AJP - GI Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 293: G484-G492, 2007. First published June 14, 2007; doi:10.1152/ajpgi.00116.2007
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/2/G484    most recent
00116.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wan, S.
Right arrow Articles by Travagli, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wan, S.
Right arrow Articles by Travagli, R. A.

NEUROREGULATION AND MOTILITY

Cholecystokinin-8s excites identified rat pancreatic-projecting vagal motoneurons

Shuxia Wan,1,2 F. Holly Coleman,1 and R. Alberto Travagli1

1Department of Neuroscience, Pennington Biomedical Research Center-Louisiana State University System, Baton Rouge, Louisiana; and 2Key Laboratory of Allergy and Immune-Related Diseases-Department of Physiology, School of Basic Medical Science, Wuhan University, Wuhan, Hubei, People's Republic of China

Submitted 7 March 2007 ; accepted in final form 12 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
It is known that cholecystokinin (CCK) acts in a paracrine fashion to increase pancreatic exocrine secretion via vagal circuits. Recent evidence, however, suggests that CCK-8s actions are not restricted to afferent vagal fibers, but also affect brain stem structures directly. Within the brain stem, preganglionic neurons of the dorsal motor nucleus of the vagus (DMV) send efferent fibers to subdiaphragmatic viscera, including the pancreas. Our aims were to investigate whether DMV neurons responded to exogenously applied CCK-8s and, if so, the mechanism of action. Using whole cell patch-clamp recordings we show that perfusion with CCK-8s induced a concentration-dependent excitation in ~60% of identified pancreas-projecting DMV neurons. The depolarization was significantly reduced by tetrodotoxin, suggesting both direct (on the DMV membrane) and indirect (on local synaptic circuits) effects. Indeed, CCK-8s increased the frequency of miniature excitatory currents onto DMV neurons. The CCK-A antagonist, lorglumide, prevented the CCK-8s-mediated excitation whereas the CCK-B preferring agonist, CCK-nonsulfated, had no effect, suggesting the involvement of CCK-A receptors only. In voltage clamp, the CCK-8s-induced inward current reversed at –106 ± 3 mV and the input resistance increased by 150 ± 15%, suggesting an effect mediated by the closure of a potassium conductance. Indeed, CCK-8s reduced both the amplitude and the time constant of decay of a calcium-dependent potassium conductance. When tested with pancreatic polypeptide (which reduces pancreatic exocrine secretion), cells that responded to CCK-8s with an excitation were, instead, inhibited by pancreatic polypeptide. These data indicate that CCK-8s may control pancreas-exocrine secretion also via an effect on pancreas-projecting DMV neurons.

brain stem; dorsal vagal complex; electrophysiology

CHOLECYSTOKININ (CCK) is released from intestinal cells following a meal containing proteins or fats (30, 36, 45). Once released, CCK is an effective endogenous gastroinhibitor and short-term anorexigenic agent. At the level of the pancreas, however, CCK acts as a potent stimulator of pancreatic exocrine secretion via vagally mediated effects (reviewed in Refs. 35, 38, 40, 41, 46, 55, 57).

CCK-immunoreactive neurons are also present in many areas of the central nervous system (CNS), including the caudal brain stem, where CCK may be considered a neurotransmitter (20, 22, 26).

Several studies have demonstrated that the vagally mediated effects of the cholecystokinin octapeptide (CCK-8s) are due almost exclusively to a paracrine action of CCK-8s on peripheral, capsaicin-sensitive C-type vagal afferent fibers (5, 18, 38, 40). A large volume of in vitro studies have emerged recently to suggest alternative sites of action for CCK; in fact, studies have demonstrated that CCK-8s can depolarize vagal afferent (A-{delta}) neurons of the nodose ganglion (50), neurons of the tractus solitarius (2–4), identified gastric-projecting neurons of the dorsal motor nucleus of the vagus (DMV) (58), area postrema neurons (51), as well as other brain stem neurons of the vagal complex not identified as to their target organ (8, 39). The available evidence appears to indicate, therefore, that the effects of CCK are not limited to a paracrine effect at peripheral, vagal C fibers, but other vagal sites and mechanisms of action are involved. Although we do not dispute the powerful and well-documented paracrine effects of CCK on peripheral vagal afferent fibers, we should like to suggest, though, that CCK also modulates pancreatic protein secretion via actions at other sites, including brain stem vagal neurons.

We have applied a well-established labeling technique (12, 13, 21, 33, 34) to label and identify pancreas-projecting vagal motoneurons for electrophysiological studies (10, 11, 54). With this in mind, this study was designed to investigate the influence of CCK-sensitive circuits on the control of putative pancreatic protein secretion.

The aims of the present study were thus to investigate 1) the mechanism of action of CCK-8s on identified pancreas-projecting DMV neurons; and, since it is well accepted that pancreatic polypeptide (PP) modulates protein secretion via brain stem vagal pathways (11, 19; reviewed in Refs. 14, 56), 2) the effects of CCK-8s and PP on identified pancreas-projecting DMV neurons.

Preliminary accounts were presented at the Digestive Disease Week meeting in Chicago (2006).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Research reported in the present manuscript fully conforms to National Institutes of Health guidelines and was approved by the Pennington Biomedical Research Center-Louisiana State University System Institutional Animal Care and Use Committee.

Retrograde tracing. Pancreas-projecting DMV neurons were labeled as described previously (10, 11). Briefly, 10- to 12-day-old Sprague-Dawley rat pups of either sex were anesthetized deeply [3% solution of isoflurane with air (400–600 ml/min); abolition of the foot-pinch withdrawal reflex]. A laparotomy was performed, following which crystals of the neuronal tracer, DiI, [1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiIC18) (3); Molecular Probes, Eugene, OR] were applied to the body of the pancreas. The application site (which covered an area ~3–5 mm2) was embedded in a fast-hardening epoxy resin that was allowed to dry for several minutes before the entire surgical area was washed with warm saline. The wound was closed with 5-0 suture and the animal allowed to recover for 10–15 days.

Electrophysiology. The brain stems were removed as described previously (10, 11, 13). Briefly, the rats were anesthetized with isoflurane before administration of a bilateral pneumothorax. The brain stem was removed and placed in oxygenated Krebs’ solution at 4°C (see Solution composition). The brain stem was used only from those animals in which the glue covering the site of DiI application was still in place at the time of the experiment. By use of a vibratome, six to eight coronal sections (2–300 µm thick) containing the dorsal vagal complex (DVC) were cut and stored in oxygenated Krebs solution at 30°C for at least 1 h before use. A single slice was transferred to a custom-made perfusion chamber (volume 500 µl) and kept in place with a nylon mesh. The chamber was maintained at 35 ± 1°C by perfusion with warmed, oxygenated Krebs’ solution at a rate of 2.5–3.0 ml/min.

Prior to electrophysiological recording, pancreas-projecting DMV neurons were identified by using a Nikon E600-FN microscope equipped with tetramethylrhodamine isothiocyanate epifluorescent filters. Once the identity of a labeled neuron was confirmed, whole cell recordings were made under bright-field illumination using DIC (Nomarski) optics.

Whole cell recordings were made with patch pipettes (2–5 M{Omega} resistance) filled with a potassium gluconate solution (see below) using an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Recordings were made only from neurons unequivocally labeled with DiI. Data were sampled every 50 µs and filtered at 2 kHz, digitized via a Digidata 1320 interface (Axon Instruments) and acquired, stored, and analyzed on a personal computer utilizing pClamp 9 software (Axon Instruments). Recordings were accepted only if the series resistance was <20 M{Omega}. In all voltage-clamp experiments the cells were held at –50 mV. Current-voltage relationship curves were constructed by stepping the membrane from –50 to –120 mV in –10-mV increments for 0.4 s. The input resistance was calculated by measuring the instantaneous current displacement obtained by stepping the membrane from –50 to –70 mV. To assess the effects of drugs, each neuron served as its own control (i.e., the results obtained after administration of a drug were compared with those before administration by the Student's paired t-test). Cells were classified as responders if CCK-8s (100 nM) induced a current ≥ ±20 pA or, in the current-clamp configuration, doubled the firing rate. Concentration-response curves were constructed from neurons in which at least three concentrations of CCK-8s were tested. At least 5 min were allowed between successive drug applications. Antagonists were applied for 10 min before reapplication of the agonist (Fig. 1). The EC50 was calculated using Statistica software (StatSoft, Tulsa, OK); for each set of responses, the results are expressed as an average. To assess the effects of CCK-8s on the action potential afterhyperpolarization (AHP) or on the calcium-dependent potassium current, neurons were current clamped at –60 mV and a single action potential was evoked. CCK-8s (100 nM) was then applied by superfusion; once the response reached plateau, the membrane potential was restored to control values by direct current injection before the protocol was repeated. In voltage clamp, neurons were held at –60 mV then step depolarized to 0 mV for 40 ms and returned to –60 mV. Only one cell per slice was tested. The time constant of decay was fit using a subroutine of the clampfit software (Axon Instruments).


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 1. Cholecystokinin-8s (CCK-8s) depolarizes a subpopulation of pancreas-projecting dorsal motor nucleus of the vagus (DMV) neurons. A: representative current-clamp traces illustrating the concentration-dependent increase in firing rate induced by perfusion of a pancreas-projecting DMV neuron with CCK-8s. A recovery period of at least 10 min was allowed between successive applications. Parallel lines indicate a 2-min interval. Holding potential = –60 mV. B: concentration response curve for the CCK-8s-induced increase in firing rate (bullet; holding potential = –60 mV) and depolarization ({square}; holding potential = –65 mV) expressed as means ± SE. Each neuron was tested with at least 3 different concentrations of CCK-8s.

 
Morphological reconstructions. At the conclusion of electrophysiological recording, Neurobiotin was injected into the DMV neuron as described previously (13, 32) and the brain stem slice was fixed overnight in Zamboni's fixative at 4°C. The fixative was cleared from the slice with multiple washes of phosphate buffer solution-Triton X-100 (PBS-TX) (see Solution composition) and the injected Neurobiotin was visualized by using a cobalt-nickel enhancement of the Avidin D-horseradish peroxidase (Avidin D-HRP) technique as described previously (13, 32).

Three-dimensional reconstructions of the individual Neurobiotin-labeled neurons, digitized at a final magnification of x600, were made using Neurolucida software (Microbrightfield, Williston, VT) as described previously (13, 32). Included in the morphological features assessed were soma area and diameter, form factor (a measure of circularity for which a value of 1 indicates a perfect circle and 0 indicates a line; form factor = 4{pi}a x 1/p2, where a is soma area and p is the perimeter of the soma in the horizontal plane), whether the cell is bipolar or multipolar, number of segments (i.e., branching of dendrites), branch order and extension in the x- and y-axes, and termination of the dendrites (i.e., with at least one dendrite ending in apposition to the central canal/fourth ventricle or not). Data analysis was performed as described previously (13, 32).

Solution composition. Krebs solution consisted of (in mM) 126 NaCl, 25 NaHCO3, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, and 11 dextrose, maintained at pH 7.4 by bubbling with 95% O2-5% CO2. Intracellular solution consisted of (in mM) 128 potassium gluconate, 10 KCl, 0.3 CaCl2, 1 MgCl2, 10 HEPES, 1 EGTA, 2 ATP, 0.25 GTP; adjusted to pH 7.35 with KOH. Zamboni's fixative consisted of 1.6% (wt/vol) paraformaldehyde, 19 mM KH2PO4, and 100 mM Na2HPO4 in 240 ml saturated picric acid-1,600 ml H2O; adjusted to pH 7.4 with HCl. PBS contained (in mM) 115 NaCl, 75 Na2HPO4·7H2O, 7.5 KH2PO4 (0.15% Triton X-100). Avidin D-HRP solution consisted of 0.05% diaminobenzidine (DAB) in PBS containing 0.5% gelatin supplemented with 0.025% CoCl2, and 0.02% NiNH4SO4.

Statistical analysis. Data are expressed as means ± SEM. We used Student's paired (to compare baseline values vs. response after treatment) or grouped (to compare the effects of drug treatments in the different experimental procedures) t-test or {chi}2 test. Significance was defined as P < 0.05.

Drugs and chemicals. Capsaicin was purchased from Tocris (Ellisville, MO), the nonsulfated form of CCK (CCK-ns) was purchased from Bachem (King of Prussia, PA); DiI was purchased from Molecular Probes (Eugene, OR); all other chemicals were purchased from Sigma (St. Louis, MO). For the in vitro experiments, drugs were made fresh immediately before use and were applied to the bath via a series of manually operated valves.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANT
 REFERENCES
 
Data were obtained only from those cells showing the brightest and most intense DiI fluorescence. The postsynaptic response to CCK-8s was tested on 154 pancreas-projecting DMV cells; the presynaptic response to CCK-8s was tested on 33 DMV neurons. The soma shape was reconstructed in 75 neurons of which we were able to obtain measures of dentritic length in 32 neurons.

Postsynaptic effects of CCK-8s. In 59% of neurons tested (91 of 154), perfusion with 100 nM CCK-8s induced an excitatory response in both current- and voltage-clamp configurations. Inhibitory or biphasic responses (i.e., inward followed by outward current, or depolarization followed by hyperpolarization) were never observed. The remaining 41% of neurons (63 of 154) showed no postsynaptic response to CCK-8s.

In the current-clamp configuration, perfusion with CCK-8s (1–300 nM) induced a concentration-dependent depolarization; the threshold for the response was ~1 nM, the EC50 was ~10 nM, and the maximal response (12 ± 1.47 mV) was obtained at 100 nM (Fig. 1). Sixteen responsive neurons tested in the current-clamp configuration were also tested in the voltage-clamp configuration (holding potential –50 mV). In these neurons, perfusion with 100 nM CCK-8s induced a –70 ± 7.1-pA inward current.

When two applications of 100 nM CCK-8s were conducted within 10 min of each other, the CCK-8s-induced response did not show tachyphylaxis. In detail, the first superfusion of CCK-8s increased the firing rate from 3 ± 1.3 to 113.4 ± 34.7 action potentials/min whereas the second superfusion of CCK-8s induced an increase from 4 ± 2.9 to 117 ± 28.8 action potentials/min (n = 5; P < 0.05). Similarly, in a further six neurons that were current clamped at –65 mV, the first perfusion with 100 nM CCK-8s induced a 9 ± 1.4-mV depolarization whereas the second perfusion of CCK-8s induced a 7.6 ± 1.9-mV depolarization (P < 0.05). Finally, when measured in the voltage-clamp configuration, the first superfusion of CCK-8s induced a –102 ± 11.2-pA inward current whereas the second superfusion of CCK-8s induced a –87 ± 17.4-pA inward current (i.e., 83 ± 11% of the first application, P > 0.05; n = 4; data not shown).

The CCK-8s-induced effects were reduced, but not abolished, following superfusion with tetrodotoxin (TTX, 1 µM) to block action potential mediated synaptic transmission. For example, in four neurons in which 100 nM CCK-8s induced a 9 ± 0.9 mV depolarization, the amplitude of the membrane displacement was reduced to 6.2 ± 1.6 mV (data not shown). Similarly, in control conditions perfusion with 100 nM CCK-8s induced a –61 ± 15.1-pA inward current that was reduced to –24 ± 5.8 pA in the presence of TTX (P < 0.05 vs. CCK-8s alone; n = 5). These data indicate that the effects of CCK-8s on pancreas-projecting DMV neurons are determined mainly by an effect on the neuronal membrane itself, but also by an effect on nerve terminals apposing the DMV cells.

To characterize the pharmacology of the CCK-8s-mediated effects, the effects of the CCK-A receptor antagonist, lorglumide (1 µM), were assessed. In five pancreas-projecting DMV neurons, perfusion with 100 nM CCK-8s induced a –71 ± 11-pA inward current. Following washout of CCK-8s and 10 min of superfusion with lorglumide, which per se did not have any effect on the holding current, the CCK-8s-induced inward current was reduced to –20 ± 6.1 pA (P < 0.05; Fig. 2). Similarly, lorglumide pretreatment reduced the CCK-8s-induced increase in the firing rate (4 ± 3.0 and 137 ± 27.1 action potentials/min in control and CCK-8s, respectively; 4 ± 2.9 and 28 ± 13.5 action potentials/min in lorglumide and lorglumide+CCK-8s, respectively; n = 6; P < 0.05; data not shown) and the CCK-8s-induced membrane depolarization (12 ± 1.9 mV in CCK-8s and 3 ± 0.8 mV in lorglumide+CCK-8s; n = 6; P < 0.05; data not shown).


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 2. Excitatory effects of CCK-8s are mediated via activation of CCK-A receptors. A: representative traces illustrating the inward current induced by perfusion with 100 nM CCK-8s on a DMV neuron voltage clamped at –50 mV (top trace). Following 10-min perfusion with the selective CCK-A antagonist lorglumide (Lorgl; 1 µM), the inward current induced by CCK-8s was reduced significantly (bottom trace). B: graphic summarizing the response to 100 nM CCK-8s alone and in the presence of 1 µM lorglumide. *P < 0.05. C: representative traces illustrating the inward current induced by perfusion with 100 nM CCK-8s on a DMV neuron voltage clamped at –50 mV (top trace). Following 10 min washout, perfusion with 1 µM of the nonsulfated form of CCK (CCK-ns) induced a small inward current (middle trace) that was completely antagonized by pretreatment with lorglumide (1 µM; bottom trace). D: graphic summarizing the response to 100 nM CCK-8s, 1 µM CCK-ns alone, and 1 µM CCK-ns in the presence of 1 µM lorglumide. *P < 0.05.

 
The effects of CCK-B receptor-preferring nonsulfated form of CCK (CCK-ns; 1 µM) were tested on 10 pancreas-projecting DMV neurons that previously responded to CCK-8s. Perfusion with 100 nM CCK-8s induced a –75 ± 6-pA inward current whereas perfusion with CCK-ns induced a –15 ± 4.1-pA inward current that was antagonized completely upon perfusion with lorglumide (Fig. 2). These data indicate that the effects of CCK-8s on pancreas-projecting DMV neurons are determined exclusively by an effect on CCK-A receptors and that the small effects of CCK-ns are due to its low affinity for the CCK-A receptor (1).

To analyze the reversal potential of the CCK-8s-induced inward current, neurons were voltage clamped at –50 mV and then step-hyperpolarized in 10-mV increments to –110 mV. In seven neurons responsive to CCK-8s, the reversal potential of the induced current was –106 ± 3.3 mV (Fig. 3). CCK-8s increased the input resistance reversibly from 330 ± 17 to 504 ± 64 M{Omega} (i.e., 151 ± 15% of control; P < 0.05; 303 ± 36 M{Omega} following washout). These data suggest that the excitatory effects of CCK-8s on the membrane of pancreas-projecting DMV neurons are determined by the closure of a potassium current.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 3. Excitatory effects of CCK-8s are mediated via the closure of a potassium conductance. A: representative traces showing the current-voltage relationship of a pancreas-projecting DMV neuron voltage clamped at –50 mV and stepped at –110 mV in 10-mV increments every 5 s; note the lack of effects of CCK-8s on the fast transient outward current. B: summary graphic showing the current-voltage relationship (means ± SE) in control and the presence of 100 nM CCK-8s. Note the reversal potential of the net current is close to EK. C: summary graphic comparing the mean input resistance in control and following perfusion with 100 nM CCK-8s. *P < 0.05.

 
The effects of CCK-8s on action potential characteristics were assessed in six pancreas-projecting DMV neurons responsive to CCK-8s as described above. CCK-8s did not affect the duration of the action potential, but both the amplitude and duration of the AHP were decreased (14 ± 1.4 mV and 53 ± 11.2 ms in control vs. 11 ± 1.9 mV and 34 ± 6.9 ms in CCK-8s, respectively; P < 0.05 for both; Fig. 4). The effects of CCK-8s on the calcium-dependent potassium current that underlies the action potential AHP were further investigated in the voltage-clamp configuration. Neurons were voltage clamped at –60 mV then step depolarized to 0 mV for 40 ms before being returned to –60 mV. In control conditions, the evoked current had an amplitude of 450 ± 47 pA and a decay constant of 43 ± 4.8 ms, whereas in the presence of 100 nM CCK-8s, the peak amplitude was 392 ± 40 pA and the decay was 35 ± 3.3 ms (n = 10; P < 0.05 for both; Fig. 4). These data suggest that the potassium current that underlies the excitatory actions of CCK-8s is likely to be a calcium-dependent potassium current (48, 53).


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 4. Excitatory effects of CCK-8s are mediated via a decrease in the afterhyperpolarization (AHP) current. A: current-clamp traces show a single action potential in a neuron excited by 100 nM CCK-8s. The holding potential of the cell was restored to control values during perfusion with CCK-8s via direct current injection. Note that the AHP amplitude and duration were decreased by CCK-8s. B: summary comparing the means ± SE. of the amplitude (left) and time constant of decay ({tau}; right) of the AHP in control and following CCK-8s perfusion. *P < 0.05. C: voltage-clamp traces illustrating the calcium-dependent potassium current (IKCa) in a neuron excited by 100 nM CCK-8s. The cell was clamped at –60 mV and stepped to 0 mV for 40 ms before being returned to –60 mV; the IKCa is the fast-decaying outward current seen at the end of the pulse. Note that the amplitude and duration of the current were decreased during perfusion with CCK-8s. D: summary comparing the means ± SE of the amplitude (left) and time constant of decay ({tau}; right) of the IKCa in control and following CCK-8s perfusion. *P < 0.05

 
In 31 pancreas-projecting DMV neurons, the effects of both CCK-8s and PP, which is known to reduce pancreatic exocrine secretion via a vagally mediated mechanism (11, 19, reviewed in Refs. 14, 56) were tested. Of the 19 neurons that were excited by CCK-8s, 12 were inhibited by perfusion with 100 nM PP whereas 7 were unresponsive. In a further 12 neurons unresponsive to CCK-8s, 5 were also unresponsive to PP whereas 7 were responsive to PP only (4 neurons were hyperpolarized by PP and 3 were depolarized; data not shown). These data indicate that the neurons responsive to CCK-8s are most likely involved in the control of pancreatic exocrine functions.

Morphological reconstructions. Of the 154 neurons in which we tested the postsynaptic effects of CCK-8s, complete morphological reconstructions were obtained for 32 cells (16 responsive and 16 nonresponsive to CCK-8s). We reported previously that pancreas-projecting neurons had either a bipolar or a multipolar soma morphology (10). In the present study, differences were not found in terms of somatic shape between responsive and nonresponsive cells (in the responsive group 14 of 16 cells were multipolar compared with 11 of 16 in the unresponsive group; P > 0.05; data not shown). Surprisingly, when considering the orientation of dendritic projections, neurons unresponsive to CCK-8s were most likely to project along the x-axis (i.e., mediolateral) and had longer segments than neurons responding to CCK-8s. There were no differences between responsive vs. nonresponsive neurons with regard to projections to the central canal or to the ependymal layer of the fourth ventricle, or with regard to their rostrocaudal distribution. Morphological measures are reported in Table 1.


View this table:
[in this window]
[in a new window]

  		Table 1. Morphological properties of pancreas-projecting DMV neurons tested with 100 nM CCK-8s

 
Presynaptic effects of CCK-8s. Spontaneous glutamatergic events were studied in 20 pancreas-projecting DMV neurons in the presence of 50 µM picrotoxin, to block GABAergic synaptic currents. In 8 of the 20 neurons, perfusion with 100 nM CCK-8s increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) from 4.3 ± 1.09 to 8.7 ± 2.1 events/s (P < 0.05), leaving the amplitude of the events unaltered (28 ± 2.7 pA in control and 29 ± 2.3 in CCK-8s; P > 0.05; Fig. 5). In three of these neurons, perfusion with CCK-8s in the presence of 1 µM TTX increased the frequency of miniature excitatory postsynaptic currents from 2.8 ± 0.6 to 3.7 ± 0.7 events/s (P < 0.05), suggesting a presynaptic site of action.


Figure 5
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 5. CCK-8s increases the frequency of spontaneous excitatory postsynaptic currents (sEPSC) via activation of CCK-A receptors. A: representative current traces showing that perfusion with 100 nM CCK-8s (top middle) but not with 1 µM CCK-ns (top right) increased the frequency of sEPSC. Perfusion with 1 µM lorglumide (left middle) did not affect the frequency of sEPSC but reduced significantly the response to CCK-8s (left bottom). Holding potential = –60 mV. B: summary graphics showing the lack of effects on the amplitude of the sEPSC. C: summary graphics showing the effects on the frequency of the sEPSC.

 
The CCK-8s induced increase in the frequency of sEPSC was antagonized by pretreatment with lorglumide (1 µM). In fact, in neurons in which CCK-8s increased the frequency of spontaneous events to 198 ± 42% of control (P < 0.05), CCK-8s had no effect on sEPSC frequency in the presence of lorglumide (96 ± 11.1% of control; n = 3; P > 0.05). Similarly, in neurons where CCK-8s increased the frequency of sEPSC from 3.4 ± 1.2 to 8.1 ± 3.2 events/min (P < 0.05), perfusion with CCK-ns had no effect (i.e., 105 ± 6% of control; n = 3; P > 0.05). These data indicate that the presynaptic effects of CCK-8s were also mediated by CCK-A receptors.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANT
 REFERENCES
 
In this study, we have shown that 1) CCK-8s induces a CCK-A receptor-mediated concentration-dependent depolarization in ~60% of identified pancreas-projecting DMV neurons; 2) the CCK-8s-induced direct depolarization of DMV neurons is mediated by the closure of potassium channels, most probably the calcium-dependent potassium conductance underlying the action potential AHP; 3) the CCK-8s-induced indirect effects are, instead, mediated by an increased release of glutamate from terminals apposing DMV neurons; and 4) if a DMV neuron responds both to CCK-8s and to PP, the response to PP is always inhibitory.

The powerful and well-documented paracrine effects of CCK on peripheral vagal afferent fibers are well accepted, but actions at other sites, including brain stem vagal neurons, must be taken into account. Given that the postprandial concentration of gut-derived CCK is in the low picomolar range (55), however, we cannot rule out the possibility that the CCK acting on brain stem vagal circuits originates from some other source, including local CCK-containing NTS neurons or CCK of peripheral origin reaching the DVC through the locally leaky blood brain barrier (20, 28, 52). Strong behavioral, immunohistochemical, and electrophysiological evidence supports a hormonelike action of CCK on CNS circuits (3, 6, 27, 41, 58). Furthermore, we report herein a threshold concentration of CCK of ~1 nM, which could potentially have a physiological effect, since it is well accepted that in vitro experimental procedures induce a rightward shift of the concentration-response curve to agonists (23).

The data from this study suggest that CCK-8s induces a direct activation of CCK-A receptors present on identified pancreas-projecting DMV neurons. Our supporting evidence is the following.

Approximately 50% of pancreas-projecting DMV neurons were depolarized by CCK-8s, similar to previous reports that showed ~40% of gastric-projecting or 43% of unidentified DMV neurons were depolarized by CCK-8s (39, 58). Pancreas-projecting DMV neurons were never hyperpolarized by CCK-8s, in line with our previous report that an insignificant proportion (3 of 267) of gastric-projecting DMV neurons were inhibited by CCK-8s (58). Our data, showing that CCK-8s induces an excitatory effect in pancreas-projecting DMV neurons, are in agreement with previously reported effects of CCK-8s in the DVC (2–4) as well as in other CNS areas (16, 17, 37, 51).

The CCK-8s-induced inward current was attenuated by pretreatment with the selective CCK-A receptor antagonist lorglumide. Conversely, the CCK-B-preferring receptor agonist, CCK8-ns, did not induce any significant effect in pancreas-projecting DMV neurons, even at high concentrations. The small effects observed upon perfusion with CCK-ns are likely due therefore to its low-affinity interaction with CCK-A receptors (1). Indeed, pretreatment with the selective CCK-A receptor antagonist lorglumide antagonized completely the effects of CCK-ns. These data suggest that the excitatory effects of CCK-8s are mediated via interaction with CCK-A receptors only, in agreement with in vivo data on pancreatic and other gastrointestinal functions (24, 29, 31, 35, 38, 41, 42). Of particular relevance are the data showing that the CCK-mediated effects at the level of the brain stem are mediated via activation of CCK-A receptors (6, 24, 42).

The CCK-8s-induced inward current reversed close to the equilibrium potential for potassium and was accompanied by an increased input resistance, suggesting that the excitatory effects of CCK-8s are mediated via closure of a potassium conductance, in agreement with results obtained in other CNS regions (7, 16, 39, 58). The present study also showed that the response to CCK-8s was reduced significantly following blockade of action potential-dependent synaptic transmission with TTX, implying that the excitatory response to CCK-8s is mediated at least partially via actions on synaptic inputs impinging onto DMV neurons, similar to observations in the parabrachial nucleus and hippocampus (9, 49). Several studies have shown an excitatory effect of CCK on brain stem areas that have robust projections to the DMV, such as the NTS and area postrema, which are preserved in our experimental conditions and might provide functional synaptic inputs to the DMV even in the slice preparation we use (2–4, 6–8, 24, 43, 44). Indeed, our data show a robust increase in glutamatergic synaptic transmission to DMV neurons mediated by CCK-A receptors.

In conclusion, the data presented herein suggest strongly that the mechanism of action of CCK requires the combined activity of CCK at different levels of brain stem vagal circuits, i.e., NTS glutamatergic terminals and DMV neurons. These effects are not surprising since portions of both the NTS and the DMV have a leaky blood-brain barrier, fenestrated capillaries, and enlarged perivascular space that allows the passage of large molecules (15, 25, 47), implying that circulating CCK may reach these neuronal circuits. Furthermore there is functional evidence that CCK can cross the blood-brain barrier to activate a solitarius-nigral pathway (27), to phosphorylate CCK-A receptors in the DVC (58), and to induce short-term satiety (6). Our electrophysiological data indicating that CCK-8s excites a subpopulation of pancreas-projecting neurons via a CCK-A mediated effect support a direct CNS-mediated effect on pancreatic function. We recognize that it is impossible to assess the physiological function of any heterogeneous neuronal population, including that comprising the pancreas-projecting DMV neurons, in a slice preparation. The fact that most of the neurons excited by CCK-8s were inhibited by PP, which is well recognized as an inhibitor of exocrine secretion (reviewed in Refs. 14, 56), supports the concept that CCK-8s-responsive motoneurons comprise the preganglionic cells that modulate pancreatic protein output.


   GRANT
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
REFERENCES
 
This research was supported by National Science Foundation Grant 0456291.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Browning and Berthoud for comments on earlier versions of the manuscript. We also thank Cesare M. Travagli for support and encouragement.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Travagli, Dept. of Neuroscience, Pennington Biomedical Research Center, Louisiana State Univ. System, 6400 Perkins Rd., Baton Rouge, LA 70808 (e-mail: alberto.travagli{at}pbrc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 

  1. Alexander SP, Mathie A, Peters JA. 7TM receptors. Br J Pharmacol 150, Suppl 1: S4–S81, 2007.[CrossRef][Medline]
  2. Appleyard SM, Bailey TW, Doyle MW, Jin YH, Smart JL, Low MJ, Andresen MC. Proopiomelanocortin neurons in nucleus tractus solitarius are activated by visceral afferents: regulation by cholecystokinin and opioids. J Neurosci 25: 3578–3585, 2005.[Abstract/Free Full Text]
  3. Baptista V, Browning KN, Travagli RA. Effects of cholecystokinin-8s in the nucleus tractus solitarius of vagally deafferented rats. Am J Physiol Regul Integr Comp Physiol 292: R1092–R1100, 2007.[Abstract/Free Full Text]
  4. Baptista V, Zheng Z, Coleman FH, Rogers RC, Travagli RA. Cholecystokinin octapeptide increases spontaneous glutamatergic synaptic transmission to neurons of the nucleus tractus solitarius centralis. J Neurophysiol 94: 2763–2771, 2005.[Abstract/Free Full Text]
  5. Blackshaw LA, Grundy D. Effects of cholecystokinin (CCK-8) on two classes of gastroduodenal vagal afferent fibre. J Auton Nerv Syst 31: 191–202, 1990.[CrossRef][Web of Science][Medline]
  6. Blevins JE, Stanley BG, Reidelberger RD. Brain regions where cholecystokinin suppresses feeding in rats. Brain Res 860: 1–10, 2000.[CrossRef][Web of Science][Medline]
  7. Branchereau P, Bohme GA, Champagnat J, Morin-Surun MP, Durieux C, Blanchard JC, Roques BP, Denavit-Saubie M. CholecystokininA and cholecystokininB receptors in neurons of the brain stem solitary complex of the rat: pharmacological identification. J Pharmacol Exp Ther 260: 1433–1440, 1992.[Abstract/Free Full Text]
  8. Branchereau P, Champagnat J, Denavit-Saubie M. Cholecystokinin-gated currents in neurons of the rat solitary complex in vitro. J Neurophysiol 70: 2584–2595, 1993.[Abstract/Free Full Text]
  9. Breukel AI, Wiegant VM, Lopes da Silva FH, Ghijsen WE. Presynaptic modulation of cholecystokinin release by protein kinase C in the rat hippocampus. J Neurochem 70: 341–348, 1998.[Web of Science][Medline]
  10. Browning KN, Coleman FH, Travagli RA. Characterization of Pancreas-projecting rat dorsal motor nucleus of the vagus neurons. Am J Physiol Gastrointest Liver Physiol 288: G950–G955, 2005.[Abstract/Free Full Text]
  11. Browning KN, Coleman FH, Travagli RA. Effects of pancreatic polypeptide on pancreas-projecting rat dorsal motor nucleus of the vagus neurons. Am J Physiol Gastrointest Liver Physiol 289: G209–G219, 2005.[Abstract/Free Full Text]
  12. Browning KN, Kalyuzhny AE, Travagli RA. Mu-opioid receptor trafficking on inhibitory synapses in the rat brainstem. J Neurosci 24: 9344–9352, 2004.
  13. Browning KN, Renehan WE, Travagli RA. Electrophysiological and morphological heterogeneity of rat dorsal vagal neurones which project to specific areas of the gastrointestinal tract. J Physiol 517: 521–532, 1999.[Abstract/Free Full Text]
  14. Chey WY, Chang T. Neural hormonal regulation of exocrine pancreatic secretion. Pancreatology 1: 320–335, 2001.[CrossRef][Web of Science][Medline]
  15. Cottrell GT, Ferguson AV. Sensory circumventricular organs: central roles in integrated autonomic regulation. Regul Pept 117: 11–23, 2004.[CrossRef][Web of Science][Medline]
  16. Cox CL, Huguenard JR, Prince DA. Cholecystokinin depolarizes rat thalamic reticular neurons by suppressing a K+ conductance. J Neurophysiol 74: 990–1000, 1995.[Abstract/Free Full Text]
  17. Crawley JN, Corwin RL. Biological actions of cholecystokinin. Peptides 15: 731–755, 1994.[CrossRef][Web of Science][Medline]
  18. Davison JS, Clarke GD. Mechanical properties and sensitivity to CCK of vagal gastric slowly adapting mechanoreceptors. Am J Physiol Gastrointest Liver Physiol 255: G55–G61, 1988.[Abstract/Free Full Text]
  19. Deng X, Wood PG, Sved AF, Whitcomb DC. The area postrema lesions alter the inhibitory effects of peripherally infused pancreatic polypeptide on pancreatic secretion. Brain Res 902: 18–29, 2001.[CrossRef][Web of Science][Medline]
  20. Dockray GJ. Immunochemical evidence of cholecystokinin-like peptides in brain. Nature 264: 568–570, 1976.[CrossRef][Medline]
  21. Doyle MW, Bailey TW, Jin YH, Appleyard SM, Low MJ, Andresen MC. Strategies for cellular identification in nucleus tractus solitarius slices. J Neurosci Methods 137: 37–48, 2004.[CrossRef][Web of Science][Medline]
  22. Fallon JH, Seroogy KB. The distribution and some connections of cholecystokinin neurons in the rat brain. Ann NY Acad Sci 448: 121–132, 1985.[CrossRef][Web of Science][Medline]
  23. Fatt P, Katz B. An analysis of the end plate potential recorded with an intracellular electrode. J Physiol 115: 320–370, 1952.[Web of Science]
  24. Glatzle J, Kreis ME, Kawano K, Raybould HE, Zittel TT. Postprandial neuronal activation in the nucleus of the solitary tract is partly mediated by CCK-A receptors. Am J Physiol Regul Integr Comp Physiol 281: R222–R229, 2001.[Abstract/Free Full Text]
  25. Gross PM, Wall KM, Pang JJ, Shaver SW, Wainman DS. Microvascular specializations promoting rapid interstitial solute dispersion in nucleus tractus solitarius. Am J Physiol Regul Integr Comp Physiol 259: R1131–R1138, 1990.[Abstract/Free Full Text]
  26. Herbert H, Saper CB. Cholecystokinin-, galanin-, and corticotropin-releasing factor-like immunoreactive projections from the nucleus of the solitary tract to the parabrachial nucleus in the rat. J Comp Neurol 293: 581–598, 1990.[CrossRef][Web of Science][Medline]
  27. Hommer DW, Palkovits M, Crawley JN, Paul SM, Skirboll LR. Cholecystokinin-induced excitation in the substantia nigra: evidence for peripheral and central components. J Neurosci 5: 1387–1392, 1985.[Abstract]
  28. Kubota Y, Inagaki S, Shiosaka S, Cho HJ, Tateishi K, Hashimura E, Hamahoka T, Tohyama M. The distribution of cholecystokinin octapeptide-like structures in the lower brain stem of the rat: an immunohistochemical analysis. Neuroscience 9: 587–604, 1983.[CrossRef][Web of Science][Medline]
  29. Li Y, Hao Y, Owyang C. High-affinity CCK-A receptors on the vagus nerve mediate CCK-stimulated pancreatic secretion in rats. Am J Physiol Gastrointest Liver Physiol 273: G679–G685, 1997.[Abstract/Free Full Text]
  30. Liddle RA. Regulation of cholecystokinin synthesis and secretion in rat intestine. J Nutr 124: 1308S–1314S, 1994.[Abstract/Free Full Text]
  31. Lloyd KCK, Raybould HE, Walsh JH. Cholecystokinin inhibits gastric acid secretion through type "A" cholecystokinin receptors and somatostatin in rats. Am J Physiol Gastrointest Liver Physiol 263: G287–G292, 1992.[Abstract/Free Full Text]
  32. Martinez-Pena y Valenzuela IM, Browning KN, Travagli RA. Morphological differences between planes of section do not influence the electrophysiological properties of identified rat dorsal motor nucleus of the vagus neurons. Brain Res 1003: 54–60, 2004.[CrossRef][Web of Science][Medline]
  33. Mendelowitz D, Kunze DL. Identification and dissociation of cardiovascular neurons from the medulla for patch clamp analysis. Neurosci Lett 132: 217–221, 1991.[CrossRef][Web of Science][Medline]
  34. Mendelowitz D, Yang M, Andresen MC, Kunze DL. Localization and retention in vitro of fluorescently labeled aortic baroreceptor terminal on neurons from the nucleus tractus solitarius. Brain Res 581: 339–343, 1992.[CrossRef][Web of Science][Medline]
  35. Moran TH, Kinzig KP. Gastrointestinal satiety signals II. Cholecystokinin. Am J Physiol Gastrointest Liver Physiol 286: G183–G188, 2004.[Abstract/Free Full Text]
  36. Moran TH, Ladenheim EE, Schwartz GJ. Within-meal gut feedback signaling. Int J Obes Relat Metab Disord 25, Suppl 5: S39–S41, 2001.[CrossRef][Web of Science]
  37. Noble F, Wank SA, Crawley JN, Bradwejn J, Seroogy KB, Hamon M, Roques BP. International Union of Pharmacology. XXI. Structure, distribution, and functions of cholecystokinin receptors. Pharmacol Rev 51: 745–781, 1999.[Abstract/Free Full Text]
  38. Owyang C, Logsdon CD. New insights into neurohormonal regulation of pancreatic secretion. Gastroenterology 127: 957–969, 2004.[CrossRef][Web of Science][Medline]
  39. Plata-Salaman CR, Fukuda A, Oomura Y, Minami T. Effects of sulphated cholecystokinin octapeptide (CCK-8) on the dorsal motor nucleus of the vagus. Brain Res Bull 21: 839–842, 1988.[CrossRef][Web of Science][Medline]
  40. Raybould HE, Tache Y. Cholecystokinin inhibits gastric motility and emptying via a capsaicin-sensitive vagal pathway in rats. Am J Physiol Gastrointest Liver Physiol 255: G242–G246, 1988.[Abstract/Free Full Text]
  41. Reidelberger RD. Cholecystokinin and control of food intake. J Nutr 124: 1327S–1333S, 1994.[Abstract/Free Full Text]
  42. Reidelberger RD, Hernandez J, Fritzsch B, Hulce M. Abdominal vagal mediation of the satiety effects of CCK in rats. Am J Physiol Regul Integr Comp Physiol 286: R1005–R1012, 2004.[Abstract/Free Full Text]
  43. Rinaman L, Hoffman GE, Dohanics J, Le WW, Stricker EM, Verbalis JG. Cholecystokinin activates catecholaminergic neurons in the caudal medulla that innervate the parventricular nucleus of the hypothalamus in rats. J Comp Neurol 360: 246–256, 1995.[CrossRef][Web of Science][Medline]
  44. Rinaman L, Verbalis JG, Stricker EM, Hoffman GE. Distribution and neurochemical phenotypes of caudal medullary neurons activated to express cFos following peripheral administration of cholecystokinin. J Comp Neurol 338: 475–490, 1993.[CrossRef][Web of Science][Medline]
  45. Ritter RC. Gastrointestinal mechanisms of satiation for food. Physiol Behav 81: 249–273, 2004.[CrossRef][Medline]
  46. Ritter RC. Increased food intake and CCK receptor antagonists: beyond abdominal vagal afferents. Am J Physiol Regul Integr Comp Physiol 286: R991–R993, 2004.[Free Full Text]
  47. Rogers RC, Hermann GE, Travagli RA. Brainstem control of gastric function. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. San Diego, CA: Elsevier, 2005.
  48. Sah P, McLachlan EM. Potassium currents contributing to action potential repolarization and the afterhyperpolarization in rat vagal motoneurons. J Neurophysiol 68: 1834–1841, 1992.[Abstract/Free Full Text]
  49. Saleh TM, Kombian SB, Zidichouski JA, Pittman QJ. Cholecystokinin and neurotensin inversely modulate excitatory synaptic transmission in the parabrachial nucleus in vitro. Neuroscience 77: 23–35, 1997.[CrossRef][Web of Science][Medline]
  50. Simasko SM, Ritter RC. Cholecystokinin activates both A- and C-type vagal afferent neurons. Am J Physiol Gastrointest Liver Physiol 285: G1204–G1213, 2003.[Abstract/Free Full Text]
  51. Sun K, Ferguson AV. Cholecystokinin activates area postrema neurons in rat brain slices. Am J Physiol Regul Integr Comp Physiol 272: R1625–R1630, 1997.[Abstract/Free Full Text]
  52. Takagi H, Kubota Y, Mori S, Tateishi K, Hamaoka T, Tohyama M. Fine structural studies of cholecystokinin-8-like immunoreactive neurons and axon terminals in the nucleus of tractus solitarius of the rat. J Comp Neurol 227: 369–379, 1984.[CrossRef][Web of Science][Medline]
  53. Travagli RA, Gillis RA, Vicini S. Effects of thyrotropin-releasing hormone on neurons in rat dorsal motor nucleus of the vagus, in vitro. Am J Physiol Gastrointest Liver Physiol 263: G508–G517, 1992.[Abstract/Free Full Text]
  54. Wan S, Coleman FH, Travagli RA. Glucagon-like peptide-1 excites pancreas-projecting preganglionic vagal motoneurons. Am J Physiol Gastrointest Liver Physiol 292: G1474–G1482, 2007.[Abstract/Free Full Text]
  55. Wang BJ, Cui ZJ. How does cholecystokinin stimulate exocrine pancreatic secretion? From birds, rodents, to man. Am J Physiol Regul Integr Comp Physiol 292: R666–R678, 2007.[Abstract/Free Full Text]
  56. Whitcomb DC, Taylor IL. A new twist in the brain-gut axis. Am J Med Sci 304: 334–338, 1992.[CrossRef][Web of Science][Medline]
  57. Woods SC. Gastrointestinal satiety signals I. An overview of gastrointestinal signals that influence food intake. Am J Physiol Gastrointest Liver Physiol 286: G7–G13, 2004.[Abstract/Free Full Text]
  58. Zheng Z, Lewis MW, Travagli RA. In vitro analysis of the effects of cholecystokinin (CCK) on rat brainstem motoneurons. Am J Physiol Gastrointest Liver Physiol 288: G1066–G1073, 2005.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
G. M. Holmes, M. Tong, and R. A. Travagli
Effects of brain stem cholecystokinin-8s on gastric tone and esophageal-gastric reflex
Am J Physiol Gastrointest Liver Physiol, March 1, 2009; 296(3): G621 - G631.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/2/G484    most recent
00116.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wan, S.
Right arrow Articles by Travagli, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wan, S.
Right arrow Articles by Travagli, R. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.