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Am J Physiol Gastrointest Liver Physiol 292: G1474-G1482, 2007. First published February 22, 2007; doi:10.1152/ajpgi.00562.2006
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NEUROREGULATION AND MOTILITY

Glucagon-like peptide-1 excites pancreas-projecting preganglionic vagal motoneurons

S. Wan,1,2 F. H. Coleman,1 and R. A. Travagli1

1Department of Neuroscience, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana; and 2Department of Physiology, Wuhan University School of Basic Medical Science, Wuhan, People's Republic of China

Submitted 11 December 2006 ; accepted in final form 12 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Glucagon-like peptide-1 (GLP-1) increases pancreatic insulin secretion via a direct action on pancreatic beta-cells. A high density of GLP-1-containing neurons and receptors is also present in brain stem vagal circuits; therefore, the aims of the present study were to investigate 1) whether identified pancreas-projecting neurons of the dorsal motor nucleus of the vagus (DMV) respond to exogenously applied GLP-1, 2) the mechanism(s) of action of GLP-1, and 3) whether the GLP-1-responsive neurons (putative modulators of endocrine secretion) could be distinguished from DMV neurons responsive to peptides that modulate pancreatic exocrine secretion, specifically pancreatic polypeptide (PP). Whole cell recordings were made from identified pancreas-projecting DMV neurons. Perfusion with GLP-1 induced a concentration-dependent depolarization in ~50% of pancreas-projecting DMV neurons. The GLP-1 effects were mimicked by exendin-4 and antagonized by exendin-(9–39). In ~60% of the responsive neurons, the GLP-1-induced depolarization was reduced by tetrodotoxin (1 µM), suggesting both pre- and postsynaptic sites of action. Indeed, the GLP-1 effects were mediated by actions on potassium currents, GABA-induced currents, or both. Importantly, neurons excited by GLP-1 were unresponsive to PP and vice versa. These data indicate that 1) GLP-1 may act on DMV neurons to control pancreatic endocrine secretion, 2) the effects of GLP-1 on pancreas-projecting DMV neurons are mediated both via a direct excitation of their membrane as well as via an effect on local circuits, and 3) the GLP-1-responsive neurons (i.e., putative endocrine secretion-controlling neurons) could be distinguished from neurons responsive to PP (i.e., putative exocrine secretion-controlling neurons).

brain stem; electrophysiology; vagus; pancreas

GLUCOSE-SENSITIVE NEURONS in the dorsal vagal complex [DVC; i.e., the nucleus tractus solitarius (NTS), the dorsal motor nucleus of the vagus (DMV), and the area postrema] react promptly to changes in glucose levels (3, 4, 8, 14, 20, 47, 52). In vivo, these changes in the activity of DVC neurons in response to blood glucose are transmitted to the pancreas, via the vagus nerve, to modulate glucose homeostasis (5–7, 26).

In response to oral ingestion of food, glucagon-like peptide-1 (GLP-1) is released into the circulation from intestinal L cells where, at the level of the pancreatic beta-cell, it induces insulin release via actions at specific receptors (16, 19, 35, 48, 49, 51). GLP-1 also interacts with pancreatic {alpha}-cells to reduce glucagon secretion (16, 35, 48), and the combination of these effects on pancreatic islet cells contributes to glycemic control.

GLP-1 also has major effects at the level of the central nervous system, penetrating the blood-brain barrier via simple diffusion (29). Centrally, GLP-1 acts via vagally mediated pathways to induce the release of insulin, decrease food intake, and delay gastric emptying. GLP-1 is additionally involved in interoceptive stress and plays a role in the attenuation of toxin-induced fever as well as lithium-induced anorexia (15, 21, 24, 31, 43–46, 50, 56, 58). GLP-1 has also been shown to increase the impulse discharge of fibers from the hepatic branch of the vagus, to induce an increase in cFos activation in the NTS, and to increase vagal efferent activity, suggesting a possible role to modulate the activity of vagal brain stem circuits (39, 40, 58).

In the brain stem, the highest density of GLP-1 binding sites and mRNA is found in the DVC, where GLP-1-immunoreactive perikarya seem to be confined to the NTS (22, 28, 33, 37, 49, 57). Most importantly, in rodents, GLP-1 does not appear to affect exocrine secretion at doses that induce insulin secretion (Travagli et al., unpublished results, and Ref. 2), suggesting that GLP-1 may potentially be capable of distinguishing vagal preganglionic neurons that control endocrine vs. exocrine secretion.

Despite its recognized role in central nervous system-mediated effects and its abundance in brain stem vagal neurons, the cellular effects of GLP-1 in brain stem vagal circuits have not yet been investigated. Our recently developed technique that allows the identification of the peripheral target of vagal preganglionic motoneurons (9–11) has resulted in the classification of distinct populations of DMV neurons innervating the pancreas, based on their electrophysiological, pharmacological [i.e., response to pancreatic polypeptide (PP)], and morphological properties. The aims of the present study were to investigate whether any of these distinct properties could be used to identify a distinct population of DMV neurons modulating the functions of the endocrine pancreas. Specifically, this study aimed to 1) investigate whether identified pancreas-projecting DMV neurons are responsive to exogenously applied GLP-1, 2) examine the mechanism(s) of action of GLP-1, and 3) determine whether the GLP-1-responsive neurons (i.e., putative endocrine secretion-controlling neurons) can be distinguished from DMV neurons responsive to PP (i.e., putative exocrine secretion-controlling neurons).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Retrograde tracers and tissue preparation. As described previously (9–11) for other visceral regions, including the pancreas, the retrograde tracer DiI was applied to the pancreas of Sprague-Dawley rats. Briefly, rats (12–14 days old) of either sex were anesthetized deeply (3% isoflurane with air, 600 ml/min) in accordance with the National Institutes for Health guidelines and with the approval of the Pennington Biomedical Research Center-Louisiana State University System Institutional Animal Care and Use Committee. A deep level of anesthesia (abolition of the foot pinch withdrawal reflex) was maintained throughout the surgical procedure. The abdominal and thoracic areas were cleaned with alcohol and Novalsan prior to performing a midline laparotomy. The spleen was reflected toward the upper right flank of the rat before gauze, soaked in sterile saline, was placed on the stomach. The pancreas was then placed gently onto the gauze, and DiI crystals were apposed to the body of the pancreas. To restrict the dye to the site of application, the neuronal tracer was embedded in place by using a fast-hardening epoxy resin that was allowed to dry for 3–5 min. The pancreas was replaced in the abdominal cavity, the gauze was removed, and the entire surgical area was washed with warmed sterile saline. The excess solution was blotted with cotton tips, the wound was closed with a 5-0 suture, and the animal was allowed to recover for 10–15 days.

The method to prepare the tissue slices has already been described (11, 53). Briefly, rats were anesthetized deeply with isoflurane (3%) before being killed by administration of a bilateral pneumothorax. The brain stem was removed and placed into oxygenated, ice-cold Krebs' solution. After being glued to a plastic support, 5–6 coronal slices (300 µm thick) containing the DMV were cut using a Vibratome. The slices were incubated and equilibrated for at least 1 h in oxygenated Krebs' solution (32 ± 1°C) prior to electrophysiological recording. In each instance, the pancreas was examined visually to ensure that the dye had not moved from its site of application and had not diffused into the abdominal milieu. A single slice was then mounted on a custom-made perfusion chamber (500 µl) and was kept in place by a nylon web. The slice was maintained at 35 ± 1°C by perfusion with Krebs' solution at 2.5 ml/min.

DMV neurons: identification and recordings. Patch-clamp recordings were made only from fluorescently labeled DMV neurons visualized with a Nikon E600FN equipped with TRITC filters. Provided that the period of illumination used for neuronal identification is brief (i.e., <5 s), carbocyanine dyes such as DiI do not cause adverse effects (11, 25, 36). Following labeling of the pancreas, typically an average of one or two unequivocally labeled neurons were observed in each brain stem slice (9, 10).

Patch-clamp recordings were made from DMV neurons by using borosilicate patch pipettes with a tip resistance of 3–7 M{Omega} when filled with a potassium gluconate intracellular solution. Recordings were done using a Axopatch 200B amplifier (Axon Instruments, Union City, CA) and were corrected manually for liquid junction potential; only those recordings having a series resistance <20 M{Omega} were used. Neurobiotin (2.5 mg/ml) was included in the recording pipette to stain the neuron for later morphological analysis. For a neuronal recording to be accepted, the membrane had to be stable at the holding potential, the action potential evoked following injection of direct current had to have an amplitude of at least 60 mV, and the membrane had to return to baseline at the end of the afterhyperpolarization.

Neurons were current-clamped at –60 mV (for the recording of firing-rate changes) or at –65 mV (for the recording of membrane depolarization/hyperpolarization) before superfusion with GLP-1 (3–1,000 nM) for a period of time sufficient for the response to reach plateau, usually 1–3 min. The longer perfusion time was necessary when the recorded neuron was deeper in the slice and GLP-1 was slower in equilibrating at the appropriate concentration. Since desensitization to the effects of GLP-1 was not observed, we assumed that the plateau effects were reported correctly. DMV neurons were considered responsive if perfusion with 100 nM GLP-1 induced a membrane shift of at least 3 mV in amplitude that recovered to baseline levels on washout; at least 10 min of recovery were allowed between successive applications of drugs. The EC50 was calculated by using Statistica software (StatSoft, Tulsa, OK) for each set of responses, the results being expressed as an average. To assess the ionic conductance(s) affected by GLP-1, the neurons were voltage-clamped at –50 mV and hyperpolarized to –110 mV in 10-mV steps (400 ms duration) before being returned to –50 mV (12). GLP-1 (100 nM) was applied by superfusion; once the response reached plateau, the protocol was repeated. Only one cell per slice was tested.

Data and statistical analysis. Data were acquired at 10 kHz, filtered at 2 kHz, digitized via a Digidata 1320 interface (Axon Instruments), and stored and analyzed on a PC using pClamp9 software (Axon Instruments). Results are presented as means ± SE. Each neuron served as its own control, i.e., the neuron was assessed before and after drug application and was analyzed by using a paired t-test with significance set at P < 0.05.

When conducting the concentration-response curve, a minimum of three different concentrations of GLP-1 were tested on the same cell at 10- to 15-min intervals.

Morphological reconstructions. At the conclusion of electrophysiological recording, Neurobiotin was injected into the DMV neuron (1-s-duration depolarizing current pulses, every 2 s for 20 min), 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 PBS-Triton X-100 (PBS-TX), 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 (11, 34).

Neurolucida software (Microbrightfield, Williston, VT) was used to make three-dimensional reconstructions of the individual Neurobiotin-labeled neurons, digitized at a final magnification of x600. 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 = soma area and p = the perimeter of the soma in the horizontal plane), whether the cell has bipolar or multipolar dendrites, 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 (11, 34).

Solution composition. Krebs' solution contained (in mM): 120 NaCl, 26 NaHCO3, 3.75 KCl, 1 MgCl2, 2 CaCl2, and 11 dextrose, maintained at pH 7.4 with 95% O2-5% CO2. Potassium gluconate intracellular solution contained (in mM): 128 K gluconate, 10 KCl, 0.3 CaCl2, 1 MgCl2, 10 HEPES, 1 EGTA, 2 ATP, and 0.25 GTP, adjusted to pH 7.35 with KOH. Zamboni's fixative contained 1.6% (wt/vol) paraformaldehyde, 19 mM KH2PO4, and 100 mM Na2HPO4·7 H2O in 240 ml saturated picric acid-1,600 ml H2O, adjusted to pH 7.4 with HCl. PBS-TX contained (in mM) 115 NaCl, 75 Na2HPO4·7 H2O, 7.5 KH2PO4, and 0.15% Triton X-100. Avidin D-HRP solution contained 0.05% diaminobenzidine in PBS containing 0.5% gelatin supplemented with 0.025% CoCl2 and 0.02% NiNH4SO4.

Chemicals. Neurobiotin and Avidin D-HRP were purchased from Vector Labs (Burlingame, CA). Permount was purchased from Fisher Scientific (Pittsburgh, PA). DiI was purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma (St. Louis, MO).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To limit spurious results, only those cells showing the brightest and most intense DiI fluorescence were used for recording. The postsynaptic response to GLP-1 was tested on 424 pancreas-projecting DMV cells (253 in current-clamp and 171 in voltage-clamp configuration).

GLP-1 depolarizes a subpopulation of pancreas-projecting DMV neurons. In the current-clamp configuration, perfusion with 100 nM GLP-1 induced a membrane depolarization in 42% of the neurons tested (106 out of 253). The remaining 58% of neurons (147 out of 253) showed no measurable response to GLP-1. During our analysis, we found no neurons that were hyperpolarized by GLP-1.

Concentration-response curves were constructed from cells in which at least three concentrations of GLP-1 (10–1,000 nM) were tested at 10- to 15-min intervals. When current-clamped at –65 mV, the depolarization induced by GLP-1 was concentration dependent and had an estimated EC50 of 80 nM. Similarly, from a membrane potential of –60 mV, the increase in firing rate induced by GLP-1 was concentration dependent and had a comparable EC50. The neuron returned to pretreatment baseline values on washout of GLP-1 (Fig. 1).


Figure 1
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  		Fig. 1. Glucagon-like peptide-1 (GLP-1) depolarizes identified pancreas-projecting dorsal motor nucleus of the vagus (DMV) neurons. A: representative traces from a pancreas-projecting DMV neuron illustrating that GLP-1 induces a concentration-dependent increase in firing rate in a neuron current-clamped at –60 mV. A recovery period of at least 10–15 min was allowed between successive applications. Parallel lines indicate a 2- to 3-min break in the recording. B: concentration-response curve for the GLP-1-induced increase in action potential firing rate in a neuron current-clamped at –60 mV. Results are expressed as absolute number of action potentials per minute. The EC50 for the GLP-1 response was ~80 nM. Each neuron was tested with at least 3 different concentrations of GLP-1. C: concentration-response curve for the GLP-1-induced depolarization in neurons current clamped at –65 mV. The EC50 for the GLP-1 response was ~80 nM. Each neuron was tested with at least 3 different concentrations of GLP-1.

 
The response to GLP-1 did not show tachyphylaxis, because 3-min perfusions with 100 nM GLP-1 repeated 10 min apart gave similar results. In fact, the first superfusion of GLP-1 depolarized the membrane by 7.3 ± 1.4 mV (P < 0.05 vs. control; n = 3) or increased the frequency of action potential firing from 2.0 ± 1.5 to 24.0 ± 2.4 action potentials/min (P < 0.05; n = 4), whereas the second superfusion of GLP-1 depolarized the membrane by 6.7 ± 1.8 mV or increased the firing rate from 2.0 ± 1.4 to 28.0 ± 10.6 action potentials/min (P < 0.05 vs. control; P > 0.05 vs. first application; Fig. 2, A and B).


Figure 2
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  		Fig. 2. Characterization of the GLP-1-induced depolarization on identified pancreas-projecting DMV neurons. A: representative current-clamp traces comparing the increase in firing rate obtained by superfusion of 100 nM GLP-1. Neurons were current-clamped at –60 mV, and GLP-1 was applied at 5- to 10-min intervals. B: summary comparing the increase in firing rate (left) or the depolarization (right) induced by GLP-1 in 2 successive applications 10–15 min apart. Note that the maximal response is not significantly reduced between the 2 applications of GLP-1. C: representative current-clamp traces comparing the increase in firing rate obtained by superfusion of 100 nM GLP-1 to that induced by exendin-4. Neurons were current-clamped at –60 mV, and drugs were applied 10–15 min apart. D: summary comparing the increase in firing rate (left) or the depolarization (right) induced by successive applications of GLP-1 and exendin-4 (E4). Note that the maximal response is similar between GLP-1 and exendin-4. E: representative current-clamp traces showing that, in a GLP-1-responsive DMV neuron current-clamped at –60 mV, the excitatory effect of GLP-1 was antagonized by pretreatment with 100 nM exendin-(9–39). F: summary comparing the increase in firing rate (left) or the depolarization (right) induced by GLP-1 alone or following 10-min pretreatment with exendin-(9–39) (E 9–39). Note that the response to GLP-1 was antagonized by exendin-(9–39).

 
The excitatory response of pancreas-projecting DMV neurons induced by GLP-1 was mimicked by perfusion with the receptor-selective agonist exendin-4 (100 nM). In fact, in eight neurons current-clamped at –60 mV, perfusion with exendin-4 increased the firing rate from 9.0 ± 3.1 to 40.0 ± 9.2 action potentials/min (P < 0.05). Similarly, in three neurons current-clamped at –65 mV, perfusion with exendin-4 depolarized the DMV membrane by 5.0 ± 1.0 mV (P < 0.05) (Fig. 2, C and D).

The increase in firing rate and the depolarization induced by GLP-1 were antagonized by the receptor-selective antagonist exendin-(9–39) (100 nM). In four neurons in which GLP-1 (100 nM) increased the firing rate from 2.0 ± 1.5 to 44 ± 7.8 action potentials/min following 10-min pretreatment with exendin-(9–39), perfusion of GLP-1 in the presence of the antagonist did not change the firing rate [0 ± 0 increase in action potentials/min in exendin-(9–39) + GLP-1 vs. exendin-(9–39) alone; P < 0.05 vs. GLP-1 alone]. Similarly, in eight neurons in which GLP-1 induced a 7.0 ± 0.8-mV depolarization, perfusion of GLP-1 in the presence of exendin-(9–39) induced a 0 ± 0.12-mV depolarization (P < 0.05 vs. GLP-1 alone; Fig. 2, E and F).

To analyze the ionic conductance(s) responsible for the excitatory effects of GLP-1, the current-voltage (I-V) curve between –50 and –110 mV in the voltage-clamp configuration was constructed. Perfusion with GLP-1 (100 nM) induced a measurable inward current in 58 of 171 neurons (i.e., 34%). The I-V curve revealed three types of responses to GLP-1. In 22 of 58 responsive neurons analyzed, GLP-1 induced a –39.0 ± 3.9-pA inward current at –50 mV. The reversal potential of this GLP-1-induced current (EGLP-1) was –104.0 ± 1.79 mV, i.e., close to the estimated reversal potential for potassium currents (EK) in our experimental conditions. In six neurons in which the reversal potential of the GLP-1-induced response was similar to that of EK, the response to GLP-1 was reexamined in the presence of 1 µM tetrodotoxin (TTX), to block action potential-dependent synaptic transmission. Under these conditions, the GLP-1-induced response was unchanged; GLP-1 induced a –34.0 ± 5.8-pA inward current at –50 mV, and EGLP-1 was –100.0 ± 4.4 mV, i.e., close to EK (P > 0.05; data not shown, but see Fig. 4, D and E).


Figure 4
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  		Fig. 4. In a neuronal subpopulation, GLP-1 induces a parallel inward shift. A: DMV neurons were voltage-clamped at –50 mV and step-hyperpolarized to –110 mV in 10-mV increments in control conditions (left) and following perfusion with GLP-1 (100 nM; right). B: DMV neurons were voltage-clamped at –50 mV and step-hyperpolarized to –110 mV in 10-mV increments in the presence of 1 µM TTX (left) and following perfusion with TTX + GLP-1 (100 nM; right). C: current-voltage plot shows that GLP-1 induced a parallel inward shift in Krebs' solution. Each data point represents the average of 16 neurons. D: current-voltage plot shows that the GLP-1-induced current reverses close to EK in TTX. Each data point represents the average of 5 neurons. E: current-voltage plot shows that the GLP-1-induced current reverses close to EK in bicuculline. Each data point represents the average of 6 neurons.

 
In contrast, in 20 of 58 neurons analyzed, GLP-1 induced a –5.0 ± 2.9-pA outward current at –50 mV and a –52.0 ± 9.7-pA inward current at –110 mV; the reversal potential for the EGLP-1 was –55.0 ± 2.1 mV, i.e., close to the estimated reversal potential for a chloride current (ECl) in our experimental conditions. In six of these neurons in which the reversal potential of the GLP-1-induced response was similar to that of ECl, the I-V curve was reconstructed in the presence of 1 µM TTX. In the presence of TTX, GLP-1 did not induce any current shift (Fig. 3, A–D). In a further four of these neurons, the I-V curve was reconstructed in the presence of the GABAA receptor-selective antagonist bicuculline (50 µM). After 10 min of pretreatment with bicuculline, GLP-1 did not induce any current shift (Fig. 3E).


Figure 3
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  		Fig. 3. In a neuronal subpopulation, the GLP-1-induced inward current has a reversal potential close to the chloride current (ECl). A: DMV neurons were voltage-clamped at –50 mV and step-hyperpolarized to –110 mV in 10-mV increments in control conditions (left) and following perfusion with GLP-1 (100 nM; right). B: DMV neurons were voltage-clamped at –50 mV and step-hyperpolarized to –110 mV in 10-mV increments in the presence of 1 µM tetrodotoxin (TTX; left) and following perfusion with TTX + GLP-1 (100 nM; right). C: current-voltage plot shows that the reversal potential for the GLP-1-induced current is close to ECl. Each data point represents the average of 20 neurons. D: current-voltage plot shows that TTX pretreatment completely antagonized the GLP-1 current. Each data point represents the average of 6 neurons. E: current-voltage plot shows that the GLP-1 current is antagonized completely in the presence of bicuculline. Each data point represents the average of 4 neurons.

 
In the remaining 16 of 58 neurons analyzed, GLP-1 induced an inward current whose amplitude was unchanged throughout the voltage range in analysis, i.e., the inward shift was parallel from –50 to –110 mV (i.e., –35 ± 5.7 and –33 ± 4.4 pA at –50 and –110 mV, respectively). In five of these neurons, in the presence of TTX, GLP-1 induced a –31.0 ± 5.0-pA inward current at –50 mV and EGLP-1 was –102.0 ± 3.7 mV, i.e., close to EK (Fig. 4, A–D). In a further six neurons, in the presence of bicuculline (50 µM), GLP-1 induced a –25.0 ± 2.1-pA inward current at –50 mV and EGLP-1 was –102.0 ± 4.9 mV, i.e., close to EK (Fig. 4E).

These data suggest that the excitatory effects of GLP-1 on pancreas-projecting DMV neurons involved multiple mechanisms of action: a direct effect on the DMV membrane (neurons in which EGLP-1 {approx} EK), a presynaptic effect involving the release of a neurotransmitter, possibly GABA (neurons in which EGLP-1 {approx} ECl), and a combination of these two effects (neurons in which GLP-1 induced a parallel inward shift).

These different GLP-1-induced mechanisms of action were confirmed in 12 neurons tested in the current-clamp configuration (–65 mV). When the data were pooled, the GLP-1-induced depolarization was 6.0 ± 0.4 mV and 3.0 ± 0.8 mV in the presence of 1 µM TTX (P < 0.05). In the presence of TTX, the GLP-1-mediated depolarization could be separated into three different types of responses. In one group, the response to GLP-1 (6.0 ± 0.7 mV depolarization) was unaffected by TTX pretreatment (6.0 ± 0.7 mV; n = 4; P > 0.05). In the second group, GLP-1 induced a 7.0 ± 0.7-mV depolarization that was completely abolished following TTX pretreatment (0 ± 0 mV; n = 4; P < 0.05), whereas the GLP-1-induced depolarization in the third group was attenuated by TTX pretreatment, i.e., from 6.0 ± 0.6 mV in control to 3.0 ± 0.2 mV in the presence of TTX (n = 4; P < 0.05).

These data support our previous conclusions that the excitatory effects of GLP-1 on pancreas-projecting DMV neurons involved multiple mechanisms of action: a direct effect on the DMV membrane (neurons in which TTX did not change the depolarizing response of GLP-1), a presynaptic effect involving the release of a neurotransmitter, possibly GABA (neurons in which TTX pretreatment abolished the response to GLP-1), and a combination of the two effects mentioned above (neurons in which TTX attenuated the GLP-1-induced depolarization).

In 50 neurons, the response of pancreas-projecting DMV neurons to perfusion of GLP-1 (100 nM) and PP (100 nM) were compared. In no instance did any neuron respond to both GLP-1 and PP. In fact, neurons were either responsive to GLP-1 (depolarization) but not to PP (n = 11), they were responsive to PP (hyperpolarization) but not GLP-1 (n = 31), or they were unresponsive to both peptides (n = 8; Fig. 5).


Figure 5
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  		Fig. 5. GLP-1 and pancreatic polypeptide (PP) modulate the membranes of different neuronal subpopulations. A: representative traces from a pancreas-projecting DMV neuron illustrating that, in a neuron current-clamped at –60 mV, GLP-1 induces an increase in action potential firing rate but PP does not have any effect. Oblique parallel lines indicate a 2- to 5-min break in the recording. B: representative traces from a pancreas-projecting DMV neuron current clamped at –60 mV illustrating that PP induces a membrane hyperpolarization but GLP-1 does not have any effect. Oblique parallel lines indicate a 2- to 5-min break in the recording.

 
Morphological reconstructions. Of the DMV neurons in which we tested the postsynaptic effects of GLP-1, the soma was reconstructed in 173 neurons (115 nonresponsive and 58 responsive to GLP-1) and complete morphological reconstructions were obtained from 61 cells (46 nonresponsive and 15 responsive to GLP-1) whose dendrites did not show retraction bulbs (which are indicative of damaged/cut terminals).

As reported previously, pancreas-projecting neurons have either a bipolar or a multipolar soma morphology (9). In the present study, differences were not found in terms of soma morphology between responsive and nonresponsive cells (in both groups ~65% of the neurons were multipolar; {chi}2 test, P > 0.05; Table 1). DMV cells responsive to GLP-1 had a smaller dendritic extent in the x-axis (mediolateral) than unresponsive neurons (214 ± 28 vs. 258 ± 14 µm, respectively, P < 0.05). When considering the orientation of dendritic projections, however, neurons unresponsive to GLP-1 were less likely to project to the ependymal layer of the central canal or of the fourth ventricle than neurons responding to GLP-1. In fact, 11 of 46 nonresponding neurons projected to the ependymal layer compared with 7 of 15 responding neurons ({chi}2 test, P > 0.05). Differences were not found, however, in the rostrocaudal distribution of GLP-1-responsive vs. nonresponsive neurons (data not shown).


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  		Table 1. Electrophysiological and morphological properties of pancreatic-projecting dorsal motor vagus neurons tested with glucagon-like peptide-1

 
The basic electrophysiological and morphological properties of pancreas-projecting neurons responsive to GLP-1 are summarized in Table 1.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have shown that 1) a subpopulation of identified pancreas-projecting DMV neurons is depolarized by exogenously applied GLP-1, 2) the effects of GLP-1 are mediated both via a direct effect on their membrane as well as via an effect on local synaptic circuits, and 3) the GLP-1-responsive neurons represent a subpopulation of DMV neurons that is distinct from the neuronal subpopulation responsive to PP.

Our data suggest that pharmacological tools (i.e., GLP-1 and PP) can be used to distinguish at least two subpopulations of pancreatic preganglionic vagal neurons. We speculate that these separate DMV neuronal subpopulations project selectively to postganglionic neurons controlling the endocrine (i.e., GLP-1-responsive) or exocrine (i.e., PP-responsive) pancreas. Our conclusions are based on the following experimental observations.

Perfusion with GLP-1 affects a subpopulation (~40%) of identified pancreas-projecting DMV cells, in which GLP-1 induced concentration-dependent depolarization of the neuronal membrane. The excitatory effects of GLP-1 on pancreas-projecting DMV neurons were mimicked by the highly potent and selective GLP-1 receptor agonist exendin-4 (also known as exenatide in clinical studies), a naturally occurring peptide isolated from the venom of the Heloderma suspectum lizard (17). The selectivity of the response was confirmed by the prevention of the GLP-1-induced effects by pretreatment with the selective GLP-1 receptor antagonist exendin-(9–39). These data indicate that the response to GLP-1 was, indeed, determined by activation of GLP-1 receptors. Indeed, the excitatory response that follows the activation of these receptors has been well described in pancreatic beta-cells (reviewed in Refs. 16, 32, and 35). The effects of GLP-1 on neuronal populations have, however, received scant attention (1, 27).

The excitatory effects of GLP-1 appear to be determined by activation of GLP-1 receptors located either on the DMV membrane itself and/or on neurons impinging on the DMV cell. More specifically, in 38% of the GLP-1-responsive DMV neurons, the inward current was unaffected by pretreatment with TTX, which blocks action potential-dependent synaptic transmission. In these neurons, the inward current induced by GLP-1 had a reversal potential close to –100 mV, consistent with an effect to block a potassium conductance. Indeed, inhibition of a potassium conductance is one of the described mechanisms of action of GLP-1 in hypocretin-containing hypothalamic neurons (1). In 35% of the neurons, however, perfusion with exogenous GLP-1 induced an inward current only at potentials negative to –50 mV. This inward current was completely antagonized by pretreatment with TTX, indicating that it was synaptically driven. Since the reversal potential of the GLP-1-induced current was close to the reversal potential of chloride-mediated currents, like those evoked by activation of the ubiquitous GABAA receptors (53–55), we tested the effects of GLP-1 in slices pretreated with the selective GABAA antagonist bicuculline. Following abrogation of GABAergic transmission, perfusion with GLP-1 did not induce any membrane displacement in these neurons, suggesting that the effects of the peptide were determined by activation of GABAergic synaptic inputs. Indeed, activation of local GABAergic inputs has also been shown to occur on hypothalamic orexin-containing neurons in response to perfusion with GLP-1 (1). In the remaining 27% of the GLP-1-responsive DMV neurons, perfusion with the peptide induced an inward current that paralleled the voltage axis in the –50 to –110 mV range. This type of response to an agonist may be determined by different factors, such as 1) more than one site of action, for example an effect on the neuronal membrane and an effect on synaptic inputs impinging on the membrane; 2) the combined and equipotent effects on different conductances on the neuronal membrane, for example the closure of a potassium conductance (with an equilibrium potential close to –100 mV) and the opening of a nonselective cationic conductance (with an equilibrium potential close to 0 mV), or 3) unsuitable voltage control over the neuronal membrane, i.e., poor space-clamp.

Our results demonstrated clearly that the parallel inward shift of the GLP-1-induced current was determined by the peptide having more than one site of action. In fact, following pretreatment with either TTX or bicuculline, these neurons responded to GLP-1 with an inward current shift that reversed close to the equilibrium potential of potassium. These data, then, indicate than in this subset of DMV neurons, the effects of GLP-1 are determined by both an action on the DMV neuronal membrane itself and an action on GABAergic circuits impinging on the DMV cell. This may be a commonplace occurrence among peptides controlling pancreatic functions, because, as we have reported recently, PP affects brain stem vagal circuits controlling pancreatic functions via actions at multiple sites (10).

That different populations of neurons can be distinguished based on their responsiveness to GLP-1 suggests that clusters of neurons may possibly be devoted to the control of specific pancreatic functions, even within the discrete subpopulation of pancreas-projecting DMV neurons. If correct, the broader implication of this hypothesis, that subsets of pancreas-projecting DMV neurons control either endocrine or exocrine pancreatic secretion, should receive even stronger experimental support. Indeed, in the present study we show that DMV neurons are responsive to either GLP-1 or PP, but never both. We speculate that these pancreas-projecting DMV neuronal populations responsive to either GLP-1 or to PP represent the preganglionic neurons controlling endocrine or exocrine pancreatic functions, respectively.

Furthermore, we hypothesize that lines of specificity exist in pancreatic vagal circuits, such that subsets of DMV preganglionic motoneurons control distinct neuronal postganglionic cells which, in turn, influence specific secretory units of the pancreas. This organization implies that each single pancreas-projecting DMV neuron functions exclusively as a preganglionic neuron to either acinar cells or to specific secretory cells (e.g., {alpha} or beta) within the islets.

This hypothesis of lines of specificity in the vagal control of pancreatic functions by preganglionic vagal neurons is supported by diverse pieces of evidence. Microinjection of PP in the DVC modulates vagal outflow to inhibit centrally mediated pancreatic exocrine secretion without affecting basal plasma insulin and glucagon secretion (2, 30, 40–42, 58). Indeed, we have shown recently (10) that PP modulates vagal motor output selectively via actions on subpopulations of pancreas-projecting DMV neurons. We refer the reader to our recently published study (10) for a rather comprehensive overview of the selective effects of PP in the DVC.

Conversely, systemic GLP-1 is a potent stimulator of insulin secretion. Although a considerable portion of this increased insulin secretion is mediated via a direct action on pancreatic beta-cells, GLP-1 appears to increase insulin also via a vagally mediated effect (reviewed in Refs. 16 and 32). Interestingly, in rodents, GLP-1 does not stimulate exocrine secretion at doses that stimulate insulin secretion (2). Furthermore, it is well accepted that systemic administration of GLP-1 increases cFos expression in the NTS and that microinjection of GLP-1 in the DVC increases insulin secretion (Refs. 19, 40, 49, and 58 and Travagli et al., unpublished data). Thus it appears that GLP-1 and PP are in a position to contribute selectively to the modulation of pancreatic secretory functions.

Small amounts of GLP-1 are released during the cephalic phase of digestion, but a larger amount of the peptide is released by L cells in the intestine following ingestion of a meal. Although this postprandial GLP-1 concentration rarely exceeds 50 pmol/l (32), it is well accepted that, in vitro, the concentration-response curve to agonists is shifted to the right by two or more orders of magnitude (18), placing the GLP-1 effects reported in the present study (i.e., threshold response in the low nanomolar range) well within the acceptable physiological range. Furthermore, GLP-1 crosses the blood-brain barrier via a simple diffusion mechanism (29), and one must keep in mind that the vasculature supplying the DVC is composed of fenestrated capillaries that allow passage from the circulation of large molecules capable of modulating the activity of DVC neurons (13, 23), providing further support to the idea that circulating GLP-1 might also act on these neuronal circuits directly. In addition to the accessibility of the DVC to circulating GLP-1, many NTS neurons also express GLP-1 immunoreactivity (38, 44, 59), suggesting that that both systemic and locally released GLP-1 modulate vagal output to the exocrine pancreas.

In summary, we have shown that identified subpopulations of pancreas-projecting DMV neurons are excited by GLP-1 via different mechanisms, and the neuronal subpopulations responding to GLP-1 are distinct from the neuronal subpopulations responsive to PP. It is then possible that GLP-1 also increases insulin secretion by affecting brain stem vagal efferent pathways, and we suggest that the separate subpopulations of pancreas-projecting DMV neurons project selectively to postganglionic neurons controlling the endocrine (i.e., GLP-1-responsive) or exocrine (i.e., PP-responsive) pancreas.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Science Foundation Grant IBN 04-56291.


   ACKNOWLEDGMENTS
 
We thank Drs. H.-R. Berthoud and K. N. Browning 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: R. A. Travagli, Dept. of Neuroscience, Pennington Biomedical Research Center, Louisiana State Univ. System, 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
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 REFERENCES
 

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