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Endocrinology Vol. 145, No. 8 3652-3657
Copyright © 2004 by The Endocrine Society

Cooperative Activation of Cultured Vagal Afferent Neurons by Leptin and Cholecystokinin

J. H. Peters, A. B. Karpiel, R. C. Ritter and S. M. Simasko

Program in Neuroscience, Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164

Address all correspondence and requests for reprints to: James H. Peters, Program in Neuroscience, Department of Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6520. E-mail: petersj{at}vetmed.wsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To test the hypothesis that leptin can directly activate vagal afferent neurons, we used fluorescence imaging to detect acute changes in cytosolic calcium after leptin application to primary cultures of vagal afferent neurons dissociated from adult rat nodose ganglia. We found that approximately 40% of vagal afferent neurons exposed to leptin (40 ng/ml) responded with rapid and reversible increases in cytosolic calcium. These responses were dependent upon extracellular calcium. As previously reported, about 35% of vagal afferents increase cytosolic calcium in response to the gut-peptide cholecystokinin (CCK). A majority (74%) of neurons that responded to CCK also exhibited increases in cytosolic calcium in response to leptin. In addition, synergistic increases in cytosolic calcium were observed when leptin and CCK were applied in combination. These results demonstrate that leptin acts directly on vagal afferent neurons to trigger acute influxes of extracellular calcium. Our results also suggest cooperation between leptin and CCK in the activation of some vagal afferent neurons. Acute activation of vagal afferents by leptin alone and in combination with CCK may contribute to modulation of visceral reflexes and control of food intake.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN IS A CYTOKINE hormone produced and released primarily by white adipose tissue (WAT) (1). Injection of recombinant leptin centrally or systemically decreases food intake and reduces adipose tissue (2, 3). Furthermore, plasma and cerebrospinal fluid concentrations of leptin are proportional to adipose mass (4, 5). Taken together, these observations suggest that leptin participates in the control of food intake and contributes to a negative feedback control of adiposity.

The mechanisms and sites of action that underlie leptin’s effects on food intake are only partially understood. However, previous reports suggest that leptin interacts with the gut peptide, cholecystokinin (CCK), to produce changes in food intake and body mass. For example, leptin-induced reduction of body weight is enhanced when CCK is coadministered with leptin (6). In addition, administration of leptin facilitates CCK-induced reduction of meal size (7) and total daily caloric intake (8). Thus, leptin and CCK cooperate in the control of food intake and body weight.

CCK is released from I-cells of the duodenum in response to protein and fat content of a meal and activates vagal afferent neurons, which contribute to the process of satiation (9, 10, 11). In addition to the well-documented role of vagal afferents in CCK-mediated satiation, vagal afferents also contribute to CCK-induced inhibition of gastric emptying (12), stimulation of intestinal motor activity (13), and activation of pancreatic exocrine secretion (14, 15).

Recently, the long form of the leptin receptor (Ob-Rb) has been found in a subpopulation of vagal afferent neurons (16, 17, 18). In addition, extracellular recordings of vagal afferent fibers revealed that exogenous leptin alters the firing rate of these fibers, and that there may be a cooperative activation of these fibers by CCK and leptin (19). Although many responses to leptin depend on transcriptional changes, requiring hours to develop (2, 20), these in vivo studies suggest that leptin might directly activate vagal afferents and that leptin and CCK may interact at the level of vagal afferent neurons. However, because the observation that leptin increases firing rate of vagal afferent fibers was made in vivo, it is not possible to rule out the possibility that leptin-induced activation of vagal afferents is an effect secondary to such actions as effects of leptin on intestinal motility (21) or alterations in enteroendocrine secretion (22).

To test the hypothesis that leptin directly and acutely activates vagal afferent neurons, we used single cell fluorescent imaging to detect acute neuronal activation by leptin in cultured vagal afferent neurons from rat nodose ganglia. We found that leptin triggers acute activation of cultured nodose neurons, as indicated by short-latency increases in cytosolic calcium. In addition, we observed that a majority of leptin-sensitive neurons also were sensitive to CCK and that in these neurons coapplication of leptin and CCK resulted in synergistic enhancement of the cytosolic calcium signal compared with exposure to either ligand individually.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Dissociated nodose neurons were obtained as previously described (23) based on the method of Lancaster et al. (24). Briefly, nodose ganglia were isolated from anesthetized (ketamine 25 mg/100 g, xylazine 2.5 mg/100 g) adult male Sprague Dawley rats (280–320 g) under aseptic conditions. The ganglia were digested for 90 min at 37 C in 3 ml of Ca2+- and Mg2+-free Hanks’ balanced salt solution containing 1 mg/ml dispase II (Roche Molecular Biochemicals, Indianapolis, IN) and 1 mg/ml collagenase type Ia (Sigma, St. Louis, MO). Cells were dispersed by gentle trituration through silanized Pasteur pipettes, washed two times with HEPES-buffered DMEM (HDMEM) (Invitrogen Life Technologies, Grand Island, NY) containing 10% fetal calf serum (Invitrogen Life Technologies) supplemented with antibiotic (penicillin-streptomycin, 100 U/ml and 100 µg/ml, respectively), and then plated onto poly-L-lysine-coated coverslips and maintained in HDMEM with 10% fetal calf serum at 37 C in a 5% CO2 atmosphere. All experiments were performed within 48 h of collecting the nodose ganglia.

Calcium measurements
Ratiometric measurements using the fluorescent calcium indicator Fura-2-AM (Molecular Probes, Eugene, OR) were performed as previously described (23). All manipulations and measurements were made at room temperature (22 C) in a physiological saline [in mM: 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 6 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH)]. Cells were loaded with 2 µM Fura-2-AM for 30 min followed by a 30-min wash. The cover slips containing the loaded cells were mounted into a closed chamber and constantly perfused with physiological saline. Neurons were easily identified and selected based on their large size and rounded profile, vs. the small and flattened profiles of glial cells. Image pairs (340 and 380 nm excitation, 510 emissions) were collected every 6 sec. Ratios of fluorescence intensity were converted to calcium concentrations using a standard curve obtained in a bath containing 10 µM fura-2, 130 mM KCl, 10 mM MOPS buffered to pH 7.4 with KOH, 10 mM EGTA, and various concentrations of CaCl2 (0–10 mM). Free calcium concentrations were calculated using the computer program EQCAL (Biosoft, Ferguson, MO). Data collection and manipulations were performed with MetaFluor Software (Universal Imaging Inc., West Chester, PA).

Experimental protocols
In all experiments, murine leptin and the sulfated form of CCK-8 were used. Leptin and CCK were dissolved directly into the physiological saline and solutions containing hormones were applied by switching solutions flowing through a common manifold upstream of the recording chamber (~15–30 sec required for new solution to reach neurons). Concentrations of hormones used are indicated in the results. Calcium responses are reported as the change in calcium level (peak response minus the basal value obtained by averaging 10 data points just before the hormone challenge). Although most neurons began the experiment with basal calcium concentrations less than 250 nM, in some neurons calcium levels between hormone challenges would begin to climb after repeated challenges to hormones. Thus, to ensure that only healthy neurons were included in the analysis, we required that neurons had to maintain a basal calcium level of less than 400 nM throughout the experiment. Furthermore, to ensure that a neuron that did not respond to CCK or leptin was capable of responding, at the end of each measurement we required that they exhibit at least a 40 nM increase in calcium in response to depolarization by 55 mM KCl (iso-osmotic reduction in NaCl). When CaCl2 was removed from the bath, it was replaced with equal molar concentration of MgCl2. Finally, in all experimental protocols, neurons from at least three separate isolations were used to ensure that the observed responses were representative of nodose neurons in general.

Statistical analyses
In the Ca2+-free experiments, each neuron could serve as its own control and thus we used a paired t test. To determine whether leptin and CCK responses occurred preferentially in the same population of cells, we used a {chi}2 analysis. To determine whether coapplication of CCK and leptin resulted in synergistic actions, we examined the response to coapplication of CCK and leptin to the summed responses to both compounds alone. In all analyses, confidence limits for significance was P < 0.05.

Chemicals
All hormones, culture media, and buffer components were purchased from Sigma unless otherwise indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We found that acute application of leptin caused rapid and reversible increases in cytosolic calcium in a subpopulation of cultured vagal afferent neurons. A dose-response protocol was used to characterize the response in 19 leptin-responsive neurons. In five of the 19, a clear dose-response relationship similar to that illustrated in Fig. 1AGo was observed. However, dose-response characterization was complicated by the occurrence of desensitization (observed in 11 of the 19 responsive neurons; example illustrated in Fig. 1BGo). We also observed significant differences in the threshold response to leptin, with some neurons responding to concentrations as low as 2 ng/ml (Fig. 1BGo), whereas other neurons did not respond until challenged with the highest leptin concentration, 32 ng/ml (example not shown).



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  		FIG. 1. Cytosolic calcium responses in cultured nodose neurons in response to bath application of leptin. The bars above the traces indicate period of ligand application. Bars labeled with numbers indicate leptin concentration (in nanograms per milliliter). The bar labeled KCl was when 55 mM KCl was applied as a test of cell viability. A, In this neuron, the leptin response is dose dependent. B, This neuron responded to 2 ng/ml but then had a prolonged period of desensitization.

 
In a second study of 20 leptin-responsive neurons, we determined whether the calcium response to leptin depended on extracellular calcium. Because of the desensitization phenomenon described above, we used two protocols to examine this question. In one, leptin was first applied in a calcium-free bath followed by a leptin challenge in the presence of bath calcium (Fig. 2AGo). In the second, protocol leptin was first applied in normal bath, followed by a leptin challenge in calcium-free bath, followed by a third leptin challenge in the normal bath (Fig. 2BGo). Although some desensitization of the response was typically observed with the third leptin challenge compared with the first response (Fig. 2BGo), the fact that a third response could still be induced indicated that the loss of response in the calcium-free baths was not due to desensitization. The average increase in cytosolic calcium in response to leptin (40 ng/ml) in the presence of bath calcium was 242 ± 32 nM. Removing bath calcium caused an almost complete abolition of the response (8 ± 2 nM increase, P < 0.0001 by a paired t test).



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  		FIG. 2. The cytosolic calcium response to leptin in nodose neurons is dependent on extracellular Ca2+. Bars labeled leptin indicate when neurons were challenged with 40 ng/ml leptin. Bars labeled Ca2+ free indicate when bath Ca2+ was replaced with Mg2+. The difference between leptin responses in Ca2+ containing and Ca2+ free baths was statistically significant (242 ± 32 nM vs. 8 ± 2 nM, paired t test; P < 0.0001, n = 20).

 
We next determined whether the cultured nodose neurons that responded to leptin also responded to CCK. In this study, 119 neurons were examined. Each neuron was exposed sequentially to leptin and CCK. In some neurons, the order of exposure was reversed. We found some neurons responded only to leptin (Fig. 3AGo), some neurons responded only to CCK (Fig. 3BGo), some neurons responded to both leptin and CCK (Fig. 3CGo), and some neurons responded to neither leptin nor CCK (example not shown). The distribution of the responsiveness of the neurons into the above four categories is illustrated in Fig. 3DGo. Overall, we found 24 of 119 responded to leptin alone (20%), 9 of 119 responded to CCK alone (8%), and 61 of 119 responded to neither leptin nor CCK (51%). A significant number of neurons (25 of 119 or 21%) were responsive to both leptin and CCK.



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  		FIG. 3. Calcium responses to leptin (40 ng/ml) and CCK (100 nM) in cultured nodose neurons. Bars above the traces indicate the period of ligand application. Cells were divided into four categories: responsive to leptin only (A), responsive to CCK only (B), response to both leptin and CCK (C), and nonresponsive (not shown). D, Relative percentage of each type of responsive neuron (n = 119).

 
We performed a {chi}2 analysis of the distribution to determine whether the occurrence of leptin and CCK responsiveness was random or clustered in the same neurons. If the distributions were random, we would expect the percentage of total leptin-responsive neurons (41%) to be the same in both CCK-responsive and -nonresponsive neurons, and vice versa; that is, the total percentage of CCK-responsive neurons (29%) should be the same in both leptin-responsive and -nonresponsive neurons. However, we observed that 74% of the CCK-responsive neurons responded to leptin (25 of 34), and 51% of the leptin-responsive neurons responded to CCK (25 of 49). This overlap of responsiveness was significantly greater than that expected if responsiveness was randomly distributed (Fig. 4Go).



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  		FIG. 4. Overlap in the distribution of leptin-responsive and CCK-responsive nodose neurons. The bars on left indicate the percentage of CCK-responsive (CCK+) and nonresponsive (CCK–) neurons that were also responsive to leptin. The bars on right indicate the percentage of leptin-responsive (lept+) and leptin-nonresponsive (lept–) neurons that were also responsive to CCK. The lines across the bars indicate the expected distribution of overlap in responsiveness if CCK responsiveness and leptin responsiveness were independent of one another. {chi}2 analysis of the distribution indicates that leptin and CCK responsiveness are not independent of one another and significantly cosegregate into the same population (P < 0.01, number of neurons represented by each bar are CCK+ 33, CCK– 86, leptin+ 49, leptin– 70).

 
Because leptin and CCK responsiveness tended to occur in the same nodose neurons, we tested for an interaction between CCK and leptin in activating calcium responses in the cultured neurons. The protocol for this experiment and sample responses are shown in Fig. 5Go. In the first set of experiments with nine responsive neurons, we used 8 ng/ml leptin and 10 nM CCK as the standard challenge. With this combination, we frequently observed quite large responses to CCK, whereas the responses to leptin were only moderate to small (Fig. 5Go, A and B). In some neurons, the combination of leptin and CCK produced an approximately additive effect (Fig. 5AGo), whereas in others there was a clear synergistic response (Fig. 5BGo). However, because of the relatively large CCK responses, the analysis of the summarized results did not reveal a synergistic interaction (Fig. 5DGo). We then repeated the experiment in another nine leptin-responsive neurons using a 1 nM CCK concentration. This CCK concentration produced only small to moderate responses to CCK itself (Fig. 5Go, C and E). Furthermore, under these conditions, neurons that had little or no response to CCK or leptin exhibited large increases in calcium when both substances were administered, suggesting a synergistic relationship for vagal afferent activation (Fig. 5Go, C and E).



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  		FIG. 5. Cytosolic calcium responses in individual cultured nodose neurons observed in response to leptin and CCK individually as well as coapplication of both ligands. The bars above the traces indicate period of hormone application. A and B, The concentration of CCK was 10 nM and leptin was 8 ng/ml. C, The concentration of CCK was reduced to 1 nM. KCl was 55 mM in all experiments. D, The average responses to leptin and CCK (10 nM) alone and coapplication of leptin and CCK are summarized. The response to coapplication was not significantly different from the sum of the individual responses (paired t test; n = 9). E, Average responses to leptin and CCK (1 nM) alone and coapplication of leptin and CCK are summarized. The response to coapplication was significantly greater than the sum of the individual responses (paired t test; n = 9; P = 0.039).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main findings of this study were first that leptin is capable of direct, acute activation of vagal afferent neurons and second that most leptin-responsive nodose neurons also are CCK responsive. Finally, we found that concentrations of leptin and CCK that were subthreshold for generating a calcium response when applied alone could strongly activate nodose neurons when applied together. This finding suggests that leptin and CCK may act synergistically to activate vagal afferents.

Previous reports indicate that leptin increases the firing rate of subpopulations of vagal afferent fibers in vivo (19, 25). These provocative in vivo extracellular recording results, taken together with the fact that leptin receptors have been identified in vagal afferent neurons (16, 17, 18), suggest that leptin might have potentially important effects on viscerosensory function. One limitation of these and other in vivo recordings, however, is that leptin receptor message and immunoreactivity also is expressed in other abdominal organs (26), such that vagal afferent responses could be secondary to leptin-induced responses in end-organ tissues. In addition, because at least some of leptin’s cellular effects are mediated via transcriptional mechanisms, the presence of leptin receptor in a neuronal population does not necessarily indicate that leptin can directly produce rapid changes in neuronal excitability. The evaluation of leptin effects in cultured vagal afferents obviates both of these limitations in the interpretation of in vivo results.

In the experiments reported here, leptin induced rapid increases in cytosolic calcium in a dose-dependent manner. However, the minimum leptin concentration for inducing a response varied from neuron to neuron, and we observed a significant rapid desensitization of the response that typically occurred after large calcium transients. The lowest concentration of leptin found to produce increased cytosolic calcium was 2 ng/ml, a concentration typical of circulating leptin levels in the rat (27, 28). However, many neurons that failed to respond to the 2 ng/ml leptin concentration responded robustly to higher doses. The highest leptin concentration we used was 40 ng/ml, a concentration that is comparable to plasma leptin levels in obese rats and humans (4, 29). Thus, the actions we observed in this in vitro preparation are likely to be relevant to the in vivo actions of leptin on these neurons.

An important consideration is the possible endogenous source of leptin that could act to regulate vagal afferent neurons. The primary source of circulating leptin is WAT and blood levels are in general proportional to fat tissue mass (30). However, leptin levels also rise during fasting and fall rapidly during refeeding after a fast (27, 28). Although it is possible that vagal afferents are responding to circulating leptin from WAT, leptin and its mRNA have also been detected in the gastric mucosa. Furthermore, both meals (31) and injection of exogenous CCK (32) appear to trigger secretion of gastric leptin. The observation of prandial secretion of gastric leptin is consistent with a paracrine mechanism for acute leptin-induced activation of vagal afferent neurons; much in the way that CCK is proposed to operate in inducing satiation (11). A paracrine mechanism of action suggests that relatively high local concentrations of leptin may act on vagal afferent terminals; thus, the responses to 40 ng/ml we observed may be physiologically consistent with such an action, even in nonobese animals. Although the total amount of leptin present in stomach is small (~1.4 ng) and suggested to be insufficient to influence circulating leptin levels significantly, a local paracrine action of leptin, coupled with the actions of other vagal stimuli, such as CCK, suggest that this source of leptin could have a significant physiological role in controlling vagally mediated physiological responses, such as satiation, gastrointestinal motility, and secretion.

The calcium response to leptin was dependent on extracellular calcium, indicating that leptin must be increasing membrane calcium conductance. Leptin has been shown to activate vagal afferent firing in situ (19, 25), and we have observed leptin-induced action potentials in the isolated nodose neuron preparation (our unpublished observation). These observations suggest that the calcium influx might be occurring as a result of action potential activity and influx through voltage-dependent calcium channels. If our previous suggestion that leptin might be acting in a paracrine manner at vagal afferent terminals is correct, then the calcium signals we observed at the cell bodies in the isolated nodose neuron preparation would likely occur in vivo as a result of action potentials invading the cell body as they transmit up the vagal afferent fiber to the brain stem. Such a calcium signal may result in calcium-induced changes at the level of the cell body, such as gene expression via calmodulin-dependent transcriptional activation.

Neurons that exhibited calcium responsiveness to leptin were significantly more likely to respond to CCK than neurons that did not respond to leptin and vice versa. The colocalization of acute responses to leptin with acute responses to CCK on vagal afferent neurons provides an anatomical substrate where leptin/CCK interaction may occur. Consistent with this idea, we found that not only did these signals occur in the same population of neurons, but also that additive and synergistic activation of vagal afferent neurons through the coapplication of CCK and leptin was present. Wang et al. (19) previously characterized vagal afferents sensitive to both leptin and CCK using a single-unit extracellular recording. Specifically, they demonstrated that CCK pretreatment enhanced leptin activation of a subpopulation of gastric vagal afferents. Additionally, Barrachina et al. (7) found that pretreatment with leptin synergistically enhanced CCK-induced satiation in mice. Our findings using cultured nodose neurons extend the in vivo evidence for additive or synergistic interactions between CCK and leptin via vagal afferents.

Leptin alters energy homeostasis by increasing energy mobilization and by decreasing food consumption (30, 33). Decreased 24-h food consumption in response to either peripheral or intracerebral leptin administration results from the reduction of individual meal size as opposed to reduction of meal frequency (30, 33). Reduction of meal size by leptin suggests that leptin sensitizes the process of satiation. Although it is possible that leptin sensitizes the satiation signals via its actions in the brain (30), our present work contributes to a growing body of evidence suggesting that leptin may influence satiation via an action directly on visceral afferents that participate in satiation through their monitoring of mechanical and humoral signals from the gastrointestinal tract. In addition, our findings are compatible with the hypothesis that activation of vagal afferent neurons by leptin alone, or in concert with CCK, directly contributes to vagally mediated satiation signals. The mechanistic basis for leptin/CCK interactions at the level of the neuronal membrane remains to be determined. However, it is likely that the primary mechanisms generating the calcium signals for leptin and CCK (23, 34) is alteration of membrane conductances, and thereby the resting membrane potential of the vagal afferent neurons. Whether they target the same conductance or different conductances will require additional experiments.

In conclusion, we have established that leptin directly activates vagal afferent neurons isolated from adult rat nodose ganglia. Additionally, we demonstrated that leptin responsiveness is highly colocalized with CCK responsiveness in nodose neurons. Our data show that coapplication of both these agents triggers enhanced increases in cytosolic calcium, supporting synergistic or cooperative effects on vagal afferent functions. Generalization of these findings suggests that a significant degree of integration of multiple visceral signals is likely to occur at the level of vagal afferent activation.


   Footnotes
 
This work was supported by National Institutes of Health Grant NS20561 and a grant from the Autzen Endowment.

Abbreviations: CCK, Cholecystokinin; HDMEM, HEPES-buffered DMEM; WAT, white adipose tissue.

Received February 19, 2004.

Accepted for publication April 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2006; 290(6): R1542 - R1543.
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J. Sanchez, P. Oliver, O. Miralles, E. Ceresi, C. Pico, and A. Palou
Leptin Orally Supplied to Neonate Rats Is Directly Uptaken by the Immature Stomach and May Regulate Short-Term Feeding
Endocrinology, June 1, 2005; 146(6): 2575 - 2582.
[Abstract] [Full Text] [PDF]

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