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The Journal of Physiology logoLink to The Journal of Physiology
. 2003 Jul 18;552(Pt 2):547–559. doi: 10.1113/jphysiol.2003.051326

Substance P presynaptically depresses the transmission of sensory input to bronchopulmonary neurons in the guinea pig nucleus tractus solitarii

Shin-ichi Sekizawa 1, Jesse P Joad 1, Ann C Bonham 1
PMCID: PMC2343393  PMID: 14561836

Abstract

Substance P modulates the reflex regulation of respiratory function by its actions both peripherally and in the CNS, particularly in the nucleus tractus solitarii (NTS), the first central site for synaptic contact of the lung and airway afferent fibres. There is considerable evidence that the actions of substance P in the NTS augment respiratory reflex output, but the precise effects on synaptic transmission have not yet been determined. Therefore, we determined the effects of substance P on synaptic transmission at the first central synapses by using whole-cell voltage clamping in an NTS slice preparation. Studies were performed on second-order neurons in the slice anatomically identified as receiving monosynaptic input from sensory nerves in the lungs and airways. This was done by the fluorescent labelling of terminal boutons after 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindocarbo-cyanine perchlorate (DiI) was applied via tracheal instillation. Substance P (1.0, 0.3 and 0.1 μM) significantly decreased the amplitude of excitatory postsynaptic currents (eEPSCs) evoked by stimulation of the tractus solitarius, in a concentration-dependent manner. The decrease was accompanied by an increase in the paired-pulse ratio of two consecutive eEPSCs, and a decrease in the frequency, but not the amplitude, of spontaneous EPSCs and miniature EPSCs, findings consistent with a presynaptic site of action. The effects were consistently and significantly attenuated by a neurokinin-1 (NK1) receptor antagonist (SR140333, 3 μM). The data suggest a new site of action for substance P in the NTS (NK1 receptors on the central terminals of sensory fibres) and a new mechanism (depression of synaptic transmission) for regulating respiratory reflex function.

Sensory nerves in the lungs and airways regulate pulmonary function and breathing pattern through CNS reflex pathways. The first CNS sites for synaptic contact of the primary afferent fibres are the second-order neurons in the nucleus tractus solitarii (NTS); it is at these synapses that the sensory input is first subjected to modulation before being transmitted through divergent pathways to generate the complex reflex outputs to the lungs, airways and respiratory muscles. Glutamate is the principle excitatory neurotransmitter at these NTS synapses, but a balance of inhibitory and excitatory modulatory mechanisms determines the net transmission. One neuromodulator of particular relevance to pulmonary and respiratory function is the neurokinin substance P. There is considerable morphological and physiological evidence to suggest that substance P is released in the intermediate and caudal NTS, where the lung and airway afferent fibres terminate: the region is enriched with substance P-containing axon nerve terminals (Brimijoin et al. 1980; Gillis et al. 1980; Helke et al. 1980; Katz & Karten, 1980; Baude et al. 1989; Kawai et al. 1989; Sykes et al. 1994; Massari et al. 1998), which have been shown to emanate from capsaicin-sensitive vagal afferent C fibres (Helke et al. 1981; Saria et al. 1988), from terminals with cell bodies perhaps originating within the NTS (Harlan et al. 1989; Gatti et al. 1995; Massari et al. 1998;) as well as from higher brain centres (Cuello & Kanazawa, 1978; Thor & Helke, 1989). A recent study using a medullary slice preparation has provided functional evidence that activation of local networks within the NTS by excitation of postsynaptic ionotropic glutamate receptors releases substance P (Colin et al. 2002).

A number of studies on the effects of substance P in the NTS on respiratory function have focused on the net modulatory effects on the reflex output, and for the most part, the data suggest an excitatory role. NTS microinjections of substance P or capsaicin, which releases neurokinins, including substance P (Buck & Burks, 1986), have been shown to augment lung C fibre-evoked lengthening of expiratory time (Mutoh et al. 2000) or to slow respiratory rate or lengthen expiratory time (Carter & Lightman, 1985; Bonham et al. 1993; Mazzone & Geraghty, 1999). Local excitation of NTS neurons by substance P (Morin-Surun et al. 1984; Henry & Sessle, 1985; Davis & Smith, 1997; Yuan & Lowell, 1997) or selective neurokinin 1 (NK1) receptor agonists (Maubach & Jones, 1997) has further confirmed an excitatory effect on neuronal activity. On the other hand, activation of NK1 receptors on vagal afferent neurons in the nodose ganglia has been shown to activate a calcium-dependent potassium current (Jafri & Weinreich, 1996) and suppress a non-inactivating hyperpolarization-activated inward current (Jafri & Weinreich, 1998), suggesting that the effects of substance P on afferent fibres could be inhibitory.

Despite the considerable amount of data on the net effects of substance P in the NTS, the specific role of the neuropeptide on the synaptic transmission between primary afferent fibres and the second-order neurons in the NTS is not known. The extent to which substance P modulates this neuron-to-neuron signalling might provide a heretofore unrecognized site for shaping the communication of the afferent signals to the NTS. Thus, the purpose of this study was to determine the effect of substance P on the synaptic transmission between primary bronchopulmonary afferent fibres and their anatomically identified second-order NTS neurons by using voltage clamping in a brainstem slice preparation to allow for isolation of presynaptic mechanisms. The effect of substance P was determined on tractus solitarius (TS)-evoked EPSCs (TS-eEPSCs), the paired-pulse ratio of two consecutive TS-eEPSCs, and the frequency and amplitude of spontaneous EPSCs (sEPSCs) and of miniature EPSCs (mEPSCs). Since most physiological responses evoked by substance P in the CNS appear to be selectively mediated via the NK1 receptors (Mazzone & Geraghty, 2000), we also studied the effect of an NK1 receptor antagonist on the substance P effects. The data suggest a new role for substance P in the NTS, that of presynaptic depression of glutamatergic transmission between bronchopulmonary afferent fibres and second-order NTS neurons.

METHODS

All experimental protocols in this work were reviewed and approved by the Institutional Animal Care and Use Committee in compliance with the Animal Welfare Act and in accordance with the Public Health Service Policy on the Humane Care and Use of Laboratory Animals.

Tracheal instillation of fluorescent dye to label bronchopulmonary central terminal boutons

Male Dunkin-Hartley guinea pigs, 45.0 ± 0.9 days old (mean ± S.E.M.) were used. To reduce airway secretion and constriction, terbutaline (0.1 mg kg−1) and atropine (0.02 mg kg−1) were injected subcutaneously 30 min prior to intubation. The animals were anaesthetized by intramuscular injection of ketamine (50 mg kg−1) and xylazine (4 mg kg−1). The oropharyngeal cavity was then anaesthetized with local application of lidocaine, and with the aid of a laryngoscope a 1.5 French endotracheal tube was inserted with its tip just below the larynx. To label the central terminal boutons of the bronchopulmonary afferent fibres, the anterogradely transported fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindocarbo-cyanine perchlorate (Fast DiI; 2 mg, dissolved in 20 μl DMSO and 180 μl surfactant (Survanta)) was instilled into the trachea through the cannula using a micropipette with a longer, gel-loading pipette tip (Christian et al. 1993). To prevent back-flow of the dye, the animal was artificially ventilated for 10 min immediately after injection, then extubated and placed on a heated pad during recovery from anaesthesia.

Brainstem slice preparation

After a period of at least 14 days following DiI instillation (to allow anterograde transport of the dye; 20.6 ± 0.7 days later; mean ± S.E.M.), the animals, which were at that point 64.6 ± 1.8 days old (mean ± S.E.M.), were anaesthetized with a combination of ketamine (35 mg kg−1) and xylazine (2 mg kg−1) and decapitated. As in previous studies (Aylwin et al. 1997; Chen et al. 2002), the brain was rapidly exposed and submerged in ice-cold (< 4 °C), high-sucrose artificial cerebrospinal fluid (aCSF) that contained (mM): 3 KCl, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 220 sucrose and 2 CaCl2 (300 mosmol kg−1); the pH was 7.4 when continuously bubbled with 95 % O2-5 % CO2. Brainstem coronal slices (250 μm thick) containing the intermediate to caudal NTS and the TS were cut with a Vibratome 1000 (Technical Products International, St Louis, MO, USA). After incubation for 45 min at 37 °C in high-sucrose aCSF, the slices were placed in normal aCSF that contained (mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose and 2 CaCl2 (300 mosmol kg−1); the pH was 7.4 when continuously bubbled with 95 % O2-5 % CO2. Four to five slices were obtained from each animal. During the experiments, a single slice was transferred to the recording chamber, held in place with a silk mesh and continuously perfused with oxygenated aCSF at a rate of approximately 4 ml min−1. All experiments were performed at 33-34 °C.

Identifying fluorescently labelled second-order neurons and whole-cell voltage-clamp recording

NTS neurons with attached boutons of the bronchopulmonary afferent fibres were identified using an Olympus BX50WI microscope (Olympus Optical, Tokyo, Japan) equipped with tetramethylrhodamine isothiocyanate epifluorescence filters, and then viewed with the aid of differential infrared contrast (Nomarski) optics for whole-cell voltage-clamp recordings. After establishing the cell-attached configuration with a seal resistance of > 1 GΩ, whole-cell currents were recorded using borosilicate glass pipettes filled with a caesium fluoride (CsF) solution containing (mM): 145 CsF, 5 NaCl, 1 MgCl2, 3 K-ATP, 0.2 Na-GTP, 10 EGTA and 10 Hepes (300 mosmol kg−1); pH 7.4 with resistances of 2.5-5 MΩ. Recordings were made with the Axopatch 1D patch-clamp amplifier (Axon Instruments, Foster City, CA, USA). Currents were filtered at 2 kHz digitized at 10 kHz with the DigiData1322A interface (Axon Instruments) and stored in an IBM-compatible computer. Data were analysed off-line using the pCLAMP8 software (Axon Instruments) and Mini Analysis program (Synaptosoft, Leonia, NJ, USA). The series resistance was < 20 MΩ. During the recordings, neurons were voltage clamped at -60 mV.

To record TS-eEPSCs in the labelled neurons, bipolar tungsten electrodes (1 μm tips separated by 80 μm) were placed in the TS ipsilateral to the recording site. To confirm that the neuron met the established criteria for synaptic activation over a monosynaptic pathway, the mean onset latency and variability of the eEPSC onset latency was determined and then two TS stimuli (2-20 V, 0.1 ms square-wave pulses) separated by 5 ms were delivered at 0.2 Hz for a total of 5-10 stimuli. An EPSC consistently evoked with an onset latency variability of < 0.5 ms or by each of the two stimuli separated by 5 ms was taken as presumptive electrophysiological evidence that the eEPSCs were evoked monosynaptically (Miles, 1986; Scheuer & Mifflin, 1998). Only neurons meeting anatomical and electrophysiological criteria for second-order neurons in bronchopulmonary afferent pathways were studied. sEPSCs were recorded, then tetrodotoxin (TTX, 5 μM) was added to the perfusate in order to record action potential-independent spontaneous mEPSCs. sEPSCs and mEPSCs were detected by MiniAnalysis Software. Time controls were performed with perfusion of aCSF without drugs.

Protocols

Presynaptic effects of substance P

To determine if substance P acted presynaptically, we measured the effect of the neuropeptide on: (1) the eEPSC peak amplitude, (2) the paired-pulse ratio, (3) the frequency and amplitude of sEPSCs and (4) the frequency and amplitude of mEPSCs (Hjelmstad & Fields, 2001; Kato & Shigetomi, 2001; Morisset & Urban, 2001; Dietrich et al. 2002; Kirischuk et al. 2002; Kombian et al. 2003). For the paired-pulse ratio, TS stimuli were delivered in pairs at interpulse intervals of 20 ms at an overall frequency of 0.2 Hz continuously over 45 s, and the peak amplitudes of the first and second eEPSCs were determined. Then sEPSCs were recorded for 2 min before (control) and continuing for 5 min after the onset of substance P perfusion. At the end of the 7 min sEPSCs recording period, the paired-pulse protocol was repeated. sEPSCs were recorded continuously during a washout period of at least 5 min, at which time the paired-pulse protocol was repeated to test for recovery of the eEPSC amplitude and paired-pulse ratio. After TTX was added to the aCSF perfusate, the frequency and amplitude of mEPSCs were recorded before, during and after washout of substance P.

An increase in the paired-pulse ratio accompanying a decrease in the first eEPSC peak amplitude of the pair and a decrease in the sEPSC and mEPSC frequencies but not amplitudes were taken as evidence for inhibition at a presynaptic locus in these studies (Debanne et al. 1996; Saitow et al. 2000; Hjelmstad & Fields, 2001; Kato & Shigetomi, 2001; Morisset & Urban, 2001; Price & Pittman, 2001; Dietrich et al. 2002; Kirischuk et al. 2002; Kline et al. 2002; Saviane et al. 2002; Shen & Johnson, 2002; Kombian et al. 2003; Lei & McBain, 2003; Shen & Johnson, 2003).

In separate studies, to determine a concentration-dependent effect of substance P, eEPSC and paired-pulse ratios were determined before and in the presence of three concentrations of substance P (0.1, 0.3 and 1.0 μM).

Pharmacology of substance P effects

To determine the extent to which the effects of substance P were mediated by NK1 receptors, the protocol was also performed in the presence of the NK1 receptor antagonist SR140333 (3 μM) plus substance P (1 μM). TS-eEPSCs, the paired-pulse ratio and sEPSC frequency and amplitude were determined during a control period, in the presence of NK1 receptor antagonist + substance P, and after washout.

Data analysis

Data are expressed as mean ± S.E.M. The level of statistical significance was set at P < 0.05. The onset latency and variability for each cell were determined from five eEPSCs delivered at 0.2 Hz. The peak amplitude of the eEPSCs was calculated as the difference between the stimulus-evoked trough current and the pre-stimulus mean current over 20 ms and averaged over 10 consecutive traces to establish the control response. The paired-pulse ratio was defined as the second eEPSC peak amplitude divided by the first eEPSC peak amplitude. To determine the concentration-dependent effects of substance P, concentration- response curves for the eEPSC peak amplitude and paired-pulse ratios (expressed as a percentage of the control value) were analysed with a one-way ANOVA. The decay trace of the eEPSCs was best fitted to a single exponential function, and its time constant was calculated. For the group data, the substance P-induced effect on eEPSC amplitude and the paired-pulse ratio was determined using a paired t test. sEPSCs and mEPSCs were detected by MiniAnalysis Software, where the threshold for detection was set just above the baseline noise of the recordings, which was < 3-5 pA, well below the average and median amplitude of the sEPSCs and mEPSCs. For the group data, the number and amplitude of sEPSCs occurring in 2 min were compared before perfusion, during perfusion (starting at 3 min after the onset of substance P perfusion) and during washout (last 2 min of washout) of substance P, by using a one-way ANOVA. mEPSCs were compared before and during perfusion of substance P by using a paired t test. The time controls for the eEPSC, paired-pulse ratio and sEPSC frequency and amplitude were also analysed using a paired t test. To determine the effects of NK1 receptor blockade on the substance P-induced effects, the peak amplitude of the first eEPSCs, the paired-pulse ratio and the frequency and amplitude of the sEPSCs were compared before and in the presence of substance P + NK1 receptor antagonist by using a paired t test.

Drugs

DiI (DiIC18(3)) was obtained from Molecular Probes (Eugene, OR, USA); substance P, CsF, EGTA, Hepes and CaCl2 were obtained from Sigma (St Louis, MO, USA) and the NK1 receptor antagonist (SR140333) was a gift from Sanofi Recherche (Montpellier, France). All other chemicals were obtained from Fisher (Pittsburgh, PA, USA). All drugs were dissolved in aCSF just prior to application.

RESULTS

All neurons that were identified by their possession of fluorescently labelled attached boutons (from instillation of dye into the lower trachea) were obtained from intermediate and caudal brainstem slices (0-1250 μm caudal to obex, and 250-500 μm lateral to midline) and met one of the presumptive electrophysiological criteria for classification of second-order neurons (n = 56). An example of such a neuron is shown in Fig. 1. The mean onset latency of the eEPSCs was 2.75 ± 0.13 ms, with a variability of 0.40 ± 0.04 ms. The longer and shorter diameters of the neurons were 16.2 ± 0.4 and 13.6 ± 0.3 μm, respectively. The whole-cell capacitance (36.1 ± 2.4 pF) had a linear relationship with the putative cell size expected by their diameters, and the input resistance averaged 470 ± 133 MΩ and mostly ranged from 150 to 700 MΩ. The peak amplitude of TS-eEPSCs averaged 152 ± 19 pA.

Figure 1. A second-order NTS neuron.

Figure 1

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A, an NTS neuron viewed with the aid of differential infrared contrast (DIC) microscopy. Calibration bar represents 20 μm. B, same neuron as in A viewed with the aid of fluorescence microscopy to visualize presynaptic bronchopulmonary afferent fibre terminal boutons. C, overlay of fluorescence and DIC images. D, patch electrode attached to the cell for whole-cell recording.

The neurons displayed a characteristic synaptic depression in response to the paired-pulse stimulation of the TS. The paired-pulse ratio of the eEPSCs averaged 0.69 ± 0.05.

Substance P decreased the eEPSC amplitude and increased the paired-pulse ratio

Substance P (1 μM) decreased the peak amplitude of the eEPSCs and increased the paired-pulse ratio (for example see Fig. 2A). Owing largely to the decrease in the first of the paired eEPSCs, the paired-pulse ratio was increased. The effects of substance P were maximal at approximately 5 min after the start of the perfusion and had partially recovered within approximately 5 min after washout.

Figure 2. Substance P decreased the eEPSC peak amplitude and increased the paired-pulse ratio.

Figure 2

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A, each trace is an average of 10 paired eEPSCs before (Control), during (SP 1 μM) and after substance P perfusion (Washout). The second eEPSC amplitude was smaller than the first, confirming the occurrence of paired-pulse depression. Substance P decreased the eEPSC peak amplitude, thereby increasing the paired-pulse ratio. B: left panel, group data confirm that substance P (SP) significantly reduced the eEPSC amplitude (*P < 0.05; n = 14); right panel, time control studies showed no decay in the eEPSC amplitude (P > 0.05, n = 11). C: left panel, in the same neurons, substance P significantly increased the paired-pulse ratio (*P < 0.05, n = 14); right panel, time control studies showed no change in the paired-pulse ratio (P > 0.05, n = 11).

The group data (Fig. 2) confirm the decrease in the eEPSC peak amplitude (Fig. 2B) and the increase in the paired-pulse ratio. Substance P (1 μM) reduced the peak amplitude of the first eEPSC by 42 ± 8 % (paired t test, P = 0.007; n = 14). The mean decay time constant of the eEPSCs, which could be fitted to a single exponential function, was not changed during treatment with substance P (4.8 ± 0.7 ms vs. 6.3 ± 1.3 ms; paired t test, P = 0.16). In the time control experiments (Fig. 2B, right panel), the eEPSC amplitude did not change (paired t test, P = 0.828; n = 11).

As shown in Fig. 2C (left panel) the group data for the same cells confirmed that substance P increased the paired-pulse ratio by 49 ± 12 % (paired t test, P < 0.001; n = 14). In the time control experiments, (Fig. 2C, right panel), the paired-pulse ratio did not change (paired t test, P = 0.296; n = 11).

Not all cells showed recovery during the washout of substance P. For the eEPSCs, in half of the neurons (7/14), the eEPSC amplitude recovered to 70 % of the control value, while in the other half the eEPSC amplitude recovered from 20 to 68 %. In those neurons displaying < 70 % recovery during washout, we increased the voltage to the TS stimulating electrode to determine whether the cell was capable of discharging an eEPSC; in all instances, the neuron discharged eEPSC, suggesting that the cell was still capable of being excited.

Concentration-dependent effects of substance P on eEPSCs and the paired-pulse ratio

The inhibitory effect of substance P on the first eEPSC was concentration dependent (Fig. 3, left panel; ANOVA, P = 0.002; Student-Newman-Keuls, P < 0.05, 1.0 μM vs. 0.1 μM substance P, 1.0 μM substance P vs. control) as was the increase in the paired-pulse ratio (Fig. 3, right panel; ANOVA, P = 0.047; Student-Newman-Keuls, P < 0.05, 1.0 μM substance P vs. control).

Figure 3. Concentration-dependent effects of substance P on eEPSC peak amplitude and paired-pulse ratio.

Figure 3

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The concentration-response curve, fitted to a sigmoid function, shows that substance P decreased the eEPSC peak amplitude (left) and correspondingly increased the paired-pulse ratio (right) in a concentration-dependent manner (*P < 0.05). Numbers in parentheses indicate the number of neurons tested.

Substance P depressed the frequency but not the amplitude of sEPSCs

Substance P (1 μM) decreased the frequency but not the amplitude of sEPSCs (Fig. 4). An example of continuous recordings of sEPSCs before and during treatment with substance P is shown in Fig. 4A. The event histogram for the same neuron (Fig. 4B) confirmed that the distribution of sEPSC amplitudes was the same, with a median amplitude of 9-10 pA before and in the presence of substance P. The cumulative probability of the sEPSC inter-event intervals was shifted to the right in the presence of substance P (Fig. 4C, left panel), indicating a decreased frequency, while the cumulative probability of the sEPSC amplitudes was not changed (Fig. 4C, right panel). The group data (Fig. 4D, left panel) confirm that substance P decreased the sEPSC frequency, decreasing the number of events per 2 min by 26.0 ± 7.5 % (Wilcoxon signed-rank test, P < 0.001; n = 13). By contrast, the sEPSC amplitude distribution before and during perfusion of substance P (Fig. 4D, right panel) was not changed in control or in substance P-treated tissue (P = 0.939). In the time control experiments, neither the sEPSC frequency nor amplitude changed (paired t test, P = 0.380 or P = 0.365, respectively; n = 11). As was the case with the eEPSCs, the recovery of the sEPSC frequency during the washout period for substance P was variable; nine of the 13 cells displayed a recovery of ≥ 70 % of the control value.

Figure 4. Substance P decreased the frequency but not the amplitude of sEPSCs.

Figure 4

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A, sample traces of continuous recordings of sEPSCs before and during substance P perfusion (SP, 1 μM). B, amplitude histogram of sEPSCs recorded from the same neuron in A, showing a decrease in the number of events with no change in the distribution of amplitudes during substance P perfusion. C, cumulative probability of the inter-event interval (left panel) and amplitude distributions (right panel) before and during substance P perfusion for the same neuron as recorded in A. Substance P increased the inter-event interval of sEPSCs, but not their amplitude. D, group data confirm that substance P decreased the average sEPSC frequency (left panel, *P < 0.05) and had no effect on the amplitude (right panel, P > 0.05; n = 13). The time controls showed no difference in either the sEPSC frequency or amplitude.

Substance P depressed the frequency but not the amplitude of mEPSCs

Substance P (1 μM) also decreased the frequency but not the amplitude of the mEPSCs (Fig. 5). An example of continuous recordings of mEPSCs before and during treatment with substance P is shown in Fig. 5A. The event histogram confirmed that the distribution of mEPSC amplitudes was the same (Fig. 5B), with the median amplitude of 5-6 pA before and in the presence of substance P. The cumulative probability of the mEPSC inter-event interval was shifted to the right in the presence of substance P (Fig. 5C, left panel), while the mEPSC amplitude was not changed (Fig. 5C, right panel).

Figure 5. Substance P decreased the frequency but not the amplitude of mEPSCs.

Figure 5

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A, sample traces of continuous recordings of mEPSCs in the presence of TTX before and during substance P perfusion (SP, 1 μM). B, amplitude histogram of mEPSCs recorded from same neuron as in A, showing a decrease in the number of events with no change in the distribution of amplitudes during substance P perfusion. C, cumulative probability of the inter-event interval (left panel) and amplitude distributions (right panel) before and during substance P perfusion for the same neuron as in A. Substance P increased the inter-event interval of sEPSCs but not their amplitude. D, group data confirm that substance P decreased the average mEPSC frequency (left panel, *P < 0.05) and had no effect on the amplitude (right panel, P > 0.05; n = 12).

The group data (Fig. 5D) confirm that substance P decreased the mEPSC frequency by 22.5 ± 9.2 % (Wilcoxon signed-rank test, P = 0.012; n = 12), but not the amplitude (P = 0.933). The substance P-induced decrease in frequency was the same for the sEPSCs and mEPSCs (Student's unpaired t test, P = 0.82). The recovery of mEPSC frequency from the effects of substance P was variable, with 8/12 cells showing a recovery of ≥ 70 % during the washout period.

NK1 receptor antagonism inhibited the effects of substance P on eEPSCs and sEPSCs

NK1 receptor antagonism abolished the presynaptic inhibitory effect of substance P on synaptic transmission, as shown in the example (Fig. 6A) and group data (Fig. 6B). When the NK1 receptor antagonist (3 μM) plus substance P (1 μM) were in the perfusate, there was no change in either the eEPSC amplitude (paired t test, P = 0.60; n = 6) or the paired-pulse ratio (paired t test: P = 0.674).

Figure 6. NK1 receptor antagonist blocked the substance P inhibitory effects on eEPSCs.

Figure 6

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A, each trace is an average of 10 eEPSCs before (Control) and during perfusion of NK1 receptor antagonist (NK1-RANT; 3 μM) + substance P (1 μM). Neither the first nor the second eEPSC amplitudes were changed during perfusion with NK1-RANT+ SP. B, group data confirm that the NK1-RANT prevented substance P effects on of eEPSC amplitude. C, in the same neurons, NK1-RANT prevented substance P effects on the paired-pulse ratio. (P > 0.05, n = 6).

NK1 receptor blockade also abolished the effects of substance P on sEPSC frequency (Fig. 7). An example of continuous traces of sEPSCs before and during treatment with NK1 receptor antagonist + substance P is shown in Fig. 7A. The event histogram (Fig. 7B) confirmed that the distribution of sEPSC amplitudes did not change. The increase in the inter-event interval observed during substance P (Fig. 4C) was not observed in the presence of the NK1 receptor antagonist (Fig. 7C, left panel); the amplitude distribution is shown in Fig. 7C, right panel. The group data (Fig. 7D) confirm that in the presence of the NK1 receptor antagonist, substance P had no effect on sEPSC frequency (P = 0.209; n = 6) or amplitude (P = 0.683).

Figure 7. Effect of NK1 receptor antagonist + substance P on sEPSCs.

Figure 7

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A, sample traces of continuous recordings of sEPSCs before (Control) and during perfusion of NK1-R antagonist + substance P (NK1-RANT+ SP). B, amplitude histogram of sEPSCs recorded from the same neuron, showing no change in the number of events or amplitude distribution before and during NK1-RANT+ substance P perfusion. C, cumulative probability of inter-event interval (left panel) and amplitude distributions (right panel). NK1-RANT+ substance P had no effect on sEPSC frequency or amplitude. D, group data confirm that substance P in the presence of NK1-RANT had no effect on the sEPSC frequency (P > 0.05) or amplitude (P > 0.05; n = 6).

Figure 8 compares the effects of substance P alone and in the presence of the NK1 receptor antagonist on eEPSC amplitude, the paired-pulse ratio and sEPSC frequency. The substance P-induced changes (n = 14) were significantly different during NK1 receptor antagonism (Student's t test; P = 0.003, eEPSCs; P = 0.024, paired-pulse ratios; P = 0.018, sEPSCs; n = 6).

Figure 8. Comparison between substance P alone and NK1 receptor antagonist + substance P on synaptic transmission.

Figure 8

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Bar plots show the mean percentage change of eEPSC amplitude (A), paired-pulse ratio (B) and sEPSC frequency (C). All graphs indicate that the effects of substance P (n = 14) were blocked by NK1 receptor antagonism (n = 6; unpaired t test: *P < 0.05).

DISCUSSION

This study presents new evidence that substance P, in a concentration-dependent manner, depresses the synaptic transmission between sensory afferent fibres and second-order NTS neurons anatomically identified as receiving monosynaptic input from sensory nerves in the lungs and airways. The findings are consistent with substance P actions at presynaptic NK1 receptors to decrease glutamate release: the neuropeptide decreased the peak amplitude of the TS-eEPSCs in conjunction with increasing the paired-pulse ratio and decreased the frequency of sEPSCs and mEPSCs without affecting the amplitude of either.

Presynaptic inhibition of eEPSCs

It is generally accepted that glutamate binding to postsynaptic ionotropic glutamate receptors transmits the sensory signals from the visceral afferent fibres, including those arising from the lungs and airways, to the second-order NTS neurons in the reflex pathways (Andresen & Kunze, 1994; Wilson et al. 1996; Aylwin et al. 1997; Smith et al. 1998). The finding that substance P decreased the first of the eEPSCs in the second-order neurons (with a resultant increase in the paired-pulse ratio) is evidence for a decreased probability of glutamate release. By contrast, had substance P acted postsynaptically to decrease the glutamate responsiveness of the second-order neuron, then the amplitudes of the first and second eEPSCs would have decreased to the same extent, resulting in an unchanged paired-pulse ratio. The presynaptic site of action is further supported by the lack of change in the eEPSC decay times. There may be several mechanisms for substance P decreasing glutamate release, but two particularly relevant studies indicate that the neuropeptide hyperpolarizes vagal afferent neurons by activating a calcium-dependent potassium current (Jafri & Weinreich, 1996) and by inhibiting a hyperpolarization-activated inward current (Ih) (Jafri & Weinreich, 1998). These complementary effects would delay action potential invasion of the central terminal, thereby depressing glutamate release.

Presynaptic inhibition of spontaneous synaptic activity

In addition to increasing the paired-pulse ratio, substance P decreased the frequency of the spontaneous synaptic currents, which signify spontaneous presynaptic neurotransmitter release. The spontaneous currents were action potential dependent (sEPSCs) and action potential independent (both sEPSCs and mEPSCs). As is the case with eEPSCs in the NTS, as shown in the pioneering work by Fortin & Champagnat (1993), the sEPSCs are also predominantly driven by glutamatergic inputs as evidenced by their abolition with ionotropic glutamate receptor blockade and insensitivity to GABAA receptor blockade (Fortin & Champagnat, 1993). In the present study, the sEPSC discharge rate averaged 7.3 ± 2.3 Hz, similar to that observed by Kato & Shigetomi (2001) in NTS neurons (6.7 ± 1.8 Hz) and, as in that study, the frequency was not significantly decreased in the presence of TTX, suggesting that a large fraction of the sEPSCs was due to the action-potential-independent release of glutamate. The sEPSC and mEPSC frequencies and amplitudes varied from cell to cell, consistent with previous findings by others (Kato & Shigetomi, 2001; Kline et al. 2002). Such variability might be expected given that NTS neurons probably have several synaptic inputs not only from the TS, but also from interneurons in the NTS and other CNS neurons. The findings that substance P decreased the frequency but not the amplitude of sEPSCs and mEPSCs is further evidence for a presynaptic locus, and raises the possibility that in addition to decreasing glutamate release from the central afferent terminals, presynaptic NK1 receptors on interneurons within the NTS may decrease spontaneous glutamate release onto the second-order neurons.

Presynaptic and postsynaptic substance P effects

While in general the effects of substance P have been reported to be excitatory in the NTS (Morin-Surun et al. 1984; Henry & Sessle, 1985; Andresen & Kunze, 1994; Davis & Smith, 1997), enhancing glutamate action (Bonham et al. 1993; Cowan et al. 2000) and reflex outputs (Mutoh et al. 2000; Seagard et al. 2000), the present experimental conditions (CsF pipette solution and whole-cell voltage clamping) were optimized to detect presynaptic effects. In the context of the new findings and the well-established excitatory effects of substance P in the NTS, the coexistence of functional presynaptic and postsynaptic NK1 receptors provides complementary mechanisms for the substance P modulation of lung-related sensory input by either limiting synaptic glutamate release or augmenting glutamate responsiveness at the postsynaptic neurons.

The lack of uniform recovery during washout of substance P is consistent with findings in other neural networks (Colin et al. 2002). Despite the variable extent of the recovery, the findings that in the time controls, there was no decay of the eEPSC amplitude, paired-pulse ratio or sEPSC frequency, that substance P exerted a concentration-dependent effect and that the substance P-induced effect was abolished by NK1 receptor antagonism, provide strong evidence that the substance P effect was real and was mediated via NK1 receptors.

Functional implications of substance P presynaptic effects

The physiological relevance of the presynaptic substance P effects may reside in the synaptic depression that is characteristic of the responses of postsynaptic NTS neurons during high-frequency afferent input (Miles, 1986; Mifflin & Felder, 1990; Scheuer et al. 1996; Schild et al. 1998; Chen et al. 1999, 2002; Kline et al. 2002). The depression, by serving as a low-pass filter, may improve signal transmission by allowing incoming signals with a wide dynamic range (which are not easily modulated) to be converted to signals with a smaller range (which are more easily modulated). There is considerable evidence that this frequency-dependent synaptic depression is mediated, at least in part, by inhibition of glutamate release from the primary afferent terminals through the activation of presynaptic receptors. For example, we have shown previously that glutamate can decrease its own release by acting at presynaptic group II and III metabotropic glutamate receptors (Chen et al. 2002). Others have shown that dopamine acting at D2 receptors (Kline et al. 2002), adenosine acting at A1 receptors (Kato & Shigetomi, 2001) and GABA acting at GABAB receptors (Brooks et al. 1992) can also depress glutamate release at these synapses. The present study adds yet another mechanism by which substance P may activate presynaptic NK1 receptors. Such redundant mechanisms confer both flexibility and dependability for regulating synaptic traffic, including that arising from the lungs and airways, at the very first synapse in the NTS. This study did not address the source of endogenous substance P release, but there is substantial evidence that substance P can be released in the NTS (Brimijoin et al. 1980; Gillis et al. 1980; Helke et al. 1980; Katz & Karten, 1980; Baude et al. 1989; Kawai et al. 1989; Sykes et al. 1994; Gatti et al. 1995; Massari et al. 1998) and in particular from capsaicin-sensitive vagal afferent C fibres (Helke et al. 1981; Saria et al. 1988), local neurons (Harlan et al. 1989; Gatti et al. 1995; Massari et al. 1998; Colin et al. 2002) or higher brain centres (Cuello & Kanazawa, 1978; Thor & Helke, 1989).

The source and conditions under which substance P is released may determine the extent to which the presynaptic and postsynaptic mechanisms are integrated to shape the neuronal output from the NTS to distal synapses in the reflex pathways. For example, certain pathophysiological conditions, such as the acute allergic inflammation associated with acute allergen exposure, results in an increase in the mRNA encoding substance P (Fischer et al. 1996), de novo substance P expression in Aδ vagal afferent fibre, likely rapidly adapting receptor, cell bodies (Undem et al. 1993), and increases in neuronal excitability, including depolarization of the membrane potential and blockade of an anomalous rectifier (Undem et al. 1999). The plasticity of substance P expression and vagal sensory neuronal excitability of the peripheral lung sensory nerves following brief allergen challenges suggests a mechanism for amplifying the synaptic input to neurons in the central network, which can, in turn, amplify the reflex output. In this case, the presynaptic NK1 receptors may provide a counterbalance to modulate an increase in afferent traffic to the NTS.

The neurons were judged to be second order by both anatomical and electrophysiological criteria. The tracheal instillation of the fluorescent dye to label the central boutons did not allow for a determination of the modality of the receptor subtypes (i.e. whether the boutons were of non-myelinated C fibres, the thinly myelinated Aδ rapidly adapting pulmonary receptors or the myelinated slowly adapting pulmonary receptors). However, the presynaptic inhibitory effect of substance P was consistent across all neurons tested, as well as in six additional unlabelled neurons, suggesting that the mechanisms operate at these first central synapses in a variety of sensory reflex pathways.

In summary, this work offers a novel mechanism by which substance P might regulate glutamate release at the first central synapses between lung afferent fibres and the second-order neurons in the lung reflex pathways. In the context of previous findings, the results further suggest that substance P takes full advantage of both presynaptic and postsynaptic mechanisms in the NTS to fine-tune the processing of lung afferent signal inputs and hence the reflex regulation of respiratory function.

Acknowledgments

This study was supported by funds from the California Tobacco-Related Disease Research Program, grant 9RT-0010. The NK1 receptor antagonist, SR140333, was a generous gift of Sanofi-Synthelabo Recherche, Montpellier, France. We are also grateful to John Bric for excellent technical assistance for tracheal instillation of the dye and to Dr Chao-Yin Chen for helpful criticisms in preparing the manuscript.

REFERENCES

  1. Andresen MC, Kunze DL. Nucleus tractus solitarius - gateway to neural circulatory control. Annu Rev Physiol. 1994;56:93–116. doi: 10.1146/annurev.ph.56.030194.000521. [DOI] [PubMed] [Google Scholar]
  2. Aylwin ML, Horowitz JM, Bonham AC. NMDA receptors contribute to primary visceral afferent transmission in the nucleus of the solitary tract. J Neurophysiol. 1997;77:2539–2548. doi: 10.1152/jn.1997.77.5.2539. [DOI] [PubMed] [Google Scholar]
  3. Baude A, Lanoir J, Vernier P, Puizillout JJ. Substance P-immunoreactivity in the dorsal medial region of the medulla in the cat: effects of nodosectomy. J Chem Neuroanat. 1989;2:67–81. [PubMed] [Google Scholar]
  4. Bonham AC, Coles SK, McCrimmon DR. Pulmonary stretch receptor afferents activate excitatory amino acid receptors in the nucleus tractus solitarius in rats. J Physiol. 1993;464:725–745. doi: 10.1113/jphysiol.1993.sp019660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brimijoin S, Lundberg JM, Brodin E, Hokfelt T, Goran N. Axonal transport of substance P in the vagus and sciatic nerves of the guinea pig. Brain Res. 1980;191:443–457. doi: 10.1016/0006-8993(80)91293-7. [DOI] [PubMed] [Google Scholar]
  6. Brooks PA, Glaum SR, Miller RJ, Spyer KM. The actions of baclofen on neurones and synaptic transmission in the nucleus tractus solitarii of the rat in vitro. J Physiol. 1992;457:115–129. doi: 10.1113/jphysiol.1992.sp019367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buck SH, Burks TF. The neuropharmacology of capsaicin. Pharmacol Rev. 1986;38:179–226. [PubMed] [Google Scholar]
  8. Carter DA, Lightman SL. Cardio-respiratory actions of substance P, TRH and 5-HT in the nucleus tractus solitarius of rats: evidence for functional interactions of neuropeptides and amine neurotransmitters. Neuropeptides. 1985;6:425–436. doi: 10.1016/0143-4179(85)90141-6. [DOI] [PubMed] [Google Scholar]
  9. Chen C-Y, Horowitz JM, Bonham AC. A presynaptic mechanism contributes to depression of autonomic signal transmission in nucleus tractus solitarius. Am J Physiol. 1999;277:H1350–1360. doi: 10.1152/ajpheart.1999.277.4.H1350. [DOI] [PubMed] [Google Scholar]
  10. Chen CY, Ling EH, Horowitz JM, Bonham AC. Synaptic transmission in nucleus tractus solitarius is depressed by Group II and III but not Group I presynaptic metabotropic glutamate receptors in rats. J Physiol. 2002;538:773–786. doi: 10.1113/jphysiol.2001.012948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Christian EP, Togo JA, Naper KE, Koschorke G, Taylor GA, Weinreich D. A retrograde labeling technique for the functional study of airway-specific visceral afferent neurons. J Neurosci Methods. 1993;47:147–160. doi: 10.1016/0165-0270(93)90031-l. [DOI] [PubMed] [Google Scholar]
  12. Colin I, Blondeau C, Baude A. Neurokinin release in the rat nucleus of the solitary tract via NMDA and AMPA receptors. Neuroscience. 2002;115:1023–1033. doi: 10.1016/s0306-4522(02)00541-9. [DOI] [PubMed] [Google Scholar]
  13. Cowan AR, Dean C, Bago M, Seagard JL. Potentiation of non-N-methyl-D-aspartate receptor-induced changes in blood pressure by substance P in rats. Neurosci Lett. 2000;278:161–164. doi: 10.1016/s0304-3940(99)00920-9. [DOI] [PubMed] [Google Scholar]
  14. Cuello AC, Kanazawa I. The distribution of substance P immunoreactive fibers in the rat central nervous system. J Comp Neurol. 1978;178:129–156. doi: 10.1002/cne.901780108. [DOI] [PubMed] [Google Scholar]
  15. Davis BJ, Smith DV. Substance P modulates taste responses in the nucleus of the solitary tract of the hamster. Neuroreport. 1997;8:1723–1727. doi: 10.1097/00001756-199705060-00031. [DOI] [PubMed] [Google Scholar]
  16. Debanne D, Guerineau NC, Gahwiler BH, Thompson SM. Paired-pulse facilitation and depression at unitary synapses in rat hippocampus: quantal fluctuation affects subsequent release. J Physiol. 1996;491:163–176. doi: 10.1113/jphysiol.1996.sp021204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dietrich D, Kral T, Clusmann H, Friedl M, Schramm J. Presynaptic group II metabotropic glutamate receptors reduce stimulated and spontaneous transmitter release in human dentate gyrus. Neuropharmacology. 2002;42:297–305. doi: 10.1016/s0028-3908(01)00193-9. [DOI] [PubMed] [Google Scholar]
  18. Fischer A, McGregor GP, Saria A, Phillipin B, Kummer W. Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic airway inflammation. J Clin Invest. 1996;98:2284–2291. doi: 10.1172/JCI119039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fortin G, Champagnat J. Spontaneous synaptic activities in rat nucleus tractus solitarius neurons in vitro: evidence for re-excitatory processing. Brain Res. 1993;630:125–135. doi: 10.1016/0006-8993(93)90650-c. [DOI] [PubMed] [Google Scholar]
  20. Gatti PJ, Shirahata M, Johnson TA, Massari VJ. Synaptic interactions of substance P immunoreactive nerve terminals in the baro- and chemoreceptor reflexes of the cat. Brain Res. 1995;693:133–147. doi: 10.1016/0006-8993(95)00728-9. [DOI] [PubMed] [Google Scholar]
  21. Gillis RA, Helke CJ, Hamilton BL, Norman WP, Jacobowitz DM. Evidence that substance P is a neurotransmitter of baro-and chemoreceptor afferents in nucleus tractus solitarius. Brain Res. 1980;181:476–481. doi: 10.1016/0006-8993(80)90633-2. [DOI] [PubMed] [Google Scholar]
  22. Harlan RE, Garcia MM, Krause JE. Cellular localization of substance P- and neurokinin A-encoding preprotachykinin mRNA in the female rat brain. J Comp Neurol. 1989;287:179–212. doi: 10.1002/cne.902870204. [DOI] [PubMed] [Google Scholar]
  23. Helke CJ, Jacobowitz DM, Thoa NB. Capsaicin and potassium evoked substance P release from the nucleus tractus solitarius and spinal trigeminal nucleus in vitro. Life Sci. 1981;29:1779–1785. doi: 10.1016/0024-3205(81)90188-0. [DOI] [PubMed] [Google Scholar]
  24. Helke CJ, O'Donohue TL, Jacobowitz DM. Substance P as a baro- and chemoreceptor afferent neurotransmitter: immunocytochemical and neurochemical evidence in the rat. Peptides. 1980;1:1–9. doi: 10.1016/0196-9781(80)90027-3. [DOI] [PubMed] [Google Scholar]
  25. Henry JL, Sessle BJ. Effects of glutamate, substance P and eledoisin-related peptide on solitary tract neurones involved in respiration and respiratory reflexes. Neuroscience. 1985;14:863–873. doi: 10.1016/0306-4522(85)90149-6. [DOI] [PubMed] [Google Scholar]
  26. Hjelmstad GO, Fields HL. Kappa opioid receptor inhibition of glutamatergic transmission in the nucleus accumbens shell. J Neurophysiol. 2001;85:1153–1158. doi: 10.1152/jn.2001.85.3.1153. [DOI] [PubMed] [Google Scholar]
  27. Jafri MS, Weinreich D. Substance P hyperpolarizes vagal sensory neurones of the ferret. J Physiol. 1996;493:157–166. doi: 10.1113/jphysiol.1996.sp021371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jafri MS, Weinreich D. Substance P regulates Ih via a NK-1 receptor in vagal sensory neurons of the ferret. J Neurophysiol. 1998;79:769–777. doi: 10.1152/jn.1998.79.2.769. [DOI] [PubMed] [Google Scholar]
  29. Kato F, Shigetomi E. Distinct modulation of evoked and spontaneous EPSCs by purinoceptors in the nucleus tractus solitarii of the rat. J Physiol. 2001;530:469–486. doi: 10.1111/j.1469-7793.2001.0469k.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Katz DM, Karten HJ. Substance P in the vagal sensory ganglia: localization in cell bodies and pericelluar arborizations. J Comp Neurol. 1980;193:549–564. doi: 10.1002/cne.901930216. [DOI] [PubMed] [Google Scholar]
  31. Kawai Y, Mori S, Takagi H. Vagal afferents interact with substance P-immunoreactive structures in the nucleus of the tractus solitarius: immunoelectron microscopy combined with an anterograde degeneration study. Neurosci Lett. 1989;101:6–10. doi: 10.1016/0304-3940(89)90431-x. [DOI] [PubMed] [Google Scholar]
  32. Kirischuk S, Clements JD, Grantyn R. Presynaptic and postsynaptic mechanisms underlie paired pulse depression at single GABAergic boutons in rat collicular cultures. J Physiol. 2002;543:99–116. doi: 10.1113/jphysiol.2002.021576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kline DD, Takacs KN, Ficker E, Kunze DL. Dopamine modulates synaptic transmission in the nucleus of the solitary tract. J Neurophysiol. 2002;88:2736–2744. doi: 10.1152/jn.00224.2002. [DOI] [PubMed] [Google Scholar]
  34. Kombian SB, Ananthalakshmi KV, Parvathy SS, Matowe WC. Substance P depresses excitatory synaptic transmission in the nucleus accumbens through dopaminergic and purinergic mechanisms. J Neurophysiol. 2003;89:728–737. doi: 10.1152/jn.00854.2002. [DOI] [PubMed] [Google Scholar]
  35. Lei S, McBain CJ. GABAB receptor modulation of excitatory and inhibitory synaptic transmission onto rat CA3 hippocampal interneurons. J Physiol. 2003;546:439–453. doi: 10.1113/jphysiol.2002.034017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Massari VJ, Shirahata M, Johnson TA, Lauenstein JM, Gatti PJ. Substance P immunoreactive nerve terminals in the dorsolateral nucleus of the tractus solitarius: roles in the baroreceptor reflex. Brain Res. 1998;785:329–340. doi: 10.1016/s0006-8993(97)01335-8. [DOI] [PubMed] [Google Scholar]
  37. Maubach KA, Jones RS. Electrophysiological characterisation of tachykinin receptors in the rat nucleus of the solitary tract and dorsal motor nucleus of the vagus in vitro. Br J Pharmacol. 1997;122:1151–1159. doi: 10.1038/sj.bjp.0701482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mazzone SB, Geraghty DP. Respiratory action of capsaicin microinjected into the nucleus of the solitary tract: involvement of vanilloid and tachykinin receptors. Br J Pharmacol. 1999;127:473–481. doi: 10.1038/sj.bjp.0702522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mazzone SB, Geraghty DP. Characterization and regulation of tachykinin receptors in the nucleus tractus solitarius. Clin Exp Pharmacol Physiol. 2000;27:939–942. doi: 10.1046/j.1440-1681.2000.03365.x. [DOI] [PubMed] [Google Scholar]
  40. Mifflin SW, Felder RB. Synaptic mechanisms regulating cardiovascular afferent inputs to solitary tract nucleus. Am J Physiol. 1990;259:H653–661. doi: 10.1152/ajpheart.1990.259.3.H653. [DOI] [PubMed] [Google Scholar]
  41. Miles R. Frequency dependence of synaptic transmission in nucleus of the solitary tract in vitro. J Neurophysiol. 1986;55:1076–1090. doi: 10.1152/jn.1986.55.5.1076. [DOI] [PubMed] [Google Scholar]
  42. Morin-Surun MP, Jordan D, Champagnat J, Spyer KM, Denavit-Saubié M. Excitatory effects of iontophoretically applied substance P on neurons in the nucleus tractus solitarius of the cat: lack of interaction with opiates and opioids. Brain Res. 1984;307:388–392. doi: 10.1016/0006-8993(84)90502-x. [DOI] [PubMed] [Google Scholar]
  43. Morisset V, Urban L. Cannabinoid-induced presynaptic inhibition of glutamatergic EPSCs in substantia gelatinosa neurons of the rat spinal cord. J Neurophysiol. 2001;86:40–48. doi: 10.1152/jn.2001.86.1.40. [DOI] [PubMed] [Google Scholar]
  44. Mutoh T, Bonham AC, Joad JP. Substance P in the nucleus of the solitary tract augments bronchopulmonary C fiber reflex output. Am J Physiol Regul Integr Comp Physiol. 2000;279:R1215–1223. doi: 10.1152/ajpregu.2000.279.4.R1215. [DOI] [PubMed] [Google Scholar]
  45. Price CJ, Pittman QJ. Dopamine D4 receptor activation inhibits presynaptically glutamatergic neurotransmission in the rat supraoptic nucleus. J Neurophysiol. 2001;86:1149–1155. doi: 10.1152/jn.2001.86.3.1149. [DOI] [PubMed] [Google Scholar]
  46. Saitow F, Satake S, Yamada J, Konishi S. β-Adrenergic receptor-mediated presynaptic facilitation of inhibitory GABAergic transmission at cerebellar interneuron-Purkinje cell synapses. J Neurophysiol. 2000;84:2016–2025. doi: 10.1152/jn.2000.84.4.2016. [DOI] [PubMed] [Google Scholar]
  47. Saria A, Martling C-R, Yan ZQ, Theodorsson-Norheim E, Gamse R, Lundberg JM. Release of multiple tachykinins from capsaicin-sensitive sensory nerves in the lung by bradykinin, histamine, dimethylphenyl piperazinium, and vagal nerve stimulation. Am Rev Respir Dis. 1988;137:1330–1335. doi: 10.1164/ajrccm/137.6.1330. [DOI] [PubMed] [Google Scholar]
  48. Saviane C, Savtchenko LP, Raffaelli G, Voronin LL, Cherubini E. Frequency-dependent shift from paired-pulse facilitation to paired-pulse depression at unitary CA3-CA3 synapses in the rat hippocampus. J Physiol. 2002;544:469–476. doi: 10.1113/jphysiol.2002.026609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Scheuer DA, Mifflin S. Identification of nucleus of the solitary tract (NTS) neurons receiving monosynaptic inputs from the vagus nerve. FASEB J. 1998;12:A60. [Google Scholar]
  50. Scheuer DA, Zhang J, Toney GM, Mifflin SW. Temporal processing of aortic nerve evoked activity in the nucleus of the solitary tract. J Neurophysiol. 1996;76:3750–3757. doi: 10.1152/jn.1996.76.6.3750. [DOI] [PubMed] [Google Scholar]
  51. Schild JH, Clark JW, Canavier CC, Kunze DL, Andresen MC. Afferent synaptic drive of rat medial nucleus tractus solitarius neurons: dynamic simulation of graded vesicular mobilization, release, and non-NMDA receptor kinetics. J Neurophysiol. 1998;74:1529–1548. doi: 10.1152/jn.1995.74.4.1529. [DOI] [PubMed] [Google Scholar]
  52. Seagard JL, Dean C, Hopp FA. Modulation of the carotid baroreceptor reflex by substance P in the nucleus tractus solitarius. J Auton Nerv Syst. 2000;78:77–85. doi: 10.1016/s0165-1838(99)00060-0. [DOI] [PubMed] [Google Scholar]
  53. Shen KZ, Johnson SW. Presynaptic modulation of synaptic transmission by opioid receptor in rat subthalamic nucleus in vitro. J Physiol. 2002;541:219–230. doi: 10.1113/jphysiol.2001.013404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Shen K, Johnson SW. Presynaptic inhibition of synaptic transmission by adenosine in rat subthalamic nucleus in vitro. Neuroscience. 2003;116:99–106. doi: 10.1016/s0306-4522(02)00656-5. [DOI] [PubMed] [Google Scholar]
  55. Smith BN, Dou P, Barber WD, Dudek FE. Vagally evoked synaptic currents in the immature rat nucleus tractus solitarii in an intact in vitro preparation. J Physiol. 1998;512:149–162. doi: 10.1111/j.1469-7793.1998.149bf.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sykes RM, Spyer KM, Izzo PN. Central distribution of substance P, calcitonin gene-related peptide and 5-hydroxytryptamine in vagal sensory afferents in the rat dorsal medulla. Neuroscience. 1994;59:195–210. doi: 10.1016/0306-4522(94)90110-4. [DOI] [PubMed] [Google Scholar]
  57. Thor KB, Helke CJ. Serotonin and substance P colocalization in medullary projections to the nucleus tractus solitarius: dual-colour immunohistochemistry combined with retrograde tracing. J Chem Neuroanat. 1989;2:139–148. [PubMed] [Google Scholar]
  58. Undem BJ, Hubbard WC, Weinreich D. Immunologically-induced neuromodulation of guinea pig nodose ganglion neurons. J Auton Nerv Syst. 1993;44:35–44. doi: 10.1016/0165-1838(93)90376-6. [DOI] [PubMed] [Google Scholar]
  59. Undem BJ, Hunter DD, Liu M, Haak-Frendscho M, Oakragly A, Fischer A. Allergen-induced sensory neuroplasticity in airways. Int Arch Allergy Immunol. 1999;118:150–153. doi: 10.1159/000024053. [DOI] [PubMed] [Google Scholar]
  60. Wilson CG, Zhang Z, Bonham AC. Non-NMDA receptors transmit cardiopulmonary C fibre input in nucleus tractus solitarii in rats. J Physiol. 1996;496:773–785. doi: 10.1113/jphysiol.1996.sp021726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yuan CS, Lowell TK. Gastric and brain stem, effects of substance P on nucleus tractus solitarius unitary responses. Peptides. 1997;18:1169–1173. doi: 10.1016/s0196-9781(97)00139-3. [DOI] [PubMed] [Google Scholar]

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