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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 295:177–182, 2000
Vol. 295, No. 1
2708/849680
Printed in U.S.A.
Gastric Effects of Cholecystokinin and Its Interaction with
Leptin on Brainstem Neuronal Activity in Neonatal Rats1
CHUN-SU YUAN, ANOJA S. ATTELE, LUCY DEY, and JING-TIAN XIE
Committee on Clinical Pharmacology (C.-S.Y.), Department of Anesthesia and Critical Care (C.-S.Y., A.S.A., L.D., J.-T.X.), and Tang Center for
Herbal Medicine Research (C.-S.Y., A.S.A., L.D., J.-T.X.), The Pritzker School of Medicine, The University of Chicago, Chicago, Illinois
Accepted for publication May 30, 2000
This paper is available online at http://www.jpet.org
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
Received for publication March 17, 2000.
1
This study was supported in part by the Brain Research Foundation and
the Clinical Practice Enhancement & Anesthesia Research Foundation.
in 13 (50%) NTS units that responded to CCK (P ⬍ .01).
Furthermore, we evaluated the combined effect of CCK and
leptin in two groups of NTS neurons. Those NTS units that
showed activation responses to both CCK (300 nM) and leptin
(10 nM) had a subadditive effect that produced a mean activation response of 338 ⫾ 12.9% compared with the control level
in all 10 (100%) neurons tested (P ⬍ .01). Eight (36%) of another
22 units that were not affected by either CCK (300 nM) or leptin
(10 nM) alone had an activation response (151 ⫾ 5.2%; P ⬍ .05)
when the same concentrations of CCK and leptin were applied
together. Subsequently, by comparing the effects of CCK and
leptin on a whole-stomach preparation to a partial-stomach
preparation, we examined the area of the stomach in which
gastric receptors contributed most to NTS unitary activity. We
showed that the distal stomach containing the pylorus determined CCK gastric activity, whereas both the proximal and
distal stomach are important for leptin’s effect. Our data suggest that leptin modulates the potency of CCK signals that
modify food intake in the neonatal rat.
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-
ABBREVIATIONS: CCK, cholecystokinin; CNS, central nervous system; NTS, nucleus tractus solitarius; PVN, paraventricular nucleus.
177
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ABSTRACT
Cholecystokinin (CCK) is a major gastrointestinal neuropeptide
that is secreted in response to food ingestion. It is involved in
the feedback regulation of gastric emptying and also modulates
food intake. Leptin, a hormone that regulates food intake and
energy balance, is secreted from adipose tissue, gastric mucosa, fundic glands, and other tissues. In a previous report we
showed that gastric effects of leptin activated the nucleus
tractus solitarius (NTS) neurons responding to gastric vagal
stimulation. In this study, using the same in vitro neonatal rat
preparation, we investigated the gastric effects of CCK and its
interaction with leptin on NTS neurons receiving gastric vagal
inputs. We observed that peripheral gastric effects of CCK (300
nM) produced a mean activation response of 271 ⫾ 3.9%
compared with control level (100%) in 33 (60%) neurons tested
(P ⬍ .01), and this response was abolished by a CCK-A receptor antagonist. A concentration-dependent effect of CCK (10
nM–1.0 ␮M) on NTS neuronal discharge frequencies was
shown. We also observed that leptin (10 nM) applied to the
stomach produced a mean activation response of 183 ⫾ 5.3%
178
Yuan et al.
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
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.
The test compounds, CCK and leptin, were dissolved in the vehicle
solution. The concentrated solution was applied to the Krebs’ solution in the gastric compartment. The final drug concentration in the
gastric compartment was calculated based on the amount of concentrated solution and the total Krebs’ volume. Drug solution was applied 5 min before any pharmacological observation to provide sufficient time for drug delivery to reach a steady-state level. To observe
CCK-leptin interaction, both solutions were added simultaneously.
After each observation, drug was washed out from the compartment.
The NTS neuronal responses observed during pretrial or pretreatment (control) were compared with post-trial (washout) to confirm
that brainstem neuronal activity returned to the control level after
washout. Tachyphylaxis was tested by reapplying the test compound
to the gastric compartment and observing whether the response to a
given concentration of the compound varied by less than 5%.
At the end of eight experiments, after the NTS neuronal responses
to gastric peptides were observed, the vagus nerve was severed at the
low thoracic level. For all eight units that responded to peptides
before vagal discontinuation, gastric effects were abolished after the
vagus was cut off. Also, at the completion of each experiment, colored
solution was applied to one compartment to make sure that there
was no leakage to the other compartment.
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
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 ␮M) on NTS neuronal discharge frequencies (Fig. 1).
The remaining 22 NTS cells showed no response to CCK
(Table 1).
To evaluate which CCK receptor subtype affects peripheral
gastric action, CCK and a CCK-A or CCK-B receptor antagonist were applied together into the gastric compartment.
When CCK (300 nM) was applied immediately after
L-364,718 (300 nM), a selective CCK-A receptor antagonist,
the gastric effect of the peptide on the NTS responses was
blocked (n ⫽ 7; not significant compared with the control).
L-365,260 (300 nM), a selective CCK-B receptor antagonist,
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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 SpragueDawley 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 ␮A 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).
Single tonic unitary discharges were recorded extracellularly in
the medial subnucleus of the NTS by glass microelectrodes filled
with 3 M NaCl, with an impedance of 10 to 20 M⍀ (unitary discharge
recordings, see Barber et al., 1995). A collision test was applied to
identify orthodromic inputs (Lipski, 1981) to ensure that only second
or higher order NTS neurons in the gastric vagal afferent system
were used in this study.
The NTS unitary discharges were amplified with high gain ACcoupled amplifiers (Axoprobe-1A; Axon Instruments, Burlingame,
CA), displayed on a Hitachi digital storage oscilloscope (model VC6525; Hitachi Denshi, Ltd., Tokyo, Japan), and recorded on a Vetter
PCM tape recorder (model 200; A.R. Vetter Co., Rebersburg, PA).
For histological identification purposes, some glass microelectrodes were filled with 2% pontamine sky blue in 0.5 M sodium
acetate solution. After each unitary recording, current was applied at
5 ␮A in 5-s on/10-s off cycles for approximately 5 min, with the
negative lead connected to the microelectrode.
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
Vol. 295
2000
Gastric CCK and Leptin on Brainstem Activity
179
TABLE 1
Percentage of activation and no effect responses of NTS neurons to
peripheral gastric effects of CCK and leptin in neonatal rats
Number in parentheses indicates the number of units recorded. Number after slash
indicates the level of activation (mean ⫾ S.E.) compared with the control (100%).
Twenty-six units tested with leptin (10 nM) were those cells that showed activation
response to CCK (300 nM). Ten units tested with CCK (300 nM) plus leptin (10 nM)
were those cells that showed activation responses to both CCK or leptin.
Result
CCK (300 nM)
Leptin (10 nM)
Activation effect
/Activation level
No effect
Total
60% (33)
/271.1 ⫾ 3.9%
40% (22)
100% (55)
50% (13)
/182.9 ⫾ 5.3%
50% (13)
100% (26)
CCK (300 nM) ⫹
Leptin (10 nM)
100% (10)
/337.8 ⫾ 12.9%
100% (10)
did not block the gastric effect of CCK on NTS response (n ⫽
8; P ⬍ .05 compared with the control). Figure 2 shows an
example. Our results suggest that CCK-A receptors in the
stomach affect the gastric activity of CCK on NTS units
receiving gastric vagal inputs. However, application of
L-364,718 (300 nM) or L-365,260 (300 nM) alone in the gastric compartment did not have significant effects on the basal
activity of NTS neurons.
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.
CCK (300 nM) and leptin (10 nM) were applied together to
the gastric compartment of 10 NTS units from the first
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.
group. As shown in Fig. 3, a subadditive effect that produced
a mean activation response of 338 ⫾ 12.9% was observed
(P ⬍ .01 compared with CCK alone, 271 ⫾ 6.9%; P ⬍ .01
compared with leptin alone, 179 ⫾ 8.3). In the second group,
8 of 22 units that did not respond to CCK or leptin application
alone (probably with subthreshold activity in extracellular
recording), showed an activation response (158 ⫾ 5.5%, P ⬍
.05 compared with the control) to the same concentrations of
CCK (300 nM) plus leptin (10 nM). Figure 4 shows a representative example, in which the discharge rate of an NTS
unit only increased after CCK plus leptin application in the
gastric compartment.
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
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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.
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Yuan et al.
Vol. 295
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).
Twenty-five NTS units that responded to CCK (300 nM)
were observed in both the whole-stomach and partial-stomach preparations. There was a significant difference between
the gastric effects of CCK on the proximal-stomach preparation (115 ⫾ 11.2%) and distal-stomach preparation (255 ⫾
9.8%) in NTS neuronal activities (P ⬍ .01). However, there
was no significant difference in NTS responses between the
whole-stomach preparation (272 ⫾ 7.9%) and distal-stomach
preparation (Fig. 5A). These results suggest that the distal
stomach containing the pylorus affects the gastric effects of
CCK on NTS neuronal activity.
Another 18 NTS units that responded to leptin (10 nM)
were observed in both the whole-stomach and partial-stomach preparations. There were no significant differences between the gastric effects of leptin on the proximal-stomach
preparation (153 ⫾ 12.0%) and distal-stomach preparation
(151 ⫾ 14.9%), and between the whole-stomach preparation
(188 ⫾ 13.7%) and proximal/distal-stomach preparations
(Fig. 5B). These results suggest that both the proximal and
distal stomach play important roles in the gastric effect of
leptin on NTS unitary activities.
Discussion
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 periph-
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.
eral 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 vagovagal 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).
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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.
2000
Gastric CCK and Leptin on Brainstem Activity
181
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 proximalstomach 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 distalstomach preparation, and between the
whole-stomach preparation and proximalstomach or distal-stomach preparations.
The control activity level is normalized to
100%. Data are presented as mean ⫾ S.E.
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 partialstomach 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
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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
182
Yuan et al.
Acknowledgments
We thank Tasha K. Lowell and Ji An Wu for technical assistance.
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Send reprint requests to: Chun-Su Yuan, M.D., Ph.D., Department of Anesthesia & Critical Care, The University of Chicago Medical Center, 5841 S.
Maryland Ave., MC 4028, Chicago, IL 60637. E-mail: cyuan@midway.
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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.
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