a DMNV or a NST neuron, we made an intracellular injection of neurobiotin. Histology was then used to locate the recorded neuron.

Intracellular injection of neurobiotin

When the location of neurons needed to be verified, the recording micropipette was advanced until the characterized neuron was impaled (passing small positive current pulses from the recording electrode facilitated this process). Penetration of the cell membrane was accompanied by a 20–40 mV drop in the voltage measured at the electrode tip, an increase in the amplitude of the action potential and a shift from a bipolar to a monopolar action potential (Renehan et al. 1995). The cells were labelled with neurobiotin by passing 2–4 nA, 250 ms positive current pulses at 2 Hz for 5–10 min. The injection was stopped if the membrane potential returned to pre-penetration levels. Receptive fields were reassessed following injection to verify that the cell characterized was the same cell that had been injected with neurobiotin. A maximum of two injections were attempted on each side.

Tissue processing

At 1–6 h following the first intracellular injection, the rats were administered a lethal dose of sodium pentobarbital (100 mg (kg body weight)−1, i.p.) and perfused through the heart with 500 ml of 0.9 % saline in 0.1 m PB (pH 7.4, room temperature). The rinse solution was followed by a 500 ml fixative containing 1 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 m PB (pH 7.4, room temperature). The brainstems were stored overnight in 0.1 m PB containing 20 % sucrose, and serial 50 μm coronal sections were cut on a sliding freezing microtome (American Optical Company, Buffalo, NY, USA). The sections were washed three times with 0.1 m PB and incubated in PB containing 1 % Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) for 1–2 h and then placed in a solution consisting of HRP-labelled avidin (Vector) in 0.4 % Triton X-100 0.1 M PB for 2 h. After three 10 min washes, a cobalt- and nickel-intensified diaminobenzidine (DAB; Sigma-Aldrich) reaction was used to visualize the HRP reaction product. The neuronal property of the recorded neuron was identified according to the morphological features (Fogel et al. 1996).

If a neuron in the DVC could not be identified as either a DMNV or a NST neuron either by their electrophysiological properties or by morphological staining, the cell was excluded from the analysis.

To verify placement of the CNA-stimulating electrode, the brain (including the CNA area) was removed and sectioned at 50 μm. The location of the stimulating electrode was documented. Only those animals in which the stimulating electrodes were located in the CNA were used.

Neurophysiological analysis

The analysis used in the present experiment is identical to that used previously (Zhang & Fogel, 2002). A 120 s trace of recorded neuronal activity was divided into four periods (30 s each). Period 1 represents basal spontaneous activity prior to the onset of stimulation. Periods 2 and 3 are the immediate and late responses to the stimulation, respectively. Period 4 represents the 30 s after the stimulus was discontinued. Five-second bins (total number of action potentials in 5 s) were used to generate a histogram and were evaluated using analysis of variance (ANOVA) with a post-hoc Bonferroni's test for multiple groups using SPSS software (SPSS, Chicago, IL, USA). Each neuron was analysed separately for a response (see our previous publication (Zhang et al. 1999) for details). Changes were considered significant only if period 2 and/orperiod 3 was statistically different from period 1 (P < 0.05).

RESULTS

Anatomical connections of the CNA and the DVC

Injection of retrograde tracer into the DVC labelled cells predominantly in the ipsilateral CNA, although some neurons were seen in the contralateral nucleus. The labelled neurons were distributed along the longitudinal axis between 1.0 and 2.8 mm caudal to the bregma. Figure 1 demonstrates the CNA distribution of the retrogradely labelled neurons. The majority of labelled neurons were located at the level of the paraventricular hypothalamic nucleus (PVN). Rostral to the PVN, labelling was heavier in the central than in the peripheral part of the CNA (Fig. 1A–E). Caudal to the PVN, labelled neurons were clustered in the medial CNA. The lateral region of the CNA had scattered labelled neurons (Fig. 1F–J). Few labelled neurons were present in the caudal CNA, but labelled fibres were prevalent, suggesting that fibres from the CNA pass through the caudal part of the CNA on the way to the DVC (Fig. 1J). No labelling was detected in the nuclei adjacent to the CNA. Figure 1K demonstrates the morphology of the labelled neurons at a higher magnification and Fig. 1L shows the injection site in the DVC.

Figure 1. Injection of the retrograde tracer Alexa-Fluor-555-conjugated cholera toxin subunit B into the left DVC produced retrogradely labelled neurons in the left CNA.

Figure 1

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These pictures were taken from 200-μm-thick brain slices with the aid of a confocal microscope (montage of confocal series). There was a transitional change from the rostral CNA to the caudal CNA (A–J). In the rostral CNA, the labelled neurons were in the central part of the CNA. The labelled neurons were generally clustered in the medial CNA at its middle level. The level of sections relative to the bregma is marked on the right upper corner. K, morphology of the labelled neurons at a higher magnification from the level of 2.2 mm caudal to the bregma. L, the site of injection of the retrograde tracer in the DVC. Both the DMNV and the NST were involved, but the nearby structures were not contaminated.

Neurophysiological study

We characterized 231 neurons that were spontaneously active in the DVC. Of the 231 neurons, 148 cells were located in the DMNV and 83 in the NST. Sixty DMNV neurons were injected intracellularly and 54 were recovered. Thirty-one NST neurons were injected and 23 recovered. NST neurons had smaller cell bodies and the dendrites were limited in the NST. Figure 2A shows a micrograph of an NST neuron.

Figure 2. Intracellularly labelled neurons from the NST (A) and the DMNV (B).

Figure 2

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A, an NST neuron that was recorded from and then injected intracellularly with neurobiotin. The neuron was located 50 μm caudal to the obex in the NST, an area where the efferent fibres from the CNA terminated. The neuron was inhibited by CNA stimulation and excited by intestinal application of HCl. B, the morphological features and position of a DMNV neuron (composed from multiple sections). The neuron was located in the middle column of the DMNV, 300 μm caudal to the obex. The dendrites reach all parts of the NST and a single axon exits the brainstem at the ventrolateral border. The neuron was excited by CNA stimulation and inhibited by intestinal distension.

DMNV neurons had three or more dendrites that extended into the ipsilateral NST and/orcontralateral NST. A single axon from the cell body projected to the ventrolateral medulla. An example of a DMNV neuron is shown in Fig. 2B.

Response of gut-related NST neurons to electrical stimulation of the CNA

We characterized the response of 83 NST neurons to electrical stimulation of the CNA and to gastrointestinal stimulation. The effects of gastrointestinal distension were similar to those reported previously (Zhang et al. 1995). Intestinal distension inhibited five neurons and excited 39 neurons. Gastric distension inhibited 12 neurons and excited 32 neurons. Twenty-seven NST neurons were affected by both gastric and intestinal distension. Electrical stimulation of the CNA changed the spontaneous activity of 54 neurons (65 % of NST neurons). The mean firing rate of the 83 NST neurons was decreased by 50 % in period 2 and by 48 % in period 3 (Fig. 3A). The spontaneous activity returned to the prestimulus level immediately after the termination of amygdala stimulation.

Figure 3. Mean responses of NST neurons to electrical stimulation of the CNA.

Figure 3

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Electrical stimulation of the CNA significantly inhibited the mean activity of NST neurons that responded to CNA stimulation (A). Among 54 NST neurons affected by CNA stimulation, 44 were significantly inhibited (B) and 10 were significantly excited (C). The mean basal activity of NST neurons inhibited by CNA stimulation was significantly higher than that of NST neurons excited by CNA stimulation (D).

The neurons that responded to CNA stimulation could be divided into two groups. Cells that were inhibited by CNA stimulation accounted for 81 % (44 out of 54 neurons) of those affected by CNA stimulation, whereas the remainder were excited by CNA stimulation. Figure 3B shows the mean response profile of NST neurons that were inhibited by CNA stimulation and Fig. 3C illustrates the mean response of the 10 NST neurons that were excited by CNA stimulation. Group analysis revealed that the mean (±s.e.m.) spontaneous firing rate of NST neurons that were excited by CNA stimulation (1.08 ± 0.36 Hz) was significantly lower than that of those that were inhibited by CNA stimulation (3.53 ± 0.47 Hz; Fig. 3D). Examples of NST neurons that were affected by CNA stimulation and gastrointestinal distension are illustrated in Fig. 4 and Fig. 5, respectively. Figure 4 shows the results from a neuron that was completely inhibited by CNA stimulation (Fig. 4A). The excitation induced by intestinal distension (Fig. 4B) was completely prevented by simultaneous CNA stimulation. Figure 5 illustrates an NST neuron that was excited by both amygdala stimulation and gastric distension.

Figure 4. The response profile of a representative NST neuron that was inhibited by CNA stimulation.

Figure 4

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The left panels represent the actual recordings made over 2 min periods. The right panels are the corresponding histograms. Electrical stimulation of the CNA completely abolished the spontaneous activity (Aa and Ab). This neuron was excited by intestinal distension (Ba and Bb). Electrical stimulation of the CNA blocked the excitatory influence of the intestinal distension (Ca and Cb).

Figure 5. The response properties of an NST neuron that was excited by CNA stimulation.

Figure 5

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Electrical stimulation of the CNA (Aa and Ab) and gastric distension (Ba and Bb) significantly increased the firing rate.

Electrical stimulation of the CNA modulated the response of NST neurons to gastrointestinal stimuli. We tested eight NST neurons that were excited either by intestinal or gastric distension. Electrical stimulation of the CNA either attenuated or eliminated the response of the NST to the gastrointestinal stimuli (Fig. 4C). In addition, we tested the response of eight NST neurons to 0.1 m HCl perfused into the intestinal loop and to electrical stimulation of the CNA. Of those eight, four were excited and one was inhibited by the HCl. Of the four neurons that were excited by HCl, electrical stimulation of the CNA completely abolished that excitatory response in two cells. Figure 6 shows the response profile of a neuron that was inhibited by CNA stimulation (Aa and Ab) and excited by HCl (Ba and Bb). Stimulation of the CNA abolished the HCl effect (Fig. 6Ca and Cb).

Figure 6. The response profile of a sample NST neuron that was inhibited by CNA stimulation and excited by intestinal HCl.

Figure 6

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Electrical stimulation of the CNA completely abolished the spontaneous activity of the neuron (Aa and Ab). The neuron was significantly excited by the intestinal 0.1 m HCl, an effect that lasted for several minutes after removal of the stimulus (Ba and Bb). Stimulation of the CNA completely blocked the excitatory response of the neuron to the intestinal HCl (Ca and Cb).

Response of gut-related DMNV neurons to electrical stimulation of the CNA

The effects of gastrointestinal distension on DMNV neurons were similar to those reported previously (Fogel et al. 1996). Among 148 DMNV neurons, intestinal distension inhibited 102 and excited 21 neurons. Gastric distension inhibited the spontaneous activity of 113 neurons, and excited that of 19 neurons. Eighty-one DMNV neurons were inhibited by both gastric and intestinal distension. Eleven neurons were excited by gastric distension and inhibited by intestinal distension. Seventeen neurons were inhibited by gastric distension and excited by intestinal distension.

Electrical stimulation of the CNA changed the spontaneous activities of 98 of the 148 DMNV neurons (66 % of DMNV neurons characterized). The overall response profile to CNA stimulation is shown in Fig. 7A. Electrical stimulation of the CNA increased the mean basal activity in periods 2 and 3 by 10 and 7 %, respectively. These changes were not statistically significant (P > 0.05).

Figure 7. Mean responses of DMNV neurons to electrical stimulation of the CNA.

Figure 7

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A, the mean response of DMNV neurons that were affected by electrical stimulation of the CNA. Although it was slightly excitatory, the change was not significant. However, among 98 DMNV neurons that were affected by CNA stimulation, 46 were significantly inhibited (B) and 52 were significantly excited (C). The mean basal activity of DMNV neurons that were inhibited by CNA stimulation was significantly higher than that of DMNV neurons that were excited by CNA stimulation (D).

Further analysis, however, indicated that the 98 DMNV neurons could be divided into two groups: 46 neurons were inhibited and 52 neurons were excited by CNA stimulation. The response profile of the 46 neurons that were inhibited by the CNA stimulation is shown in Fig. 7B. The response properties of the 52 DMNV neurons that were excited by electrical stimulation of the CNA is demonstrated in Fig. 7C. The mean basal activity of the neurons excited by CNA stimulation was significantly lower than that of the neurons inhibited by the CNA stimulation (P < 0.05). The mean activity of DMNV neurons that were excited by CNA stimulation was 1.73 ± 0.19 Hz, whereas the mean activity of DMNV neurons that were inhibited by CNA stimulation was 2.41 ± 0.21 Hz (Fig. 7D).

Figure 8 shows the data from a neuron that was inhibited by CNA stimulation as well as distension of the stomach or duodenum. An example of the response profile of DMNV neurons that were excited by electrical stimulation of the CNA is shown in Fig. 9.

Figure 8. The response profile of a sample DMNV neuron that was inhibited by electrical stimulation of the CNA.

Figure 8

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CNA stimulation significantly suppressed the spontaneous activity (Aa and Ab). The neuron was also slightly inhibited by gastric distension (Ba and Bb) and was completely inhibited by intestinal distension (Ca and Cb).

Figure 9. The response profile of a sample DMNV neuron excited by CNA stimulation.

Figure 9

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Electrical stimulation of the CNA significantly increased the activity of this neuron (Aa and Ab). The spontaneous activity was completely abolished by both gastric (Ba and Bb) and intestinal (Ca and Cb) distension. In addition, electrical stimulation of the CNA partially blocked the inhibitory influence of intestinal distension on this neuron (Da and Db).

DISCUSSION

The results of the present study demonstrate the anatomical and functional relationship between the CNA and the DVC. CNA neurons projecting to the DVC have a topographic distribution. At the rostral CNA, labelled neurons were distributed in the central region. At the middle level of the CNA, cells were clustered predominantly in the medial part, with a few neurons also present in the lateral area. A few labelled neurons were detected in the caudal CNA. Electrical stimulation of the CNA modulated the basal activity of gut-related DMNV and NST neurons. The spontaneous activity of 65 % of gut-related NST neurons was altered following CNA stimulation. The predominant effect was inhibition of NST neurons. In contrast, there was no dominant effect on DMNV neurons. CNA stimulation excited 53 % and inhibited 47 % of DMNV neurons. In addition, electrical stimulation of the CNA attenuated the response of DVC neurons to gastrointestinal stimuli. These results suggest the existence of a neural circuit for the regulation of gastrointestinal function by the CNA.

Anatomical connections between the CNA and the DVC

Using the retrograde tract-tracing method, we have confirmed the existence of direct projections from the CNA to the DVC and provided additional data regarding a topographic distribution of CNA neurons that project to the DVC. Labelled neurons have been identified in the bilateral CNA following injection of retrograde tracers into the DVC (Schwaber et al. 1980; van der Kooy et al. 1984). The CNA neurons projecting to the DVC were located predominantly in the medial CNA (Veening et al. 1984), However, Thompson & Cassell (1989) found labelled neurons throughout the medial, lateral and ventral subdivisions of the CNA. Our data revealed that CNA neurons that project to the DVC are not evenly distributed.

We used Alexa-Fluor-555-conjugated cholera toxin subunit B and confocal microscopy to characterize the detailed projections from the CNA to the DVC. Cholera toxin B subunit is more sensitive and reveals more projections than either horseradish peroxidase (HRP) or the fluorescent tracers used in the other studies (Schwaber et al. 1980; van der Kooy et al. 1984; Veening et al. 1984). HRP and fluorescent tracers enter neurons via the passive process of endocytosis, whereas the B submit of cholera toxin induces active, receptor-mediated uptake (van der Want et al. 1997; Vercelli et al. 2000). Serial sections and confocal microscopy were used to identify CNA neurons that project to the DVC. This kind of preparation included most of the CNA neurons that project to the DVC and provided a topographical distribution of the CNA neurons that project to the DVC.

The efferent projections from the CNA in the DVC have been studied extensively (Hopkins & Holstege, 1978; van der Kooy et al. 1984; Veening et al. 1984; Danielsen et al. 1989; Liubashina et al. 2000; Saha et al. 2000, 2002). Efferent fibres from the CNA terminate predominantly the mNST, although some fibres innervate the DMNV and other subnuclei of the NST. The mNST also receives information from the gastrointestinal tract (Altschuler et al. 1989; Zhang et al. 1995, 2000) and sends ascending projections to the CNA (Ricardo & Koh, 1978; Zhang et al. 1995; Petrov et al. 1996; Jia et al. 1997). These anatomical data suggest a bidirectional connection between the neurons receiving gastrointestinal input and the CNA. The CNA may receive information regarding the condition of the gastrointestinal tract and use this information to modulate the responsiveness of NST neurons and the vago-vagal reflex.

The effect of the CNA on gut-related NST neurons

There is a controversy regarding the effects of CNA stimulation on NST neurons. Liubashina and colleagues (2002) reported that electrical stimulation of the CNA inhibits NST neurons in rats. In contrast, Cox and colleagues (1986) reported that stimulation of the CNA markedly excited NST neurons that receive afferent innervation from either the aortic and/orthe vagus nerve in rabbits. Our results demonstrate that CNA stimulation inhibits the majority of gut-related NST neurons and modulates the response of NST neurons to gastrointestinal stimuli. Stimulation of the CNA blocked the excitatory response to intestinal distension (mechanical stimulation) and intestinal HCl (chemical stimulation).

Why do some reports indicate an excitatory response to CNA stimulation whereas others show an inhibitory response? These differences may reflect the different species studied. Alternatively, the stimulation electrodes may have been in different parts of the CNA. Since the CNA neurons projecting to the DVC are unevenly distributed, stimulation of different regions of the CNA may have different effects on DVC neurons. A third possibility is that the response of gut-related NST neurons is different from that of the NST neurons involved in cardiovascular reflex.

Finally, it is possible that the electrical stimulation of the CNA activated either other nuclei or fibres of passage. We consider this possibility unlikely. The CNA is a relatively isolated structure. Injection of retrograde tracer into the DVC did not label neurons in the surrounding nuclei. Unlike the PVN and the lateral hypothalamus, which have many fibres of passage, there is no evidence of significant fibres passing through the CNA to the DVC. Consequently, we believe that the observed effect on the DVC neurons is due to the activation of CNA neurons.

Although the majority of gut-related NST neurons were inhibited by CNA stimulation, a subset of NST neurons was excited. Whether the excitatory response of the NST to the CNA stimulation is direct or indirect remains unclear. We believe that both scenarios are possible. Direct excitation is supported by the heavy neural projections to the NST. A subgroup of CNA neurons may directly excite NST neurons.

Indirect excitation, however, cannot be excluded. Many NST neurons send terminals to other neurons in the NST (Rogers & McCann, 1993; Zhang et al. 1995). These terminals may inhibit the downstream NST neurons by releasing GABA (Jia et al. 1996). CNA stimulation inhibits these GABAergic NST neurons and produces an excitatory response in another group of NST neurons that receive GABAergic innervation.

We noticed that NST neurons inhibited by CNA stimulation had a significantly higher basal rate than those excited by CNA stimulation (Fig. 3D). The implication of this difference is unclear. The basal activity of NST neurons is regulated by input from vagal afferents as well as other central nuclei. The higher basal rate shown by the neurons inhibited by the CNA may result from tonic excitatory input from other sources (Loewy, 1991). We must point out, however, that the present result is generated in anaesthetized animals. We do not know if the difference in spontaneous activity between the two groups of neurons would be present in normal conditions.

Although we did not study the neurotransmitters that mediate the influence of the CNA on DVC neurons, the inhibitory influence of the CNA on NST neurons may be mediated by GABA. Double tract tracing and immunocytochemistry revealed that NST neurons receive heavy GABAergic innervation (Pickel et al. 1995, 1996; Jia et al. 1997). Saha and colleagues (2000) found that the majority (93.4 %) of terminals in the DVC from the CNA are GABA positive. In addition to GABA, a substantial proportion (20 %) also contain somatostatin (Higgins & Schwaber, 1983; Saha et al. 2002). Somatostatin has been shown to inhibit DVC neurons in brain slice preparations (Oomura & Mizuno, 1986). In addition, some CNA neurons that project to the DVC also contain neurotensin and corticotropin-releasing factor (Veening et al. 1984). The roles of these neurotransmitters in the CNA-DVC axis need to be clarified. The proposed circuits and possible neurotransmitters that are involved in the CNA-DVC axis are illustrated in Fig. 10.

Figure 10. The drawing illustrates the proposed circuits of interaction between the CNA and the DVC.

Figure 10

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The thicker lines represent the major effects and the thinner lines indicate the minor effects. The CNA mainly inhibits gut-related NST neurons to modulate the gastrointestinal vago-vagal reflex. The attenuated vago-vagal reflex induced by the activity of the CNA will result in hyperactivity of some gut-related DMNV neurons and consequently increase the motility and secretion of the gastrointestinal tract (stress ulcer, distress of the gastrointestinal tract). SOM, somatostatin; NG, nodose ganglion; (+) excitatory effect; (−), inhibitory effect.

The effect of the CNA on gut-related DMNV neurons

In comparison to the effect of the CNA on NST neurons, the effect of the CNA on gut-related DMNV neurons was almost equally divided between excitatory and inhibitory effects. The effects of CNA stimulation on DMNV neurons may be mediated by both direct and indirect pathways. Whether the direct effect of CNA stimulation on a DMNV neuron is excitatory or inhibitory cannot be predicted from our data. However, we postulate that the majority of the inhibitory responses of DMNV neurons to CNA stimulation are due to a direct influence. As we have discussed previously, the inhibitory response of DMNV neurons to gastrointestinal stimuli may be mediated by gut-related NST neurons. Inhibition of NST neurons by CNA stimulation will result in increased DMNV neuron activity.

Similar to NST neurons, DMNV neurons that were inhibited by CNA stimulation had a significantly higher basal rate than those that were excited by CNA stimulation (Fig. 7D). The implication of this difference needs to be explored further.

Influence of the CNA–DVC axis on gastrointestinal function

Several lines of evidence indicate that the amygdala regulates various gastrointestinal activities and may play a role in the development of the functional symptoms seen in irritable bowel syndrome (Henke et al. 1991; Monnikes et al. 2001). Stimulation of some areas of the CNA increased gastric acid secretion and motility and produced gastric erosion, whereas stimulation of other regions inhibited gastric motility and acid secretion. Both excitatory and inhibitory changes were blocked by vagotomy (Henke et al. 1991).

Our data indicate that the CNA is able to modulate the gastrointestinal vago-vagal reflex induced by mechanical and chemical stimulation of the gastrointestinal tract. The vago-vagal reflex is a negative feedback loop that is important in regulation of the gastrointestinal motility, acid secretion and absorption (Blackshaw et al. 1987; Fogel et al. 1991, 1996; Zhang et al. 1992, 1995, 1998; Chu et al. 1993; Rogers & McCann, 1993; Schwartz et al. 1993).

In Fig. 10, we present a schematic representation of the proposed circuitry. Stimulation of the CNA inhibits the majority of gut-related NST neurons. This inhibition will (1) reduce the inhibitory input from NST neurons to DMNV neurons and (2) prevent information ascending from the gut to the higher brain centres including the amygdala. The effect of CNA stimulation on individual DMNV neurons may be the result of either a direct projection or, alternatively, may be mediated by NST neurons that project to the DMNV.

Acknowledgments

These studies were supported by NIH grants DK53159 (X.Z.) and NS30083 (R.F.) as well as funds from the Henry Ford Health System to X.Z. and R.F. We thank Mrs Yisheng Cui for her excellent technical assistance.

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