Tachykinins mediate slow excitatory postsynaptic transmission in guinea pig sphincter of Oddi ganglia

Brian P. Manning and Gary M. Mawe

Department of Anatomy and Neurobiology, The University of Vermont College of Medicine, Burlington, Vermont 05405


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular recording techniques were used to test whether tachykinins could be mediators of slow excitatory postsynaptic potentials (EPSPs) in guinea pig sphincter of Oddi (SO) ganglia. Application of the tachykinin substance P (SP) onto SO neurons caused a prolonged membrane depolarization that was reminiscent of the slow EPSP in these cells. Pressure ejection of the neurokinin 3 (NK3) receptor-specific agonist senktide caused a similar depolarization; however, no responses were detected on application of NK1 or NK2 receptor agonists. The NK3 receptor antagonist SR-142801 (100 nM) significantly inhibited both SP-induced depolarization and the stimulation-evoked slow EPSP, as did NK3 receptor desensitization with senktide. Capsaicin, which causes the release of SP from small-diameter afferent fibers, induced a depolarization that was similar to the evoked slow EPSP in both amplitude and duration. The capsaicin-induced depolarization was significantly attenuated in the presence of SR-142801. These data indicate that tachykinins, released from extrinsic afferent fibers, act via NK3 receptors to provide slow excitatory synaptic input to SO neurons.

motility; neurokinin receptors; myenteric ganglia; innervation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE SPHINCTER OF ODDI (SO) is a muscular valve that consists of smooth muscle bundles that ensheathe the common bile and pancreatic ducts as they pass through the duodenal wall. The SO regulates the flow of bile and pancreatic fluids into the duodenum, as well as the filling of the gallbladder, and prevents the reflux of duodenal contents into the biliary tree. Regulation of these processes requires coordinated changes in the tone of the SO musculature. Between meals, SO resistance increases and bile flows retrogradely into the gallbladder, rather than into the intestine. During a meal, the resistance through the SO decreases, allowing bile and pancreatic fluids to flow out of the biliary tree and into the duodenum (for review, see Ref. 30). However, this simple mechanism does exhibit some species-specific traits (for example, see Ref. 2), and the relative importance of neural and hormonal signaling in modulating SO muscle tone is not completely understood. In all models studied, however, the wall of the SO contains a ganglionated nerve plexus that lies in the same plane as the myenteric plexus of the remainder of the gastrointestinal tract. This location suggests that the neurons residing in SO ganglia may be connected to other neurons residing in the gastrointestinal tract, most notably the duodenum, and suggests that these neurons comprise a portion of an intrinsic neural circuit that may act to modulate SO function.

Synaptic inputs to ganglionated plexuses in the enteric nervous system provide a mechanism for modulation of physiological functioning of digestive organs. Studies by Mawe and colleagues (for example, see Refs. 20, 28, and 33) have shown that synaptic inputs to SO neurons include fast and slow excitatory postsynaptic potentials (EPSPs) as well as inhibitory postsynaptic potentials (IPSPs). By using low-frequency stimulation of interganglionic fiber bundles, fast excitatory inputs to SO neurons have been shown to involve presynaptic release of acetylcholine acting on nicotinic receptors on SO neurons to depolarize the postsynaptic cell membrane (32). Inhibitory postsynaptic potentials are evoked by high-frequency stimulation of fiber bundles and involve the release of norepinephrine acting on alpha 2 receptors (33). Slow excitatory postsynaptic potentials can be evoked in SO neurons by high-frequency fiber tract stimulation and result in prolonged depolarization of the membrane, often resulting in bursts of action potentials. However, the signal and receptor responsible for generating the slow EPSP in SO neurons is currently unknown.

Likely candidates for modulation of slow synaptic potentials in SO neurons include tachykinins. Tachykinins are a class of neuroactive peptides that have been shown to act as neuromodulators in many tissue types, including primary and accessory digestive organs, including the gastric corpus (26), ileum (4, 12), and colon (24). Within the guinea pig gallbladder, application of exogenous tachykinins onto gallbladder neurons causes a prolonged depolarization (18, 19). Further studies in this system have shown that the response is mediated by neurokinin 3 (NK3) receptors and that slow EPSPs in gallbladder ganglia involve the release of tachykinins (19). The ganglionated plexus of the SO contains a large number of neurons and nerve fibers that are tachykinin immunoreactive (29, 34), suggesting a role for this family of peptides in neurotransmission within the SO.

This study was designed to test whether tachykinins have a role in generation of slow excitatory postsynaptic potentials in SO neurons. The objectives of this study were to determine whether SO neurons respond to tachykinins and, if so, to determine which neurokinin receptor subtype(s) are responsible for these effects. In addition, we tested whether SO neurons respond to the release of endogenous tachykinins by capsaicin and whether the slow EPSP in SO ganglia is diminished by neurokinin receptor antagonists and/or by receptor desensitization.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue Preparation

Adult guinea pigs of either sex, weighing between 250 and 300 g, were anesthetized with halothane and exsanguinated. This method has been reviewed and approved by the Institutional Animal Care and Use Committee of the University of Vermont (protocol no. 97-125). The duodenum and SO were removed and immediately placed in an iced Krebs solution (in mM: 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 8 glucose, aerated with a 95% O2-5% CO2 gas jet). Nifedipine (5 µM) and atropine (200 nM) were added to the Krebs solution to eliminate smooth muscle contraction. The duodenum/SO preparation was dissected by pinning both the free end of the bile duct and the distal portion of the duodenum, then carrying a longitudinal incision along the mesenteric border opposite the entrance of the bile duct into the duodenum. The tissue was then pinned flat in a Sylgard-polymer lined dish while being continuously perfused by circulating iced Krebs. The mucosal and submucosal layers of the duodenum were removed under a dissecting microscope, exposing the underlying muscularis externa of the duodenum and the SO itself. A second longitudinal incision was created through the opening of the bile duct and SO musculature. The SO was also pinned flat, mucosal side up, and the mucosa and underlying circular muscle bundles were removed. This exposed the ganglionated plexus of the SO. This dissected tissue was then placed into a tissue chamber for electrophysiological recording.

Electrophysiological Recordings

The methods for electrophysiological recording were similar to those previously described (20, 32). The SO preparation was pinned flat in a low-volume (6 ml) tissue chamber and continuously perfused with warm Krebs solution (37°C, aerated by 95% O2-5% CO2). Ganglia were visualized by using Hoffman Modulation Contrast microscopy.

Intracellular recordings were made using an Axoclamp 2B electrometer (Axon Instruments, Foster City, CA) with 2 M KCl-filled microelectrodes. These microelectrodes had resistances between 60 and 80 MOmega for voltage recording. Capacitance neutralization was adjusted as needed for optimum current injection and voltage recording. Results were monitored on a separate oscilloscope, and data acquisition and analysis were performed by using MacLab Chart software to emulate oscilloscope readings (MacLab hardware and software; AD Instruments, Castle Hills, NSW, Australia). Results were also recorded on both a thermal array chart recorder (Astro-Med, West Warwick, RI) and magnetic tape for subsequent data reacquisition. Electrodes used to stimulate fiber tracts were extracellular monopolar Teflon-coated platinum wires (25-µm diameter) placed against interganglionic fiber bundles and were used to stimulate synaptic transmission to impaled cells. Activation of slow EPSPs involved 3-s trains of 0.5-ms pulses at a frequency of 20-30 Hz.

Compounds to be tested on SO neurons were applied either by pressure microejection or addition to the circulating Krebs solution. Pressure ejection of compounds involved glass micropipettes (15- to 20-µm tip diameter) placed 50-100 µm from the target ganglia and were induced by pulses of inert nitrogen gas (300 kg/cm2; 10-1,000 ms). Distance of pressure ejection micropipette was maintained at 50-100 µm from the tissue if selection of a new target ganglion was necessary. Washout periods (>= 20 min) were done between subsequent administrations of test compounds to allow for the recovery of responses in the event of receptor desensitization (19). Most test compounds were dissolved in Krebs solution as 1 mM stock solution and serially diluted in Krebs, as necessary. Capsaicin was dissolved in absolute ethanol as a stock concentration of 10 mM and diluted to the working concentration in Krebs solution. The low-Ca2+ Krebs solution used for some experiments consisted of (in mM): 121 NaCl, 5.9 KCl, 0.5 CaCl2, 5.0 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 8 glucose.

Numerical Analysis

Averaged values are presented as means ± SE. Statistical analysis was performed by using a paired Student's t-test to determine the difference between control and experimental values. Because of the rapid desensitization of vanilloid receptors, experiments involving capsaicin application in the presence of the NK3 receptor-specific antagonist SR-142801 were compared with control experiments done in separate tissue preparations. Thus, in these experiments, results were compared by using an unpaired Student's t-test to compare different populations of cells. Differences were considered to be significant if P <=  0.05.

Sources of Compounds

All solutions and compounds were obtained from Sigma Chemical (St. Louis, MO) with the exception of SR-142801 and SR-140333, which were the gifts of Sanofi Recherche (Montpellier, France).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular recordings were obtained from a total of 74 neurons from 64 preparations. Impaled neurons exhibited passive and active properties that were comparable to earlier findings within guinea pig SO (5, 9, 32). Four types of neurons exist within the ganglionated plexus of the SO: tonic, phasic, afterhyperpolarizing (AH), and quiescent. Tonic neurons exhibit multiple action potentials following the onset of a depolarizing current pulse and often fire spontaneously while the membrane is at rest. Phasic neurons fire only one or two action potentials at onset of a depolarizing current pulse and are not spontaneously active. AH neurons exhibit a shoulder during the repolarizing phase of an action potential and a prolonged afterhyperpolarization (lasting seconds) following the repolarization of the cell membrane. Quiescent cells exhibit no action potentials on depolarization, exhibit no anodal break spike at offset of a hyperpolarizing current pulse, and do not produce spontaneous activity while the membrane is at rest. In the current study, out of a total of 74 neurons, 12 neurons were tonic (16.2%), 32 were phasic (43.2%), 1 was AH (1.4%), and 29 were quiescent (39.2%). Because no difference was observable between the cell classification and its response to electrical stimuli or responses to test compounds, data were pooled for all impaled neurons.

Responses of SO Neurons to Substance P and Other Tachykinins

Substance P-mediated depolarization. In the initial experiments, application of substance P (SP; 100 µM) was performed by pressure microejection (500-ms duration). The rapid washout of the peptide with this method minimizes receptor desensitization. All of the 10 cells tested responded to SP with a prolonged membrane depolarization. The mean amplitude of the depolarization was 10.8 ± 1.7 mV [range 4.3-23.5 mV; mean resting membrane potential (Vm) = -57.3 ± 2.2 mV; Fig. 1]. The duration of the SP-induced depolarization was determined by measuring the length of time between onset of depolarization and the return of the membrane potential to half of the maximal depolarization amplitude. As such, the half-maximal duration (dur50) of SP depolarization in these neurons was 23.4 ± 3.9 s.


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Fig. 1.   Pressure ejection of neurokinin receptor agonists onto sphincter of Oddi (SO) neurons. A: application of either substance P (SP) or the neurokinin 3 (NK3) receptor-specific agonist senktide (both 100 µM in pipette) results in appreciable membrane depolarization. Application of either NK1 or NK2 receptor-specific agonists {[Sar9,Met(O2)11]SP or [beta -Ala8]neurokinin A, respectively; 100 µM each in pipette} results in only slight membrane depolarization. B: representative traces of SO neuronal response to SP or NK1, NK2, or NK3 receptor-specific agonists. Arrows indicate point of agonist application.

Actions of low-Ca2+ Krebs and TTX on SP- and fiber tract-stimulated depolarizations. To test if the SP-induced depolarization was a direct response of the impaled neuron, SP was applied to SO neurons following a 20-min equilibration period in the presence of low-Ca2+ Krebs containing TTX (0.3 µM) to block transmitter release from nerve terminals in the preparation. In the seven cells tested, a control application of 100 µM SP resulted in a mean depolarization of 18.1 ± 3.5 mV. When SP was added to SO neurons in a low-Ca2+ Krebs solution, the resulting depolarization amplitude was not significantly different from control (16.6 ± 4.0 mV; P > 0.1). Furthermore, the dur50 of the depolarization was not significantly different in control vs. low-Ca2+ conditions (control 25.2 ± 2.8 s; low-Ca2+ 22.3 ± 4.1 s; P > 0.05).

In contrast, replacement of normal circulating Krebs with low-Ca2+ Krebs dramatically attenuated fiber tract stimulation-induced synaptic responses, indicating that neurotransmission was eliminated under these conditions. Control fiber tract stimulations elicited slow EPSPs with a mean amplitude of 7.7 ± 1.3 mV and with a mean dur50 of 13.4 ± 2.3 s. However, following a 20-min equilibration period in low-Ca2+ Krebs, fiber tract-stimulated depolarizations were significantly inhibited in both amplitude (1.5 ± 0.3 mV; P <=  0.005; n = 7) and dur50 (6.7 ± 0.9 s; P <=  0.025; n = 7).

Actions of neurokinin receptor-specific agonists. To determine which neurokinin receptor was responsible for the SP-mediated depolarization, neurokinin receptor-specific agonists were applied to impaled SO neurons. Either the NK1 receptor-specific agonist [Sar9, Met(O2)11]SP, the NK2 receptor agonist [beta -Ala8]neurokinin A(4-10), or the NK3 receptor-specific agonist senktide was applied (pressure microejection 100 µM in pipette, 500-ms duration or superfusion 100 nM). Pressure microejection of the NK3 receptor agonist senktide resulted in a depolarization that was similar in amplitude to fiber tract-stimulated slow EPSPs (slow EPSP, 6.2 ± 3.8 mV, Vm = -54.1 ± 1.3 mV, n = 16; senktide, 6.8 ± 3.8 mV, Vm = -54.5 ± 2.4 mV, n = 10; Fig. 1). The senktide-induced depolarization was associated with a high-frequency burst of action potentials in tonic cells and was frequently associated with an onset of action potential generation in phasic cells. Neither the NK1 nor the NK2 receptor agonist caused a detectable change in membrane depolarization or excitability (NK1 receptor agonist, control resting membrane potential -51.8 ± 2.4 mV; after agonist -51.6 ± 3.1 mV, n = 5; NK2 receptor agonist, control resting membrane potential -49.4 ± 5.3 mV; after agonist -49.9 ± 5.7 mV, n = 5; Fig. 1).

Effects of Neurokinin Receptor Antagonists on Tachykinin Responses

Actions of NK3 receptor-specific antagonist on SP-mediated depolarization. To further verify that SP depolarizes SO neurons by acting on the NK3 receptor, SP was applied to impaled neurons by pressure microejection (100 µM in pipette, 500-ms duration) in the presence of the NK3 receptor antagonist SR-142801 (100 nM, continuous perfusion). Before application of SR-142801, impaled SO neurons responded to SP with a prolonged depolarization [amplitude 10.8 ± 1.7 mV; dur50 23.4 ± 3.9 s (as described in Substance P-mediated depolarization), n = 10]. After at least 5 min of continuous presentation of the NK3 receptor antagonist, the SP-induced depolarization was significantly diminished in both amplitude (2.2 ± 1.3 mV, P <=  0.0005, n = 10) and dur50 (3.6 ± 2.0 s, P <=  0.0005, n = 10; Fig. 2).


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Fig. 2.   SP elicits a prolonged depolarization that is attenuated in the presence of a NK3 receptor-specific antagonist. A: representative response of SO neuron to pressure ejection application of SP (500 ms; 100 µM; arrow). B: response of the same SO neuron to pressure ejection application of SP (500 ms; 100 µM) in the presence of the NK3 receptor antagonist SR-142801 (100 nM). Application of SR-142801 resulted in a significant reduction in the SP response (P <=  0.005, n = 10).

To further clarify the role of the NK3 receptor in SP-mediated depolarization, senktide (1 µM) was applied by continuous superfusion to desensitize the NK3 receptor. Ten minutes before senktide application, impaled SO neurons responded to SP pressure ejection (100 µM) with the expected prolonged depolarization (amplitude 11.6 ± 2.1 mV; dur50 26.9 ± 4.5 s; n = 15). After 10 min of continuous senktide application, the response to SP was almost completely eliminated (amplitude 3.5 ± 1.5 mV, P <=  0.01, n = 9; dur50 10.1 ± 1.1 s, P <=  0.025, n = 8; Fig. 3, A and B). A period of 20 min elapsed between control applications of SP and applications in senktide-desensitized preparations to allow for full recovery of SP responses.


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Fig. 3.   NK3 receptor desensitization by continuous senktide application results in attenuation of SP and fiber tract stimulation-induced depolarizations. Depolarization to SP (500 ms; 100 µM) is significantly inhibited in both amplitude (A) and half-maximal duration (B) following 10 min of continuous senktide application compared with control. Fiber tract stimulation-induced depolarizations are significantly inhibited in both amplitude (C) and half-maximal duration (D) following senktide application compared with control. *P <=  0.01; **P <=  0.025. EPSP, excitatory postsynaptic potentials.

Effects of Capsaicin on Impaled SO Neurons

Capsaicin depolarizes impaled SO neurons exhibiting slow excitatory synaptic inputs. To determine if release of endogenous SP from intracellular stores can depolarize impaled SO neurons, capsaicin (1 µM) was applied to SO neurons that responded to high-frequency fiber tract stimulation with a prolonged depolarization typical of a slow excitatory postsynaptic input. Before capsaicin application, impaled neurons were tested for the presence of slow excitatory synaptic inputs by electrically stimulating interganglionic fiber tracts with a high-frequency impulse (20-30 Hz, 2-3 s train duration). In the nine cells tested, fiber tract stimulation resulted in a prolonged depolarization with an amplitude of 9.2 ± 1.7 mV and a dur50 of 9.7 ± 1.8 s (Vm = -60.0 ± 2.0 mV).

Capsaicin was applied by pressure microejection (1 µM in pipette, 500 ms duration). In the same nine cells, capsaicin application resulted in a prolonged depolarization that was statistically similar to that elicited by fiber tract stimulation in both amplitude (9.3 ± 1.9 mV, P > 0.4, n = 9) and dur50 (11.7 ± 3.7 s, P > 0.4, n = 9, Vm = -60.6 ± 1.8 mV; Fig. 4). Subsequent applications of capsaicin to a given SO preparation resulted in little or no observable depolarization of the impaled SO neuron cell membrane, suggesting that desensitization of the vanilloid receptors had occurred. Rapid desensitization is a characteristic of vanilloid receptors (11).


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Fig. 4.   Pressure ejection of capsaicin (500 ms; 1 µM) elicits a membrane depolarization that is similar to evoked slow EPSPs, which is attenuated in the presence of NK3 receptor antagonists. A: no significant difference exists between depolarization amplitude elicited by either fiber tract stimulation or acute capsaicin application (1 µM; n = 9). However, capsaicin-induced depolarization is significantly reduced in the presence of the NK3 receptor antagonist SR-142801 (100 nM; n = 6). *P <=  0.01 by unpaired Student's t-test. B: acute capsaicin application results in a membrane depolarization that is inhibited in the presence of the NK3 receptor-specific antagonist SR-142801 (100 nM). Arrows indicate application of capsaicin.

Actions of capsaicin in the presence of the NK3 receptor-specific antagonist SR-142801. To determine if capsaicin-induced depolarization involves the release of tachykinins and the activation of NK3 receptors on SO neurons, capsaicin (1 µM) was applied to impaled SO neurons in the presence of the NK3 receptor-specific antagonist SR-142801 (100 nM). Because a single application of capsaicin rapidly desensitizes the vanilloid receptors, these data were compared with capsaicin responses in the absence of the antagonist in a separate population of cells. In six cells studied, application of capsaicin subsequent to application of SR-142801 resulted in a mean depolarization amplitude of 1.5 ± 0.2 mV and a mean dur50 of 7.0 ± 1.8 s (Fig. 4). These results show a significant decrease in the depolarization amplitude compared with cells presented with capsaicin in the absence of SR-142801 (mean amplitude 9.3 ± 1.9 mV, P <=  0.01, n = 6; Fig. 4). As in the experiments involving the effects of capsaicin in the absence of the receptor antagonist, multiple applications of capsaicin to a single preparation resulted in no observable depolarization.

Effects of Neurokinin Antagonists on Slow EPSPs

NK3 but not NK1 receptor-specific antagonists block fiber tract-stimulated slow excitatory postsynaptic potentials. Because the initial findings of this study indicated that NK3 receptors mediate tachykinin responses in SO ganglia, we tested whether the slow EPSP in this system was sensitive to NK3 receptor blockade and/or desensitization. At least 10 min after recording of a stimulus-evoked control slow EPSP, the NK3 antagonist SR-142801 (100 nM) was added to the bathing solution. The slow EPSP was significantly attenuated following a 10-min application of SR-142801 (control, 5.4 ± 1.1 mV; SR-142801, 2.9 ± 0.6 mV; P <=  0.025, n = 7; Fig. 5). The possibility of receptor desensitization was minimized by allowing ~20 min to pass between control fiber tract stimulus and stimulus in the presence of receptor antagonists.


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Fig. 5.   Interganglionic fiber tract stimulation generates a prolonged depolarization that is attenuated in the presence of NK3 receptor-specific antagonists. A: representative response of SO neuron to fiber tract stimulation (20 Hz, 3 s) exhibiting a prolonged depolarization. B: evoked slow EPSP is significantly attenuated in the presence of SR-142801 (100 nM).

Recordings were also performed in the presence of the NK1 receptor antagonist SR-140333 (100 nM). Before application of SR-140333, control stimulus-evoked slow EPSPs exhibited a mean depolarization amplitude of 4.8 ± 0.4 mV and a dur50 of 15.1 ± 2.9 s (Vm = -50.0 ± 1.3 mV; n = 5). Following at least 10 min in the presence of the receptor antagonist, evoked slow EPSPs were not noticeably affected (amplitude 4.4 ± 0.6 mV; dur50 17.4 ± 3.6 s; Vm = -51.7 ± 0.7 mV; P > 0.1, n = 5). As mentioned in Actions of NK3 receptor-specific antagonist on substance P-mediated depolarization, a period of at least 20 min elapsed between control fiber tract stimulus and evoked stimulus in the presence of the receptor antagonist to account for receptor desensitization following subsequent agonist applications.

Desensitization of the NK3 receptor by senktide attenuates the slow EPSP. To test whether desensitization of the NK3 receptor also results in an attenuation of the stimulus-evoked slow EPSP, the NK3 receptor-specific agonist senktide (1 µM) was continuously perfused over SO preparations. Control fiber tract stimulations of six cells 10 min before senktide application resulted in a mean depolarization of 6.1 ± 1.7 mV, with a dur50 of 11.4 ± 2.7 s. After 10 min of continuous senktide perfusion, the evoked slow EPSP was significantly inhibited in amplitude (0.9 ± 0.9 mV vs. control, P <=  0.025, n = 6) and dur50 (3.2 ± 3.2 s vs. control, P <=  0.01, n = 6; Fig. 3, C and D).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was conducted to test the hypothesis that tachykinins released from extrinsic sensory fibers contribute to the slow EPSP in SO ganglia. The data reported here support this hypothesis. SO neurons were depolarized by the naturally occurring tachykinin SP and the NK3 receptor-specific agonist senktide but not by agonists of NK1 or NK2 receptors. Furthermore, antagonism of NK3 receptors, or desensitization by prolonged agonist exposure, diminished the agonist-induced depolarization and evoked slow EPSPs. Finally, capsaicin, which releases tachykinins from small-diameter afferent fibers, caused a prolonged depolarization that was attenuated by the NK3 receptor antagonist. These data suggest that peripheral axon reflexes involving small-diameter afferent fibers can play a role in regulating SO ganglionic activity and therefore SO tone.

Within viscera, tachykinin receptors are present within both intrinsic autonomic ganglia and visceral smooth muscle. It has been established that NK1, NK2, and NK3 receptors are present throughout both central and peripheral nervous systems, as well as being located in close proximity with visceral smooth muscle (see Ref. 14 for review). Furthermore, tachykinin receptors are known to mediate excitatory synaptic inputs in respiratory (23) and cardiovascular systems (7) as well as the gut and gallbladder (19, 22, 26). Within the gut, tachykinins can modulate muscular contraction and peristalsis through both direct and indirect means by acting on smooth muscle and neuronal neurokinin receptors (see Ref. 15 for review). In the bowel, NK1 and NK2 receptors are present on most smooth muscle cells and can be activated by tachykinin release in the form of SP and neurokinin A, which are coreleased from intrinsic excitatory motor neurons (3, 16, 35). By contrast, NK3 receptors are present predominately on neurons and can act to increase or decrease muscle contraction indirectly by causing the release of acetylcholine/tachykinins or nitric oxide from excitatory and inhibitory motor neurons, respectively (12, 17). Recently, NK1 receptors have also been identified on select subpopulations of myenteric neurons and submucosal neurons (12, 20, 21, 24).

Data reported in this study demonstrate that tachykinins can cause a prolonged depolarization of SO neurons and that the subtype of neurokinin receptor that mediates this response is the NK3 receptor. Activation of tachykinin receptors by application of either the general receptor agonist SP or the NK3 receptor-specific agonist senktide activated a prolonged depolarization that was similar in amplitude and duration to the evoked slow EPSP in SO ganglia. The peak responses of SO neurons to SP and senktide were similar in amplitude and duration to the depolarization following a high-frequency stimulation of an attached interganglionic fiber tract. Furthermore, application of the NK3 receptor-specific antagonist SR-142801 attenuated both the slow EPSP elicited by fiber tract stimulation and the depolarization created by capsaicin application. The finding that the NK3 receptor antagonist significantly attenuated, but did not abolish, the evoked slow EPSP suggests that tachykinins are a principal mediator of slow EPSPs in SO ganglia but that additional neuroactive compounds may contribute as well. Candidate compounds that are present in SO ganglia and may contribute to the evoked postsynaptic depolarization include 5-HT acting on 5-HT1P receptors (8) or calcitonin gene-related peptide (CGRP) (6).

Previous studies have shown that tachykinin-immunoreactive fibers are abundant throughout the ganglionic plexus of the SO (29, 34). Most of these tachykinin-immunoreactive fibers are also immunoreactive for CGRP (34), suggesting that a dense network of extrinsic afferent fibers is present within the ganglionated plexus of the SO. Within the SO, tachykinin immunoreactivity has also been detected in cholinergic SO neurons (29), suggesting that these neurons may be excitatory motor neurons. Thus there are two potential sources of the tachykinins that contribute to the NK3-mediated component of the slow EPSP in SO ganglia: extrinsic afferent fibers and intrinsic SO neurons. However, since most SO neurons project from the ganglia to the muscle, and rarely to other SO neurons (32), sensory fibers probably represent the major source of tachykininergic input to SO neurons.

To test whether extrinsic afferent fibers contribute to the slow EPSP in SO ganglia, experiments were conducted to investigate the actions of the vanilloid receptor agonist capsaicin, which selectively releases neuroactive compounds, including tachykinins, from small-diameter afferent fibers (1, 10, 28). In the SO, capsaicin application resulted in a prolonged depolarization that was similar in amplitude and duration to that of the evoked slow EPSP, suggesting that peripheral release of SP by capsaicin occurs from nerve terminals synapsing on SO neurons. The capsaicin-induced depolarization was absent in the presence of the NK3 receptor antagonist SR-142801, indicating that tachykinins released from small-diameter afferent fibers in SO ganglia can reach and activate NK3 receptors on SO neurons.

Previous studies have demonstrated that nicotinic, fast excitatory synaptic input to SO neurons arises from cholinergic neurons in the myenteric neurons of the duodenum (20). These processes represent a local sensory reflex that connects the duodenal mucosa to SO neurons and allows for rapid alteration in SO muscular tone via these nicotinic fast EPSPs. That study also excluded duodenal neurons as a source of slow excitatory inputs to SO neurons. Another type of synaptic response in SO neurons, the IPSP, is mediated by sympathetic inputs acting on alpha 2 receptors (33).

The results of the current study indicate that slow excitatory inputs to SO neurons arise from processes of capsaicin-sensitive, extrinsic afferent fibers that pass through SO ganglia. When activated, these fibers release tachykinins, which activate NK3 receptors, creating a prolonged membrane depolarization. Thus capsaicin-mediated release of tachykinins from extrinsic afferents may elicit a response in SO ganglia that is similar to activation of primary afferents by sensory stimuli. Further studies will be required to determine if slow excitatory inputs originate from primary afferent fibers intrinsic or extrinsic to the SO. The endogenous tachykinins that contribute to the NK3 receptor-mediated depolarization are likely to include both SP and neurokinin A. The antiserum that was used to detect tachykinin immunoreactivity in SO ganglia can be preabsorbed with either SP or neurokinin A, but immunoassaying is not altered by neurokinin B preabsorbtion (34). Furthermore, the preprotachykinin mRNAs that encode for both SP and neurokinin A, but not neurokinin B, are expressed by peripheral neurons (27, 31).

Without knowledge of which neurons in the SO ganglia receive tachykininergic slow excitatory synaptic input, it is difficult to make definitive conclusions regarding the physiological role of synaptic transmission between extrinsic sensory fibers and SO ganglia. However, the following three possible functions come to mind: 1) if the sensory fibers provide input to excitatory motor neurons, this circuit may serve to increase resistance to bile flow at times when biliary tract pressure is increasing; 2) if sensory fibers synapse on inhibitory motor neurons, this pathway could provide a means of relieving pressure; and 3) since we know that these extrinsic sensory fibers can be activated within the duodenal myenteric plexus (20), this pathway may provide a means of communication between the duodenum and the SO to modulate SO resistance when duodenal pressure is increased. This latter function would serve to prevent the movement of duodenal luminal contents into the biliary tree.

In summary, the data reported here, in combination with previously published observations, indicate that tachykinins are likely to be neurotransmitters in SO ganglia. Immunoreactivity for tachykinins is abundant in extrinsic afferent fibers and in a subset of neurons within SO ganglia. Application of exogenous SP depolarizes SO neurons by activating NK3 receptors. Release of tachykinins from endogenous stores by application of capsaicin or by stimulation of interganglionic fiber tracts causes a depolarization that is inhibited by an antagonist of the NK3 receptor. These data support the concept that SO neuronal activity, and therefore SO tone, can be modulated by a local axon reflex involving the release of tachykinins from extrinsic afferent fibers.


    ACKNOWLEDGEMENTS

We thank Lisa M. Ellis for editorial assistance and Dr. Rodney L. Parsons for valuable discussion.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-45410 and NS-26995.

Address for reprint requests and other correspondence: G. M. Mawe, Given C 423, Dept. of Anatomy and Neurobiology, Univ. of Vermont, Burlington, VT 05405 (E-mail: gmawe{at}zoo.uvm.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.

Received 8 January 2001; accepted in final form 29 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(2):G357-G364
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