Introduction

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Afferent sensory fibers are the primary neuroanatomical link between the gastrointestinal tract and the central neural substrates that mediate the control of food intake (Altschuler et al., 1989; Berthoud et al., 1990). The vagus is a major visceral sensory nerve conveying information from the gastrointestinal tract to the brainstem. Enteric neuropeptides [e.g., cholecystokinin (CCK)] and hormones (e.g., leptin) can signal the central nervous system (CNS) via gastric vagal afferents in their role of regulating digestive functions (Lee et al., 1994; Wang et al., 1997).

CCK is expressed in endocrine cells of the intestine and in nerve fibers distributed to all parts of the gastrointestinal tract (Schultzberg et al., 1980; Dockray, 1987; Shulkes and Baldwin, 1997). CCK produced satiety, decreased food intake, and slowed gastric motility, partly by its direct effect on the gastrointestinal tract, and partly by its action in the CNS. Previous studies indicate that CCK produces distinct peripheral and central effects, and that the stomach and vagus are peripheral sites of CCK action (Barber et al., 1990; Lee et al., 1994). One component of the satiety effect of CCK is mediated by CCK-A receptors at the periphery through activation of vagal afferents (Forster et al., 1990; Reidelberger, 1992).

Leptin, the secreted product of the obese (ob) gene, regulates food intake and energy balance. Leptin is not only expressed in adipose tissue (Zhang et al., 1994) but also in gastric mucosa and fundic glands in rats (Bado et al., 1998) and humans (Mix et al., 1999). Adipose tissue-secreted leptin acts as a feedback signal on specific hypothalamic nuclei, which, in turn, modulate the action of brain neuropeptides (Erickson et al., 1996).

Previous investigations showed that leptin synergistically interacts with CCK to increase firing frequency of gastric vagal terminals (Wang et al., 1997), and leads to suppression of food intake, involving CCK-A receptors and capsaisin-sensitive afferents (Barrachina et al., 1997). Recently, we observed that gastric effects of leptin can activate nucleus tractus solitarius (NTS) neurons responding to gastric vagal stimulation (Yuan et al., 1999). Although the precise function of the gastric leptin pool is still unknown, it is responsive to feeding as well as to peripheral CCK administration (Bado et al., 1998). It appears that gastric leptin can interact with CCK, and modulates food-related satiety signals. In this study, we evaluated the peripheral gastric effect of CCK on unitary activity in the NTS by using an in vitro neonatal rat brainstem-stomach preparation, and then investigated gastric interaction between CCK and leptin on brainstem neurons.

	Materials and Methods

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Animal and Surgical Preparation. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Chicago. Experiments were performed on 32 Sprague-Dawley neonatal rats 1 to 5 days old. After the animal was deeply anesthetized with halothane, a craniotomy was performed and the forebrain was ablated at the caudal border of the pons by transection. The caudal brainstem and cervical spinal cord were isolated by dissection in modified Krebs' solution that contained 128.0 mM NaCl, 3.0 mM KCl, 0.5 mM NaH₂PO₄, 1.5 mM CaCl₂, 1.0 mM MgSO₄, 21 mM NaHCO₃, 1.0 mM mannitol, 30.0 mM glucose, and 10 mM HEPES. The stomach, connected to the esophagus, with the vagus nerves linking it to the brainstem, was kept and all the other internal organs were removed. The preparation was then isolated and pinned, with the dorsal surface up, on a layer of Sylgard resin (Corning Inc., Acton, MA) in a recording chamber. The preparation was isolated and superfused with Krebs' solution at 23 ± 1°C. The bathing solution was aerated continuously with a mixture of 95% O₂, 5% CO₂ and adjusted to pH 7.35 to 7.45 (Barber et al., 1995; Yuan et al., 1998).

Stimulation and Recording Methods. A suction microelectrode was placed on the gastric vagal branch from the subdiaphragmatic vagi for electrical stimulation and units in the medial subnucleus of the NTS receiving gastric vagal inputs were evaluated in this study. The gastric vagal fibers were stimulated with single or paired pulses of 200 µA for 0.2 ms at a frequency of 0.5 Hz by a Grass stimulator (model S8800) coupled to a stimulus isolation unit (SIU 5B; Grass Instruments, Quincy, MA). This current provided a stimulus intensity 1.5 to 2.0 times that required to produce maximal amplitude of the C-wave in the vagal nerve action potential (Yuan et al., 1998).

Experimental Protocols. CCK and leptin may have both peripheral and central actions. To investigate the peripheral gastric effects of the peptides on brainstem neurons without interfering with CNS functions, a partition was made at the mid-thoracic level of the preparation. An agar seal separated the recording bath chamber into a brainstem compartment and a gastric compartment. Peptides were applied only to the gastric compartment and their effects on the NTS neuronal activity were evaluated.

Drugs. Drugs used in this study were CCK or sulfated CCK-8 (Research Biochemicals International, Natick, MA), L-364,718 and L-365,260 (Merck Sharp and Dohme, West Point, PA), and leptin or the methionyl murine leptin (Amgen, Thousand Oaks, CA).

Data and Statistical Analysis. The data from the NTS unitary activity were expressed as mean ± S.E. and analyzed on the basis of action potential discharge rate and drug concentration-related effects. The number of action potentials in a given duration was measured under pretrial, trial, and post-trial conditions. The control data (pretrial) were normalized to 100%, and the NTS neuronal activities during and after trials were compared with the control data. Data were analyzed by using ANOVA for repeated measures and Student's t test with P < .05 considered statistically significant.

	Results

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A total of 120 tonic, gastric vagally evoked NTS units were recorded. Their mean basal firing rate was 0.9 ± 0.3 Hz. There was no significant difference in basal firing rate between units that responded and did not respond to gastric CCK and/or leptin.

Peripheral Gastric Effects of CCK. Peripheral gastric effects of CCK (300 nM) produced a mean activation response of 271 ± 3.9% compared with control level (100%) in 33 of 55 neurons tested. The difference in the NTS neuronal discharge frequency between the control recording and the recording after CCK (300 nM) applications was significant (P < .01). There was a concentration-dependent effect of CCK (10 nM-1.0 µM) on NTS neuronal discharge frequencies (Fig. 1). The remaining 22 NTS cells showed no response to CCK (Table 1).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Concentration-related peripheral gastric effect of CCK on seven NTS units receiving gastric vagal input. The minimal effective concentration is 30 nM (P < .01 compared with the control). The EC50 is 68 nM. The control activity level is normalized to 100%. Data are presented as mean ± S.E.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Sequential spike frequency histogram of a representative unit recorded in the NTS. Each histogram represents 10 consecutive samples of the unit in each test condition. A, control. B, some increase of the neuronal activity after CCK (30 nM). C, significant increase in discharge rate after CCK (300 nM). D, after washout (data not shown), L-364,718 (300 nM) antagonizes the CCK (300 nM) effect. E, after washout (data not shown), L-365,260 (300 nM) does not change CCK (300 nM) effect.

Peripheral Gastric Effects of Leptin. Twenty-six units that showed activation responses to CCK in the preceding section also were tested after leptin application. As shown in Table 1, peripheral effects of leptin (10 nM) produced a mean activation response of 183 ± 5.3% of control level in 13 neurons tested. The difference in the NTS neuronal activity between the control and the recording after leptin was significant (P < .01). The remaining 13 units that responded to CCK were not affected by leptin.

Gastric Interaction between CCK and Leptin on NTS Unitary Activity. To evaluate the potential synergistic effect between CCK and leptin, two groups of NTS neurons were tested. The first group consisted of 10 units that showed activation responses to both CCK (300 nM) and leptin (10 nM), which were reported above. The second group consisted of 22 units that were not affected by either CCK or leptin at the same concentrations.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Subadditive effect of gastric CCK and leptin on 10 NTS units receiving gastric vagal inputs. All these units responded to both CCK (300 nM) and leptin (10 nM). A subadditive effect is observed when CCK (300 nM) and leptin (10 nM) are applied together. Control is normalized to 100%. Data are presented as mean ± S.E. *P < .01 compared with CCK alone. **P < .01 compared with leptin alone.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Sequential spike frequency histograms of an NTS unit with 10 consecutive samples of the recording. A, control. B, no change in discharge frequency of the unit after leptin (10 nM) application in the gastric compartment. C, no change in discharge frequency of the same unit after CCK (300 nM) application. D, significant increase in discharge rate after CCK (300 nM) plus leptin (10 nM) application. E, after drug washout.

Site of CCK and Leptin Actions in the Stomach. To investigate the distribution of the gastric CCK and leptin receptors that affect NTS neuronal activity, a whole-stomach preparation and a partial-stomach preparation were used in this part of the study. The gastric mucosal structure of the proximal and the distal stomach under the dissecting microscope appear distinctly different. This mucosal structure difference was used as a landmark to make the partial-stomach preparation. Peripheral gastric effects of peptides were observed first in the whole-stomach preparation. Next, the proximal part or the distal part (containing the pylorus) of the stomach was carefully removed, while unitary recording in the NTS was maintained. The peptides' effects on the same NTS cell were then observed in the proximal-stomach and distal-stomach preparations (Yuan, 1996).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Peripheral gastric effects of CCK and leptin on NTS neuronal activity in the whole-stomach preparation versus the partial-stomach preparation. A, there is a significant difference between the gastric effects of CCK (300 nM) on the proximal-stomach preparation and distal-stomach preparation in NTS neuronal activities; *P < .01. However, there is no significant difference between the whole-stomach preparation and distal-stomach preparation. B, there are no significant differences between the gastric effects of leptin (10 nM) on the proximal-stomach preparation and distal-stomach preparation, and between the whole-stomach preparation and proximal-stomach or distal-stomach preparations. The control activity level is normalized to 100%. Data are presented as mean ± S.E.

	Discussion

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In this study, gastric effects of CCK and its interaction with leptin on NTS units processing gastric vagal inputs were investigated. A neonatal rat brainstem-stomach preparation was used, in which we have previously demonstrated gastric neurochemical effects on gastric vagally evoked brainstem neuronal activity (Barber et al., 1995; Yuan et al., 1998). CCK and leptin are peptides that have central and peripheral effects. This preparation allows us to restrict CCK and leptin to the gastric compartment and to observe peripheral effects without interfering with brainstem functions. The development of obesity in rodent models is concomitant with effects from hormonal and metabolic changes on leptin homeostasis (Saladin et al., 1995). Our experiments were performed on nonobese preweaned animals to avoid the complicating effects of metabolic patterns on leptin activity as seen in adults.

Our results demonstrated that neurons located in the medial subnucleus of the NTS are responsive to gastric CCK and leptin. The medial NTS is the first relay station for vagal afferents that form the sensory limb of gastrointestinal vago-vagal reflexes. The majority of neurons responded to CCK that was applied to the gastric compartment in our experiment by increasing their poststimulus neuronal discharge frequency by 271%. Moreover, the CCK responses were concentration dependent, and confirmed previous results that CCK-A receptors were involved (Reidelberger, 1992; Barrachina et al., 1997). Many of the vagal afferents in the gastrointestinal tract, including the gastric antrum, that mediate gastric-distension are sensitive to exogenous CCK (Forster et al., 1991). Physiologically, there is an increase of plasma CCK level postprandially (Reidelberger et al., 1989). In addition, CCK that is expressed in gastric vagal afferents also may be released in response to stimulation by gastric distension. Our data also showed that the activity of some NTS neurons that were responsive to CCK was increased by gastric effects of leptin. Although leptin is derived mainly from adipose tissue (Zhang et al., 1994), leptin mRNA and leptin protein are also present in the rat gastric epithelium (Bado et al., 1998). In addition, many vagal afferents innervating the gastrointestinal lumen are polymodal, with sensitivities for numerous chemical and mechanical stimuli. Our results suggest that gastric leptin, like the gut hormone CCK, can activate peripheral terminals of visceral afferent neurons and initiate an acute action through vago-vagal reflexes.

Our results indicated that gastric effects of leptin increase the excitability of NTS cells responsive to gastric CCK. NTS units that showed activation responses to CCK (300 nM) and leptin (10 nM) had a subadditive effect that produced a mean activation response of 338% when the peptides were applied together. In addition, approximately 36% of units that were not affected by either CCK or leptin alone had an activation response of 158% when the same concentrations of CCK and leptin were applied together. Previous studies have shown synergism between leptin and CCK. Wang et al. (1997) reported that i.a. injections of leptin significantly increased the poststimulus spike count of some gastric vagal terminals responsive to CCK. Other investigators have demonstrated CCK-leptin interactions with intraventricularly injected leptin (Emond et al., 1999), although gastric interaction between CCK and leptin on brainstem neurons has not been reported before this study. However, in our experimental paradigm, we cannot tell whether leptin or CCK-sensitive and leptin or CCK-insensitive neurons were located in a particular region of the medial subnucleus of the NTS.

The regulation of body weight by circulating leptin appears to depend on its interaction with leptin receptors in the arcuate nucleus within the hypothalamus. The arcuate nucleus projects to the paraventricular nucleus (PVN), and roles for both neuropeptide Y and melanocortin in mediating the actions of leptin through this pathway have been proposed (Halaas et al., 1995; Erickson et al., 1996). Alternatively, leptin may transduce signals to the PVN from gastric vagal afferents via the brainstem. These interactions lead to a long-term reduction in food intake and an increase in energy expenditure. The synergistic interaction between CCK and leptin that we observed suggests the presence of a second pathway of leptin action in the brain. It is possible that ascending signals from NTS neurons responsive to vagally mediated CCK-leptin interaction may project to cells within the PVN that are independently activated by circulating leptin. The behavioral effects of such CCK-leptin interaction might go beyond the effects of CCK to reduce the size of an individual meal. This CCK-leptin interactive pathway may convey meal-related signals to hypothalamic nuclei that are then integrated with adipose tissue-related signals conveyed by leptin.

Data from this study, using the whole-stomach and partial-stomach preparation, demonstrated that the distal stomach, not the proximal stomach, is important in the CCK gastric effects on NTS neuronal activity. However, the results of an earlier study showed that CCK inhibited gastric emptying by acting on both the proximal stomach and pylorus in the adult dog (Yamagishi and Debas, 1978). Differences in the criteria used to define the proximal and distal stomach, and species differences may account for the disparity under Results between the two studies. Robinson et al. (1987) and Schwartz et al. (1990) studied the distribution of CCK-binding sites autoradiographically at different stages of the development in rat upper gastrointestinal tract. They showed that CCK binding was present in the gastric mucosa, the muscular wall of the distal part of the stomach, and the muscle of the gastroduodenal junction in rat fetus. In addition, 3 to 10 days after birth, the antral muscle binding and pyloric binding progressively increased. These results support our electrophysiological observation that the distal stomach plays a key role in the CCK gastric activity in neonatal rats. Our experiments also were aimed at identifying the site of action of gastric effects of leptin and indicated that both the proximal and distal areas of the stomach were important sites of gastric effects of leptin action.

We previously showed that a physiological role for gastric effects of leptin is to activate gastric vagal afferent signals to the brain (Yuan et al., 1999). The results of our present study revealed that gastric effects of leptin increase the potency of CCK-derived vagal afferents to the CNS. Wang et al. (1998) reported a synergy between CCK and i.p.-injected leptin in reducing food intake, and further examined brain sites that mediate this result. Their data showed that, in fasted lean mice, the induction of c-Fos was only localized to the hypothalamic PVN, a central target of leptin (Tartaglia et al., 1995; Lee et al., 1996). A similar study in which leptin was centrally injected 1 h before CCK, however, showed induction of c-Fos in the NTS as well as the PVN (Emond et al., 1999). The implicit suggestion for induction of c-Fos in the NTS was the presence of a leptin-activated descending pathway from the PVN, altering NTS cell response to peripheral CCK. Electrophysiologically, our results showed that NTS neurons can be activated by the gastric CCK-leptin synergy. From the NTS, ascending pathways may convey the signal to the PVN and are integrated into centrally mediated leptin signals. In addition, axonal projections to the dorsal motor nucleus, an area of preganglionic parasympathetic motor neurons that provide vagal outflow to the viscera (Van Giersbergen et al., 1992), are also possible. To identify other potential brain sites besides the NTS that might mediate a behavioral response to gastric CCK-leptin synergy, c-Fos immunohistochemical studies need to be conducted.

We used 1- to 5-day-old rats to demonstrate synergism between gastric effects of leptin and CCK on neurons in the medial subnucleus of the NTS. In a series of retrograde transynaptic neuronal viral infection studies of rats in this age group, Rinaman et al. (1999, 2000) demonstrated synaptic connectivity between gastric vagal afferents, neurons in the medial subnucleus of the NTS, and preganglionic vagal motor neurons. In rats, the leptin system, with respect to the ob gene expression and leptin production, is operational 1 day after birth (Rayner et al., 1997). In our recent study we showed that i.p.-injected leptin modulated feeding behavior that led to a significant decrease in weight gain in 1- to 5-day-old rats (Yuan et al., 2000). Thus, our experimental model seems to be appropriate for investigating the physiological roles of leptin.

In summary, we observed peripheral gastric effects of CCK and its interaction with leptin on brainstem neuronal activity. Our results support the hypothesis that gastric leptin interacts with CCK at the level of the stomach to increase afferent neural signals to the NTS. Our data also showed that gastric effects of leptin synergistically increased the NTS neuronal response to gastric effects of CCK, and suggest that leptin modulates potency of CCK signals that modify food intake in the neonatal rat.