Effects of pancreatic polypeptide on pancreas-projecting rat dorsal motor nucleus of the vagus neurons
Kirsteen N. Browning,
F. Holly Coleman, and
R. Alberto Travagli
Department of Neuroscience, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana
Submitted 21 December 2004
; accepted in final form 21 March 2005
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ABSTRACT
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We investigated the pre- and postsynaptic effects of pancreatic polypeptide (PP) on identified pancreas-projecting neurons of the rat dorsal motor nucleus of the vagus in thin brain stem slices. Perfusion with PP induced a TTX- and apamin-sensitive, concentration-dependent outward (22% of neurons) or inward current (21% of neurons) that was accompanied by a decrease in input resistance; PP was also found to affect the amplitude of the action potential afterhyperpolarization. The remaining 57% of neurons were unaffected. PP induced a concentration-dependent inhibition in amplitude of excitatory (n = 22 of 30 neurons) and inhibitory (n = 13 of 17 neurons) postsynaptic currents evoked by electrical stimulation of the adjacent nucleus of the solitary tract, with an estimated EC50 of 30 nM for both. The inhibition was accompanied by an alteration in the paired pulse ratio, suggesting a presynaptic site of action. PP also decreased the frequency, but not amplitude, of spontaneous excitatory (n = 6 of 11 neurons) and inhibitory currents (n = 7 of 9 neurons). In five neurons, chemical stimulation of the area postrema (AP) induced a TTX-sensitive inward (n = 3) or biphasic (outward and inward) current (n = 2). Superfusion with PP reversibly reduced the amplitude of these chemically stimulated currents. Regardless of the PP-induced effect, the vast majority of responsive neurons had a multipolar somata morphology with dendrites projecting to areas other than the fourth ventricle or the central canal. These results suggest that pancreas-projecting rat dorsal motor nucleus of the vagus neurons are heterogeneous with respect to their response to PP, which may underlie functional differences in the vagal modulation of pancreatic functions.
brain stem; parasympathetic; gastrointestinal
CHOLINERGIC NEURONS OF THE dorsal motor nucleus of the vagus (DMV) control parasympathetic function to the gastrointestinal tract by impinging on postganglionic excitatory cholinergic or inhibitory nonadrenergic, noncholinergic (NANC) neurons (2, 18, 51). The DMV also provides the parasympathetic innervation to the pancreas; microinjection of the retrograde transneuronal Bartha virus into the pancreas of sympathectomized rats results in labeling of two morphologically different types of neurons in the DMV; one group of neurons has a multipolar soma morphology, whereas the other group of cells has bipolar soma (14, 21, 28, 29, 46). The pancreas-projecting DMV neurons can further be distinguished on the basis of their dendritic arborization with one group projecting toward, whereas the other group projects to regions other than, the central canal or fourth ventricle (8, 42, 46).
Vagal activation affects directly both the exocrine as well as the endocrine pancreas (6, 24, 27, 38, 39, 41). Indeed, it has been shown that different frequencies of stimulation affect either endocrine or exocrine secretion (3, 5), raising the possibility that selective types of stimulation or different vagal preganglionic cells can affect these functions differently.
After ingestion of a meal, pancreatic polypeptide (PP) is released from the pancreas via a vagally-dependent, atropine-sensitive mechanism (reviewed in Ref. 40); circulating PP then potently inhibits exocrine secretion (reviewed in Refs. 23, 55). This inhibition of pancreatic exocrine secretion is not, however, due to a direct effect of PP on the pancreatic acini. In fact, PP does not affect secretion in an in vitro acinar preparation, and there are no PP receptors on the acinar or ductal cells (30, 37, 41, 45, 55). Rather, circulating PP acts centrally to enhance gastric secretion and motility via actions at the level of efferent vagal fibers. Physiologically identified gastric-projecting neurons are excited by PP (3234), and recent studies in rats have shown that PP (Y4) receptors are located in the dorsal vagal complex [DVC, i.e., DMV and nucleus of the solitary tract (NTS)] (17, 56), where they are distinguishable from the neuropeptide Y (NPY) and peptide YY (PYY) binding sites (Y1 and Y2 receptors) (25, 54). As with the effects of PP on gastric function, the reduction of exocrine pancreatic secretion induced by systemic administration of PP occurs via actions in the DVC and is dependent on an intact vagal innervation (17, 43, 55). The response of identified pancreas-projecting DMV neurons to PP, however, has not been assessed.
The study of the effects of PP on synaptic activity within the DVC is also of physiological relevance, because the in vivo inhibitory effects of PP are enhanced by treatments, such as 2-deoxy-glucose or CCK, known to increase pancreatic baseline secretion (39). Recently, it has been hypothesized that synaptic connections within the DVC and, in particular, between the area postrema and the DMV play a relevant role in the physiological actions of PP (17). The effects of PP on the synaptic responses of identified pancreas-projecting DMV neurons, however, have not been assessed.
The aims of this study were 1) to investigate the in vitro effects of PP on the membrane of identified rat pancreas-projecting DMV neurons 2) to determine the effects of PP on excitatory and inhibitory synaptic transmission within the DVC to identified pancreatic-projecting DMV neurons, and 3) to determine whether cells responding to PP have distinguishing membrane characteristics.
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METHODS
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Retrograde tracers and tissue preparation.
As described previously for other visceral regions including the pancreas (8, 11), the retrograde tracer DiI was applied to the pancreas of Sprague-Dawley rats. Briefly, rats (1214 days old) of either sex were anesthetized deeply (3% Isoflurane with air, 600 ml/min) in accordance with the National Institutes of Health guidelines and the Pennington Biomedical Research Center-Louisiana State University System 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 70% ethanol before performing a 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 on top of the gauze, and DiI crystals were apposed to the body of the pancreas. To restrict it to the site of application, the neuronal tracer was embedded in place using a fast-hardening epoxy resin that was allowed to dry for 35 min before the pancreas was replaced; then 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 50 suture, and the animal was allowed to recover for 1015 days.
The method to prepare the tissue slices has already been described (11, 49). Briefly, rats were anesthetized deeply with isofluorane (5%) before being killed by severing the major blood vessels in the chest. The brain stem was removed and placed into oxygenated, ice-cold Krebs solution (see below). After being glued to a plastic support, five to six 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 (volume 500 µl) and 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 the period of illumination used for neuronal identification is brief, carbocyanine dyes (such as DiI) do not cause adverse effects (11, 20, 35). After labeling of the pancreas, typically an average of 12 unequivocally labeled neurons were observed in each brain stem slice.
Patch-clamp recordings were made from DMV neurons using borosilicate patch pipettes with a tip resistance of 37 M
when filled with a potassium gluconate intracellular solution (see below). Recordings were corrected manually for liquid junction potential, and only those recordings having a series resistance of <15 M
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 (DC) 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 voltage clamped at 50 mV before superfusion with PP (0.31,000 nM) for a period of time sufficient for the response to reach plateau. DMV neurons were classified as PP responders if perfusion with 100 nM PP induced a current of at least 20 pA in amplitude that recovered to baseline levels on washout; at least 10 min recovery were allowed between successive applications of drugs. 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 PP on action potential characteristics, neurons were current clamped at 60 mV, and either a single action potential was evoked or the frequency of action potential firing in response to longer DC injections was assessed as described previously (12, 26). PP (100 nM) was then applied by superfusion; once the response reached plateau, the membrane potential was restored to control values by injection of DC before the protocols were repeated. Only one cell per slice was tested.
Evoked and spontaneous synaptic currents.
Tungsten bipolar stimulating electrodes (WPI, Sarasota, FL) placed in the adjacent NTS (subnucleus centralis, medialis, or commissuralis) were used to evoke excitatory or inhibitory postsynaptic currents (EPSCs or IPSCs, respectively) in the recorded DMV neuron. When evoking EPSCs, neurons were current clamped at 60 mV (i.e., close to ECl), when evoking IPSCs, neurons were current clamped at 50 mV, and the perfusing solution contained 1 mM kynurenic acid (to block glutamatergic currents, 50).
Spontaneous IPSCs were recorded in neurons voltage clamped at 50 mV using a perfusing solution containing 1 mM kynurenic acid and intracellular solution containing KCl; conversely, spontaneous excitatory events were recorded at 60 mV holding potential with a potassium gluconate intracellular solution.
Data and statistical analysis.
Data were filtered at 2 kHz, digitized via a Digidata 1320 interface (Axon Instruments, Union City, CA), stored, and analyzed on a PC using the pClamp8 software (Axon Instruments). Results are represented as means ± SE. Each neuron served as its own control, i.e., the neuron was assessed before and after drug application and analyzed using a paired t-test with significance set at P < 0.05.
Morphological reconstructions.
At the conclusion of electrophysiological recording, Neurobiotin was injected into the DMV neuron as described previously (11, 31), 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-TX (see below), and the injected Neurobiotin was visualized using a cobalt-nickel enhancement of the Avidin D-horseradish peroxidase (Avidin D-HRP) technique as described previously before mouting in Permount (11, 31).
As described previously (11, 31), 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. Each reconstruction was verified using the software for "mathematical completeness" with optical and physical compression of the slice corrected by rescaling the section to the original thickness at the time of sectioning (300 µm).
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
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, termination of the dendrites (i.e., with at least 1 dendrite ending in apposition to the central canal/4th ventricle or not). Data analysis was performed as described previously (11, 31).
Solutions composition.
Krebs solution consisted of (in mM) 120 NaCl, 26 NaHCO3, 3.75 KCl, 1 MgCl2, 2 CaCl2, and 11 dextrose, maintained at pH 7.4 with O2-CO2 (955%). Potassium gluconate intracellular solution consisted of (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. Potassium chloride intracellular solution consisted of (in mM) 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 EGTA, 2 ATP-Na, and 0.25 GTP-Na, adjusted to pH 7.35 with HCl. Zamboni's fixative consisted of 1.6% (wt/vol) paraformaldehyde, 19 mM KH2PO4, and 100 mM Na2HPO4·7H20 in 240 ml saturated picric acid-1,600 ml H2O, adjusted to pH 7.4 with HCl. PBS-TX consisted of (in mM) 115 NaCl, 75 Na2HPO4·7H2O, 7.5 KH2PO4, and 0.15% Triton X-100. Avidin D-HRP solution consisted of 0.002% Avidin D-HRP in PBS, 1% Triton X-100, and 0.05% DAB 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); rat-PP was purchased from Bachem (King of Prussia, PA); all other chemicals were purchased from Sigma (St. Louis, MO).
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RESULTS
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To limit spurious results, only those cells showing the brightest and most intense DiI fluorescence were used for recording. The postsynaptic response to PP was tested on 142 pancreas-projecting DMV cells; in 73 neurons, we obtained the full spectrum of electrophysiological parameters, and 50 neurons were filled sufficiently with Neurobiotin to provide the full spectrum of morphological parameters. The presynaptic effects of PP were tested on 74 pancreas-projecting rat DMV neurons; morphological reconstructions were obtained in 15 of these neurons.
Postsynaptic effects of PP.
In 22% of neurons tested (32 of 142), PP induced an outward current, whereas a further 20% of neurons (29 of 142) responded with an inward current. Biphasic responses (i.e., inward followed by outward current or vice versa) were never observed. The remaining 57% of neurons (81 of 142) showed no postsynaptic response to PP. The inward and outward currents induced by PP were concentration dependent (10300 nM) and had similar estimated EC50 concentrations of 30 nM (Fig. 1, A and B). The maximal amplitude of the PP-induced outward current, however, was larger than that of the PP-induced inward current (41.6 ± 9.34 vs. 24.0 ± 2.57 pA, respectively; P < 0.05).

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Fig. 1. Effects of pancreatic polypeptide (PP) on identified pancreas-projecting dorsal motor nucleus of the vagus (DMV) neurones. Representative traces from a pancreas-projecting DMV neuron illustrating the concentration-dependent nature of the outward current induced by PP. The cell was voltage clamped at 50 mV. A recovery period of at least 5 min was allowed between successive applications. Concentration-response curve for the PP-induced outward and inward current expressed as a percentage of the maximal response. The EC50 for the PP response was 30 nM. Each neuron was tested with at least 3 different concentrations of PP. Summary comparing the mean outward (n = 3) or inward (n = 4) current amplitude of 100 nM PP in the absence and presence of TTX (P > 0.05 vs. control).
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The PP-induced outward and inward currents were unaffected by superfusion with the synaptic blocker, TTX (1 µM; Fig. 1C). For example, in control conditions, perfusion with 100 nM PP induced a 36.0 ± 6.02-pA outward current that reversed following washout. After 10 min perfusion with Krebs solution containing TTX, PP induced a 37.0 ± 4.9-pA outward current (P > 0.05 vs. PP alone; n = 3). Similarly, in four neurons in which 100 nM PP induced an inward current, the amplitude of the current was unaffected by perfusion with TTX (28.2 ± 1.1 pA in control conditions vs. 25.7 ± 2.5pA in the presence of TTX; P > 0.05).
In neurons in which perfusion with 100 nM PP induced an outward current, the input resistance (measured between 50 and 60 mV) was 516 ± 96.5 M
in control and 379 ± 74.7 M
following perfusion with PP (73.9 ± 5.1% of control; P < 0.05; n = 4). The reversal potential of the outward current induced by 100 nM PP was assessed in four neurons voltage clamped at 50 mV and subjected to 200 ms-long steps, in 10-mV increments, every 10 s up to 120 mV, in the presence and absence of PP. The reversal potential of the PP-induced outward current was between 90 and 100 mV, i.e., close to the potassium equilibrium potential (Ek; Fig. 2). In neurons in which perfusion with 100 nM PP induced an inward current, the input resistance was 393 ± 49.8 M
in control and 262 ± 46.2 M
following perfusion with PP (66.7 ± 7.2% of control; P < 0.05; n = 5) with an estimated reversal potential close to 0 mV.

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Fig. 2. Current-voltage relationship for the PP-induced current. Left: representative traces showing the control response of a pancreas-projecting DMV neuron voltage clamped at 50 mV and stepped to 120 mV in 10-mV increments for 200 ms every 5 s. Right: same protocol as left but in the presence of 100 nM PP. Note that PP induced an outward current. Current-voltage relationship for the traces depicted above. Note that the reversal potential for the PP-induced current is 100 mV, i.e., close to EK.
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The basic electrophysiological and morphological properties of pancreas-projecting neurons responsive to PP are summarized in Table 1. Briefly, neurons that were inhibited by PP were found to have a broader action potential than nonresponsive neurons but a smaller action potential afterhyperpolarization. Neurons responsive to PP (inward or an outward current) differed only in the amplitude of their action potential afterhyperpolarization (see Table 1).
The effects of PP on action potential characteristics were assessed in 11 responsive neurons, six of which were depolarized and the remaining five were hyperpolarized. In all neurons, measurements of the action potential characteristics in the presence of PP were conducted after returning the membrane potential to baseline values via DC injection. In neurons in which PP induced a membrane depolarization, the action potential duration was unaffected (2.22 ± 0.21 and 2.37 ± 0.18 ms in control and PP, respectively, P > 0.05), but both the afterhyperpolarization (AHP) amplitude and duration were decreased (18.8 ± 2.3 mV and 79.0 ± 10.9 ms in control vs. 16.2 ± 2.1 mV and 67.6 ± 9.5 ms in PP, respectively; P < 0.05 for both), whereas a greater number of action potentials was evoked following DC injection (Fig. 3). Conversely, in neurons hyperpolarized by PP, the action potential and AHP duration were unaffected (2.38 ± 0.24 and 90.0 ± 11.0 ms in control vs. 2.18 ± 0.20 and 102.6 ± 18.0 ms in PP, respectively; P > 0.05), but the AHP amplitude was increased from 18.0 ± 1.8 to 21.0 ± 1.6 mV in control and PP, respectively (P < 0.05), whereas fewer action potentials were evoked following DC injection (Fig. 3).

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Fig. 3. Effects of PP on action potential characteristics. Frequency-response curves for pancreas-projecting DMV neurons depolarized (solid line) or hyperpolarized (dotted line) by PP. Note the changes in firing frequency in the presence of PP. Holding potential = 60 mV. *P < 0.05 vs. control. B, top trace: current-clamp trace shows a single action potential in a neuron excited by PP. The holding potential of the cell was restored to control values during perfusion with 100 nM PP. Note that the afterhyperpolarization amplitude and duration were decreased by perfusion with PP. Bottom trace: current-clamp trace shows a single action potential in a neuron inhibited by PP. The holding potential of the cell was restored to control values during perfusion with 100 nM PP. Note that the afterhyperpolarization amplitude, but not its duration, was increased by perfusion with PP. Holding potential = 60 mV. C, top traces: representative traces illustrating an inward current induced by perfusion with 300 nM PP in a neuron voltage clamped at 50 mV. After 10 min perfusion with 100 nM apamin, the inward current induced by reapplication of PP was reduced significantly. Bottom traces: representative traces illustrating an outward current induced by perfusion with 300 nM PP in a neuron voltage clamped at 50 mV. After 10 min perfusion with 100 nM apamin, the outward current induced by reapplication of PP was reduced significantly. D: summary comparing the mean outward (n = 4) and inward (n = 2) current amplitudes in response to 300 nM PP in the presence and absence of 100 nM apamin. *P < 0.05 vs. control.
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Because the effects of PP on pancreas-projecting neurons seems to involve an action on the AHP, which, in DMV neurons is composed, at least in part, of an apamin-sensitive calcium-dependent potassium current (44, 50), we tested the response to PP in the presence and absence of apamin. In six cells that responded to PP (4 with an outward and 2 with an inward current), 10 min of perfusion with 100 nM apamin decreased the amplitude of the PP-induced current to 19.1 ± 10.81% of control (P < 0.05; Fig. 3, C and D).
Presynaptic effects of PP.
EPSCs were evoked by electrical stimulation of the subnuclei centralis, medialis, and/or commissuralis of the NTS and recorded in 30 identified pancreas-projecting DMV neurons. Perfusion with PP (31,000 nM) induced a concentration-dependent inhibition in evoked EPSC amplitude in 22 of the 30 neurons tested (i.e., 73%; Fig. 4A) with an estimated IC50 of 30 nM (Fig. 4C). At 300 nM, the maximal inhibition of EPSC amplitude was 36.3 ± 5.6% (n = 4). The effect of PP did not appear to desensitize during the perfusion time, and tachyphylaxis was not observed when a second perfusion with PP was conducted following 10- to 15-min washout.

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Fig. 4. PP induced a concentration-dependent inhibition in evoked current amplitude. A: representative traces showing the concentration-dependent inhibition of evoked excitatory postsynaptic current (EPSC) amplitude by PP. Full recovery was achieved between successive applications of PP. Each trace is the average of 3 EPSCs. Holding potential = 60 mV. B: representative traces showing the concentration-dependent inhibition of evoked inhibitory postsynaptic current (IPSC) amplitude by PP. Full recovery was achieved between successive applications of PP. Each trace is the average of 3 IPSCs. Holding potential = 50 mV. C: graphical representation of the concentration-dependent effects of PP expressed as percentage of maximum response (PP, 100 nM). Note that PP induced inhibitions with similar estimated IC50 values. Each data point represents the average of up to 6 neurones for IPSCs and up to 16 neurones for EPSCs.
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IPSCs were evoked by electrical stimulation of the NTS in 17 DMV neurons. Perfusion with PP (31,000 nM) induced a concentration-dependent inhibition in evoked IPSC amplitude in 13 of the 17 neurons tested (i.e., 76%; Fig. 4B) with an estimated IC50 of 30 nM (Fig. 4C). At 300 nM, the maximal inhibition of IPSC amplitude was 22.7 ± 4.2% (n = 3). The effect of PP did not appear to desensitize during the perfusion time, and tachyphylaxis was not observed when a second perfusion with PP was conducted 1015 min later.
The ratio of the amplitude of two postsynaptic currents evoked a few milliseconds apart is used to determine whether a drug is acting at a pre- or postsynaptic site, with a change in the ratio being taken as indicative of a presynaptic site of action (9, 16, 52). When two EPSCs were evoked 50200 ms apart, in cells where PP had an effect on the evoked currents, PP decreased the amplitude of the first current (C1) more relative to that of the second current (C2) such that the paired pulse ratio (C2/C1) increased. For example, in the presence of 100 nM PP, the paired pulse ratio of evoked EPSCs increased from 0.76 ± 0.05 to 0.98 ± 0.08 (n = 15, P < 0.05; data not shown). Similarly, in the presence of 100 nM PP, the paired pulse ratio of evoked IPSCs increased from 0.72 ± 0.12 to 0.77 ± 0.14 (n = 4, P < 0.05; data not shown).
Spontaneous glutamatergic events were studied in 11 DMV neurons. In 6 of the 11 neurons, perfusion with 100 nM PP reduced the frequency of spontaneous EPSCs from 6.95 ± 1.43 to 4.46 ± 1.34 events/s (i.e., 58.1 ± 7.85% of control; P < 0.05), leaving the amplitude of the events unaltered (38.6 ± 5.46 pA in control and 39.1 ± 5.32 pA in PP; P > 0.05; Fig. 5, A and C).

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Fig. 5. PP decreases the frequency but not the amplitude of spontaneous postsynaptic currents. Representative traces showing that perfusion with 100 nM PP decreased the frequency of spontaneous EPSCs; the frequency of spontaneous EPSCs returned to baseline levels following washout of PP. Holding potential = 60 mV. B: computer-generated graphics from the same neuron as in A showing that PP altered the frequency (left) but not the amplitude (right) of spontaneous EPSCs. C: graphic highlighting the decrease in spontaneous event frequency induced by PP (left) but the lack of effect on spontaneous event amplitude (right). *P < 0.05 vs. control.
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Spontaneous GABAergic events were studied in nine DMV neurons. In seven of the nine neurons, perfusion with 100 nM PP reduced the frequency of spontaneous IPSCs from 3.29 ± 1.17 to 1.53 ± 0.59 events/s (i.e., 50.9 ± 8.8% of control; P < 0.05), leaving the amplitude of the events unaltered (84.4 ± 12.9 pA in control and 74.1 ± 10.12 pA in PP; P > 0.05; Fig. 5C).
The effects of PP (100 nM) on the response induced by chemical stimulation of the area postrema were studied in five pancreas-projecting DMV neurons. A pipette containing KCl was placed over the area postrema, and a picospritzer (Parker Instruments, Fairfield, NJ) was used to apply pulses of KCl sufficient to induce a synaptic response in the recorded neuron. In three neurons, stimulation of the area postrema evoked only an inward current, whereas in the remaining two neurons, area postrema stimulation evoked a biphasic response (outward followed by an inward current). Perfusion with the synaptic blocker TTX (1 µM) reduced the induced inward current by 88.4 ± 11.4% (i.e., 238 ± 122 to 37 ± 14 pA; n = 3). In all five neurons, perfusion with PP (100 nM) reduced the inward current induced by stimulation of the area postrema by 38.2 ± 7.7% (238 ± 84 to 171 ± 75 pA; n = 5; P < 0.05) and reduced the induced outward current by 49.6 ± 8.6% (524 ± 2.5 to 264 ± 44 pA; n = 2; Fig. 6).

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Fig. 6. PP decreases the amplitude of currents induced by chemical stimulation of the area postrema. A: representative traces showing that in a pancreatic-projecting DMV neuron voltage clamped at 50 mV, chemical stimulation of the area postrema was able to induce a TTX- and PP-sensitive inward current. B: graphic highlighting the decrease in the amplitude of both the outward and inward currents evoked by chemical stimulation of the area postrema. *P < 0.05 vs. control.
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The basic membrane characteristics (input resistance, action potential duration at threshold, afterhyperpolarization amplitude and kinetics of decay, and action potential firing frequency) were compared between pancreas-projecting neurons with PP-responsive (n = 43) and -nonreponsive (n = 20) synaptic inputs (Table 2). No differences were observed in the basic membrane properties of these two groups of neurons.
Morphological reconstructions.
Of the 142 neurones in which we tested the postsynaptic effects of PP, we were able to obtain complete morphological reconstructions of 50 cells (25 nonresponsive and 25 responsive to PP). We reported previously (8) that pancreas-projecting neurons had either a bipolar or a multipolar somata morphology. In the present study, differences were not found in terms of morphological differences between responsive and nonresponsive cells (in both groups, 17 of 25 neurones were multipolar;
2-test P > 0.05; Fig. 7A). Surprisingly, when considering the orientation of dendritic projections, neurons unresponsive to PP were most likely to project to the ependymal layer of the central canal or fourth ventricle than neurons responding to PP (independently of whether they responded with an inward or an outward current). In fact, 11 of 25 nonresponding neurons projected to the ependymal layer compared with 4 of 24 responding neurons (
2-test P < 0.05; note: 1 neuron had its dendrites severed and was thus excluded from the count). Differences were not found, however, in the rostrocaudal distribution of PP responsive vs. nonresponsive neurons (Fig. 7B).

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Fig. 7. Morphology and localization of pancreas-projecting DMV neurons responsive to PP. Representative computer-aided morphological reconstructions illustrating a multipolar pancreas-projecting neuron that responded to PP with an outward current (left trace) and a bipolar pancreas-projecting neuron that was unresponsive to PP (right trace). B: localization of pancreas-projecting DMV neurons in the horizontal plane. Note that neurons unresponsive to PP (squares) and neurons responsive to PP (inward current; filled circles; outward current, triangles) were evenly distributed throughout the rostrocaudal extent of both the left and right portions of the DMV. For the sake of simplicity, the unresponsive neurons are all represented on the left DMV, whereas the responsive neurons are represented on the right DMV, the rostrocaudal localization is, however, preserved. C: horizontal scheme of the rostrocaudal extent of the DMV showing the location of the neurons in which the synaptic inputs were tested with PP [note that the neuronal location has been arbitrarily set in either the left (IPSC) or right (EPSC) side; the rostrocaudal position was, however, preserved]. , Neurons with EPSCs inhibited by PP; , neurons with EPSCs unaffected by PP; , neurons with IPSCs inhibited by PP; , neurons with IPSCs unaffected by PP. Note the lack of a discrete rostrocaudal distribution in responsive or nonresponsive neurons.
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Of the 74 pancreas-projecting neurons in which the responsiveness of synaptic inputs to PP was assessed, a complete morphological analysis was obtained in 15 cells. Of those, PP inhibited synaptic input to 11 neurons with the remaining four neurons showing no effect. Of the 11 neurons in which PP inhibited synaptic transmission, the majority (10 of 11) had a multipolar rather than a bipolar morphological shape with dendrites that projected away from rather than toward the central canal or fourth ventricle (7 of 10 neurons). Differences in morphological properties were not observed in the distribution along the rostrocaudal axis of neurons with PP-responsive vs. nonresponsive synaptic inputs (n = 32, 10 neurons with inputs nonresponsive to PP, 22 neurons with inputs responsive to PP; Fig. 7C).
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DISCUSSION
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In this study, we have shown that, in an in vitro preparation of juvenile rats, 1) perfusion of identified pancreas-projecting neurons with PP induces either an inward (21%) or an outward (22%) current in distinct subsets of neurons, leaving the remaining 57% unaffected; 2) perfusion with PP decreases synaptic transmission between the NTS and the DMV and between the area postrema and the DMV; and 3) the effects of PP are more likely to occur in pancreas-projecting DMV neurons with multipolar somata morphology and dendrites projecting in directions other than the ependymal layer of the fourth ventricle.
On the basis of our data, we suggest that portions of the vagal-mediated effects of PP on pancreatic functions are prompted by both the direct modulation of subsets of DMV preganglionic neurons as well as by inhibition of discrete brain stem circuits. The pharmacological diversity of the responses of DMV neurons may indicate functional differences in the vagal control of pancreatic functions. Our conclusions are based on the following evidence.
Perfusion with PP affects a subpopulation (
40%) of identified pancreas-projecting DMV cells in which PP induced concentration-dependent excitation or inhibition of the neuronal membrane. The inhibitory effects of PP seem to be mediated by an increase in a potassium conductance, because the PP-induced outward current was associated with a decrease in input resistance, had a reversal potential close to equilibrium potential (EK), and the amplitude of the fast, apamin-sensitive, calcium-dependent AHP that develops at the termination of the action potential was increased significantly. Indeed, pretreatment with apamin reduced significantly the amplitude of the outward PP-induced current.
The PP-induced inward current was similarly sensitive to apamin, suggesting the involvement of the closure (or at least the lack of opening) of a calcium-dependent potassium conductance, most likely the fast, apamin-sensitive AHP that has already been described in DMV neurons (11, 44). The PP-induced inward current, however, was also associated with a decrease in input resistance, which, coupled with an extrapolated reversal potential positive to 0 mV, would also imply the involvement of either a nonselective cationic conductance or the opening of channels (e.g., calcium or sodium) that have a positive reversal potential. Unfortunately, the low occurrence of PP-induced inward currents prevented us from conducting a more thorough study of the underlying ionic mechanisms.
In a subpopulation of pancreas-projecting DMV neurons, PP inhibits, in a concentration-dependent manner, EPSCs and IPSCs evoked by electrical stimulation of the adjacent NTS. When pairs of EPSCs or IPSCs were evoked, PP induced an alteration in the ratio of the current amplitudes and, additionally, PP reduced the frequency but not amplitude of spontaneous EPSCs or IPSCs. Both effects are strongly suggestive of a presynaptic site of action, i.e., on synaptic terminals apposing DMV neurons.
Recently, we have suggested that pancreas-projecting preganglionic parasympathetic neurons within the DMV can be distinguished from neurons innervating other areas of the gastrointestinal tract by a combination of their electrophysiological and morphological properties (8). Our present results show that the electrophysiological properties of the subgroup of PP-responsive pancreas-projecting neurons are not different from neurons unresponsive to PP (with the exception of neurons hyperpolarized by PP, which showed a broader action potential and a smaller AHP). Furthermore, their distribution throughout the rostrocaudal extent of the brain stem does not differ from neurons unresponsive to PP. We observed, however, that the majority of DMV neurons responsive to PP had a multipolar somata shape with dendritic projections oriented away from the central canal or fourth ventricle.
Although this observation is rather puzzling, because PP effects are supposedly determined by circulating PP rather than peptide release by local nerve terminals, one should consider that large portions of the DVC are either outside (i.e., the area postrema) or have a leaky blood-brain barrier (i.e., DMV and NTS) (15), which can be crossed by PP (1). It is thus possible that both DMV neurons and the synaptic circuits modulated by PP comprise one or more components that are accessible to circulating PP.
Our results, showing that perfusion with PP reduces significantly the synaptic currents evoked by stimulation of the area postrema, support recent in vivo data by Deng and colleagues (17). In their work, Deng et al. (17) reported that ablation of the area postrema and, likely, of some of the synaptic connections between NTS and DMV significantly reduced the effects of PP on basal pancreas exocrine secretion. Interestingly, the same work also reported that PP increased its inhibitory effects on 2-deoxyglucose (2-DG) stimulated pancreas secretion in rats that underwent area postrema ablation. This observation would suggest that synapses other than the area postrema-DMV circuit are also involved in the modulatory effect of PP on pancreatic exocrine functions. Indeed, in the present work, we showed that PP also modulates both excitatory as well as inhibitory transmission between the NTS and the DMV.
We have shown previously that the actions of several neurotransmitters or neuromodulators including other members of the pancreatic polypeptide family, i.e., NPY and PYY, do not inhibit GABAergic synaptic transmission to gastric-projecting DMV neurons unless the activity of the cAMP-protein kinase A pathway is stimulated either by direct activation of adenylate cyclase (e.g., forskolin) or by neurotransmitters positively coupled to adenylate cyclase (e.g., CCK, thyrotropin-releasing hormone) (13, 48, 51). It is interesting to note, then, that PP can inhibit GABAergic synaptic transmission to pancreas-projecting DMV neurons in naïve brain stem slices, without the prior pharmacological enhancement of cAMP levels. PP acts exclusively at Y4 receptors that, as with the other receptors of the PP family, are negatively coupled to adenylate cyclase through actions at Gi/o (36). It remains to be seen whether these inhibitory actions of PP on GABAergic synapses reflect intrinsic differences in the cAMP levels of GABAegic nerve terminals impinging on pancreatic-projecting neurons or whether it reflects intrinsic differences in the coupling of the Y4 receptor.
Physiological significance.
It has been well established that the frequency of vagal stimulation determines the type of response in the stomach; different frequencies of stimulation, for example, release acetylcholine, nitric oxide, or vasoactive intestinal peptide selectively (47). Similarly, it is also well established that the vagus nerve controls the pancreas in a heterogeneous manner (3, 5, 38, 39, 41, 53) and, similar to gastric functions, the influence of the vagus nerve on pancreatic exo- or endocrine function may depend on the frequency of experimental stimulation or on the frequency of action potential firing the DMV neuron can sustain. Indeed, Berthoud et al. (5) have shown that, when comparing gastric acid secretion to insulin and glucagon secretion, different parameters of vagal stimulation have divergent effects. Furthermore, electrical stimulation of the vagal gastric and hepatic branches increased insulin secretion in an independent and additive manner (3), suggesting that differences may exist either in the particular vagal fibers (and, by consequence, in the original vagal soma) or in the vagal neuroeffector coupling. In this study, we have provided evidence for the presence of at least three pharmacological subgroups of pancreas-projecting neurons, i.e., those that respond to PP with either an excitation, with an inhibition, and those that are insensitive to PP. It is tempting to speculate that these differing groups of pancreas-projecting DMV neurons represent classes of parasympathetic preganglionic neurons that regulate either exocrine (i.e., the neurons responsive to PP) or endocrine (the neurons unresponsive to PP) pancreatic functions selectively.
Because the overall effect of in vivo administration of PP on pancreatic functions is inhibitory (23, 55), our data suggest that these opposing effects of PP on the DMV membrane (excitatory and inhibitory) are likely mediated by different actions on cholinergic DMV neurons; PP would inhibit the DMV neurons contributing the preganglionic cells of the excitatory cholinergic pathway while exciting the neurons that contribute the preganglionic cells of the inhibitory NANC pathway.
Although the presence of a dual vagal innervation (cholinergic and NANC) to the pancreas has not been investigated specifically, a great deal of circumstantial evidence supports its presence. In fact, pretreatment with the selective muscarinic antagonist atropine blocked protein output induced by vagal or chemical stimulation (19, 22). Atropine, however, was ineffective in preventing the vagally stimulated increase in flow of pancreatic juices as well as bicarbonate secretion (19). The effects on these exocrine functions were, in contrast, antagonized by pretreatment with the ganglionic nicotinic antagonist hexamethonium (19). These data would suggest the involvement of more than one type of postganglionic pancreatic vagal innervation. Similarly, Okumura and colleagues (39) showed that the inhibitory actions of PP injected in the DVC could be blocked by vagotomy and suggested that rather than acting to inhibit the tonic excitatory cholinergic vagal fibers, PP may activate specific inhibitory fibers, further implicating the existence of an inhibitory NANC pathway to the pancreas. Also, Berthoud and colleagues showed that vagal electrical stimulation increases both c-Fos and pCREB expression in a large percentage of pancreatic ganglionic neurons, the majority of which were also NADPH positive (4), suggesting the presence of a nitrergic innervation to the pancreas. Finally, Deng and colleagues (17) showed that peripheral perfusion of PP increases cFos expression in the DMV and area postrema, providing further evidence that the inhibitory actions of PP are, at least in part, mediated by activation of a vagal (inhibitory) circuit. In their elegant paper, these authors also show that cFos expression in DMV is enhanced following lesions of the area postrema. Interestingly, whereas the inhibitory effects of PP on basal exocrine pancreatic secretion were abolished by lesions of the area postrema, such lesions enhanced the inhibitory effects of PP on 2-DG-stimulated secretion (17). These data suggest that pancreatic exocrine secretion is modulated both by the actions of PP directly on the membrane of DMV neurons as well as on the synaptic circuitry impinging on the DMV cells themselves, a hypothesis supported by the present study.
Although we recognize the limitations of in vitro tissue studies and the dangers of extrapolating such data to ascribe in vivo functions, the brain stem slice preparation used in the present study, as well as many others, provides an excellent system in which to study vagal reflexes, because 1) many of the synaptic connections to NTS as well as DMV neurons are still intact, and 2) it is possible to exercise control over the extracellular, and even intracellular, neuronal environment (for reviews, see Refs. 24, 43, 51). Thus, in the present study, we have provided evidence that the rat DMV comprises pharmacologically heterogeneous subpopulations of pancreas-projecting neurons and PP modulates vagal pancreatic functions both via a direct effect on the membrane of DMV neurons as well as indirectly via modulation of the synaptic currents impinging on them. On the basis of these results, we hypothesize that distinct DMV cells groups form clusters of neurons that selectively control pancreatic functions.
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GRANTS
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This work was supported by National Science Foundation Grant 0456291.
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ACKNOWLEDGMENTS
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We thank Dr. R. C. Rogers and M. Ruiter for critical comments on earlier versions of the manuscript. We thank Cesare M. Travagli for support and encouragement.
Portions of this manuscript have been published in abstract form (7).
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FOOTNOTES
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Address for reprint requests and other correspondence: R. Alberto 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.
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REFERENCES
|
---|
- Banks WA, Kastin AJ, and Jaspan JB. Regional variation in transport of pancreatic polypeptide across the blood-brain barrier of mice. Pharmacol Biochem Behav 51: 139147, 1995.[CrossRef][ISI][Medline]
- Berthoud HR, Blackshaw LA, Brookes SJ, and Grundy D. Neuroanatomy of extrinsic afferents supplying the gastrointestinal tract. Neurogastroenterol Motil 16, Suppl 1: 2833, 2004.[CrossRef]
- Berthoud HR, Fox EA, and Powley TL. Localization of vagal preganglionics that stimulate insulin and glucagon secretion. Am J Physiol Regul Integr Comp Physiol 258: R160R168, 1990.[Abstract/Free Full Text]
- Berthoud HR, Patterson LM, and Zheng H. Vagal-enteric interface: vagal activation-induced expression of c-fos and p-CREB in neurons of the upper gastrointestinal tract and pancreas. Anat Rec 262: 2940, 2001.[CrossRef][ISI][Medline]
- Berthoud HR and Powley TL. Characteristics of gastric and pancreatic responses to vagal stimulation with varied frequencies: evidence for different fiber calibers? J Auton Nerv Syst 19: 7784, 1987.[CrossRef][ISI][Medline]
- Berthoud HR and Powley TL. Identification of vagal preganglionics that mediate cephalic phase insulin response. Am J Physiol Regul Integr Comp Physiol 258: R523R530, 1990.[Abstract/Free Full Text]
- Browning KN, Coleman FH, and Travagli RA. Pancreatic polypeptide (PP) targets selective populations of pancreatic-projecting brainstem vagal motoneurons. Digestive Disease Week, May 1520, 2004 (New Orleans, LA).
- Browning KN, Coleman FH, and Travagli RA. Characterization of pancreas-projecting rat dorsal motor nucleus of the vagus neurons. Am J Physiol Gastrointest Liver Physiol 288: G950G955, 2005.[Abstract/Free Full Text]
- Browning KN, Kalyuzhny AE, and Travagli RA. Opioid peptides inhibit excitatory but not inhibitory synaptic transmission in the rat dorsal motor nucleus of the vagus. J Neurosci 22: 29983004, 2002.[Abstract/Free Full Text]
- Browning KN, Kalyuzhny AE, and Travagli RA. Mu-opioid receptor trafficking on inhibitory synapses in the rat brainstem. J Neurosci 24: 93449352, 2004.
- Browning KN, Renehan WE, and Travagli RA. Electrophysiological and morphological heterogeneity of rat dorsal vagal neurones which project to specific areas of the gastrointestinal tract. J Physiol 517: 521532, 1999.[Abstract/Free Full Text]
- Browning KN and Travagli RA. Characterization of the in vitro effects of 5-hydroxytryptamine (5HT) on identified neurones of the rat dorsal motor nucleus of the vagus (DMV). Br J Pharmacol 128: 13071315, 1999.[CrossRef][ISI][Medline]
- Browning KN and Travagli RA. Neuropeptide Y and peptide YY inhibit excitatory synaptic transmission in the rat dorsal motor nucleus of the vagus. J Physiol 549: 775785, 2003.[Abstract/Free Full Text]
- Buijs RM, Chun SJ, Niijima A, Romijn HJ, and Nagai K. Parasympathetic and sympathetic control of the pancreas; a role for the suprachiasmatic nucleus and other hypothalamic centers that are involved in the regulation of food intake. J Comp Neurol 431: 405423, 2001.[CrossRef][ISI][Medline]
- Cottrell GT and Ferguson AV. Sensory circumventricular organs: central roles in integrated autonomic regulation. Regul Pept 117: 1123, 2004.[CrossRef][ISI][Medline]
- Debanne D, Guerineau NC, Gahwiler BH, and Thompson SM. Paired-pulse facilitation and depression at unitary synapses in rat hippocampus: quantal fluctuation affects subsequent release. J Physiol 491: 163176, 1996.[Abstract]
- Deng X, Wood PG, Sved AF, and Whitcomb DC. The area postrema lesions alter the inhibitory effects of peripherally infused pancreatic polypeptide on pancreatic secretion. Brain Res 902: 1829, 2001.[CrossRef][ISI][Medline]
- Grundy D and Schemann M. The interface between the enteric and central nervous system. In: Innvervation of the Gut: Pathophysiological Implications, edited by Tache Y, Wingate DL, and Burks TF. Boca Raton, FL: CRC, 1993, p. 157166.
- Holst JJ, Schaffalitzky de Muckadell OB, and Fahrenkrug J. Nervous control of pancreatic exocrine secretion in pigs. Acta Physiol Scand 105: 3351, 1979.[ISI][Medline]
- Honig MG and Hume RI. DiI and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing. TINS 12: 333341, 1989.[CrossRef][ISI][Medline]
- Jansen ASP, Hoffman JL, and Loewy AD. CNS sites involved in sympathetic and parasympathetic control of the pancreas: a viral tracing study. Brain Res 766: 2938, 1997.[CrossRef][ISI][Medline]
- Jung G, Louie DS, and Owyang C. Pancreatic polypeptide inhibits pancreatic enzyme secretion via a cholinergic pathway. Am J Physiol Gastrointest Liver Physiol 253: G706G710, 1987.[Abstract/Free Full Text]
- Katschinski M. Nutritional implications of cephalic phase gastrointestinal responses. Appetite 34: 189196, 2000.[CrossRef][ISI][Medline]
- Krowicki ZK. Role of selected peptides in the vagal regulation of gastric motor and endocrine pancreatic function. J Physiol Pharmacol 47: 399409, 1996.[ISI][Medline]
- Larsen PJ and Kristensen P. The neuropeptide Y (Y4) receptor is highly expressed in neurones of the rat dorsal vagal complex. Brain Res Mol Brain Res 48: 16, 1997.[ISI][Medline]
- Lewis MW, Hermann GE, Rogers RC, and Travagli RA. In vitro and in vivo analysis of the effects of corticotropin releasing factor on rat dorsal vagal complex. J Physiol 543: 135146, 2002.[Abstract/Free Full Text]
- Li Y, Hao Y, and 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: G679G685, 1997.[Abstract/Free Full Text]
- Loewy AD, Franklin MF, and Haxihu MA. CNS monoamine cell groups projecting to pancreatic vagal motor neurons: a transneuronal labeling study using pseudorabies virus. Brain Res 638: 248260, 1994.[CrossRef][ISI][Medline]
- Loewy AD and Haxihu MA. CNS cell groups projecting to pancreatic parasympathetic preganglionc neurons. Brain Res 620: 323330, 1993.[CrossRef][ISI][Medline]
- Louie DS, Williams JA, and Owyang C. Action of pancreatic polypeptide on rat pancreatic secretion: in vivo and in vitro. Am J Physiol Gastrointest Liver Physiol 249: G489G495, 1985.[Abstract/Free Full Text]
- Martinez-Pena Y, Valenzuela IM, Browning KN, and 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: 5460, 2004.[CrossRef][ISI][Medline]
- McTigue DM, Edwards NK, and Rogers RC. Pancreatic polypeptide in dorsal vagal complex stimulates gastric acid secretion and motility in rats. Am J Physiol Gastrointest Liver Physiol 265: G1169G1176, 1993.[Abstract/Free Full Text]
- McTigue DM, Hermann GE, and Rogers RC. Effect of pancreatic polypeptide on rat dorsal vagal complex neurons. J Physiol 499: 475483, 1997.[Abstract]
- McTigue DM and Rogers RC. Pancreatic polypeptide stimulates gastric motility through a vagal-dependent mechanism in rats. Neurosci Lett 188: 9396, 1995.[CrossRef][ISI][Medline]
- Mendelowitz D and Kunze DL. Identification and dissociation of cardiovascular neurons from the medulla for patch clamp analysis. Neurosci Lett 132: 217221, 1991.[CrossRef][ISI][Medline]
- Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, Quirion R, Schwartz T, and Westfall TC. XVI international union of pharmacology reccommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev 50: 143150, 1999.[ISI]
- Mulholland MW, Lally K, and Taborsky GJ Jr. Inhibition of rat pancreatic exocrine secretion by neuropeptide Y: studies in vivo and in vitro. Pancreas 6: 433440, 1991.[ISI][Medline]
- Nishizawa M, Nakabayashi H, Kawai K, Ito T, Kawakami S, Nakagawa A, Niijima A, and Uchida K. The hepatic vagal reception of intraportal GLP-1 is via receptor different from the pancreatic GLP-1 receptor. J Auton Nerv Syst 80: 1421, 2000.[CrossRef][ISI][Medline]
- Okumura T, Pappas TN, and Taylor IL. Pancreatic polypeptide microinjection into the dorsal motor nucleus inhibits pancreatic secretion in rats. Gastroenterology 108: 15171525, 1995.[ISI][Medline]
- Owyang C. Negative feedback control of exocrine pancreatic secretion: role of cholecystokinin and cholinergic pathway. J Nutr 124: 1321S1326S, 1994.[Medline]
- Putnam WS, Liddle RA, and Williams JA. Inhibitory regulation of rat exocrine pancreas by peptide YY and pancreatic polypeptide. Am J Physiol Gastrointest Liver Physiol 256: G698G703, 1989.[Abstract/Free Full Text]
- Rinaman L and Miselis RR. The organization of vagal innervation of rat pancreas using cholera toxin-horseradish peroxidase conjugate. J Auton Nerv Syst 21: 109125, 1987.[CrossRef][ISI][Medline]
- Rogers RC, McTigue DM, and Hermann GE. Vagal control of digestion: modulation by central neural and peripheral endocrine factors. Neurosci Biobehav Rev 20: 5766, 1996.[CrossRef][ISI][Medline]
- Sah P and McLachlan EM. Potassium currents contributing to action potential repolarization and the afterhyperpolarization in rat vagal motoneurons. J Neurophysiol 68: 18341841, 1992.[Abstract/Free Full Text]
- Sheikh SP, Roach E, Fuhlendorff J, and Williams JA. Localization of Y1 receptors for NPY and PYY on vascular smooth muscle cells in rat pancreas. Am J Physiol Gastrointest Liver Physiol 260: G250G257, 1991.[Abstract/Free Full Text]
- Streefland C, Maes FW, and Bohus B. Autonomic brainstem projections to the pancreas: a retrograde transneuronal viral tracing study in the rat. J Auton Nerv Syst 74: 7181, 1998.[CrossRef][ISI][Medline]
- Takahashi T and Owyang C. Vagal control of nitric oxide and vasoactive intestinal polypeptide release in the regulation of gastric relaxation in rat. J Physiol 484: 481492, 1995.[Abstract]
- Travagli RA and Browning KN. The State of Activation of NTS Neurons Determines the Effects of Pancreatic Polypeptides on Inhibitory Synaptic Transmission Within the Rat Dorsal Vagal Complex. Society for Neuroscience, 2001.
- Travagli RA, Gillis RA, Rossiter CD, and Vicini S. Glutamate and GABA-mediated synaptic currents in neurons of the rat dorsal motor nucleus of the vagus. Am J Physiol Gastrointest Liver Physiol 260: G531G536, 1991.[Abstract/Free Full Text]
- Travagli RA, Gillis RA, and 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: G508G517, 1992.[Abstract/Free Full Text]
- Travagli RA, Hermann GE, Browning KN, and Rogers RC. Musings on the Wanderer: What's New in our Understanding of Vago-Vagal Reflexes? III. Activity-dependent plasticity in vago-vagal reflexes controlling the stomach. Am J Physiol Gastrointest Liver Physiol 284: G180G187, 2003.[Abstract/Free Full Text]
- Travagli RA and Williams JT. Endogenous monoamines inhibit glutamate transmission in the spinal trigeminal nucleus of the guinea pig. J Physiol 491: 177185, 1996.[Abstract]
- Wang J, Zheng H, and Berthoud HR. Functional vagal input to chemically identified neurons in pancreatic ganglia as reveled by Fos expression. Am J Physiol Endocrinol Metab 277: E958E964, 1999.[Abstract/Free Full Text]
- Whitcomb DC, Puccio AM, Vigna SR, Taylor IL, and Hoffman GE. Distribution of pancreatic polypeptide receptors in the rat brain. Brain Res 760: 137149, 1997.[CrossRef][ISI][Medline]
- Whitcomb DC and Taylor IL. A new twist in the brain-gut axis. Am J Med Sci 304: 334338, 1992.[ISI][Medline]
- Whitcomb DC, Taylor IL, and Vigna SR. Characterization of saturable binding sites for circulating pancreatic polypeptide in rat brain. Am J Physiol Gastrointest Liver Physiol 259: G687G691, 1990.[Abstract/Free Full Text]
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