All animal procedures were conducted with the approval of the institutional Animal Care and Use Committee. These procedures were in accordance with the National Veterinary Research and Quarantine Service guidelines of the Republic of Korea. Efforts were made to minimize the number of animals used and their suffering.

Coronal brain stem slices were taken from 9- to 12-week-old male Sprague–Dawley (SD) rats (KRIB&B, Korea). The rats were deeply anesthetized with isoflurane (5%). The anesthesia level was confirmed by the absence of the flexor withdrawal reflex, after which the chest was compressed to stop the heart. The brainstem was removed and placed in 4°C artificial cerebrospinal fluid (ACSF) composed of (in mM) 125 NaCl, 3 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, 10 dextrose, and 2 CaCl₂ and bubbled with 95% O₂–5% CO₂. Acidic solutions (pH 6.8) were made by adding HCl. Coronal slices (300 μm thick) containing the DMV were cut using a microtome (VT-1200; Leica, Germany).

Slices were secured with a polyethylene mesh in a perfusion chamber and perfused with ACSF at 34–36°C and 307 mOsm. Recordings were then made from DMV neurons. Whole-cell recording pipettes were visually guided to mDMV neurons using infrared illumination and differential interference contrast optics (40X water immersion lens) on a BX51WI microscope (Olympus, Tokyo, Japan) coupled to an infrared-sensitive ZEISS Axiocam 503 mono camera (Figure 1). Whole-cell patch-clamp recordings were carried out using a MultiClamp 700B amplifier (Molecular Devices, Sunnyvale, CA, United States). Voltage-clamp and current-clamp recordings were performed in the conventional whole-cell configuration. Recording electrodes (3–3.5 MΩ) were filled with an intracellular solution containing the following (in mM): 10 NaCl, 40 KCl, 90 K gluconate, 11 EGTA, 1 CaCl₂, 2 MgCl₂, 10 HEPES, 2 Na₂ATP, and 0.2 Na₂GTP, pH 7.3 (295 mOsm). Under these ionic conditions, Cl^– selective currents at the holding potential (-50 mV) will be inward (E_Cl- = -22.4 mV). All recordings were corrected for liquid junction potential. In voltage-clamp mode, 70–75% series resistance compensation was achieved using the series resistance compensation feature of the amplifier. Input resistance measurements were performed by measuring the currents induced by hyperpolarizing steps to -60 and -100 mV. Whole-cell recordings were obtained from 87 visually identified mDMV neurons from different brain slices. Only one DMV slice was made from one animal.

All drugs were administered via bath ACSF perfusate. A selective NK-1 receptor antagonist, sendide, was obtained from Enzo (Farmingdale, NY). A selective NK3 receptor antagonist SB223412 (SB); a selective TASK-1 channel blocker ML365, a selective TASK-3 channel blocker, PK-THPP, and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzoquinoxaline-7-sulfonamide (NBQX), 3-([(3,4-dichlorophenyl)methyl]amino]propyl)diethoxymethyl)phosphinic acid (CGP), D-2-amino-5-phosphonovalerate (AP5), SR95531 (gabazine), tetrodotoxin (TTX), and bicuculline were obtained from Tocris Bioscience (Ellisville, MO, United States). SP and picrotoxin were purchased from Sigma–RBI (Natick, MA, United States). Different concentrations of SP were perfused for 6 to 20 min at a flow rate of 1.8–2 ml/min. The synaptic responses over the final 5-min period of each concentration exposure were analyzed.

All spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs) were detected and analyzed from digitized waveforms using MiniAnalysis (Synaptosoft, Decatur, GA), as described previously (Kim et al., 2018). Briefly, except for the determination of frequency rates, events < 4 pA and those with multiple peaks were excluded from waveform analyses and rate calculations. Baseline currents were measured over a 2 ms section of the recorded traces prior to every detected event. For statistical comparisons of sEPSCs or sIPSCs, waveform amplitude, event frequency and baseline values across each group were averaged over the last 5 min of each cumulative concentration step. Cumulative distributions of spontaneous postsynaptic current response amplitudes, frequencies, and baseline currents were compared using Kolmogorov–Smirnov (K–S) non-parametric analysis. For comparing two groups, t test were used. Statistical comparisons of three or more groups were made with one-way ANOVA or repeated-measures ANOVAs (RM-ANOVA) followed by Bonferroni/Dunn post hoc testing, when appropriate (Statview 4.57; Abacus Concepts, SAS Campus Drive, Cary, NC). The chi-squared test was used to compare the effect of 0.1 and 1 μM SP on the proportion of firing neurons. All data presented are means ± SDMs. Differences were considered statistically significant at P values of less than 0.05.

All recorded neurons had sIPSCs and sEPSCs, as reported earlier (Browning and Travagli, 1999; Lewis and Travagli, 2001). We first examined the effect of SP on GABAergic sIPSCs in the presence of the glutamate receptor antagonist NBQX. Under these conditions, the mean frequency of the sIPSCs was 0.39 ± 0.15 Hz. Application of submicromolar (0.1 and 0.3 μM) concentrations of SP had no effect on sIPSC frequency or amplitude but shifted the holding current more inward (Figures 2A,B). Increasing the SP concentration to micromolar levels (1 and 3 μM) increased the inward level of the holding current and markedly increased the sIPSC frequency. These responses did not fully recover to control levels even after a 25 min washout with ACSF. As a result, brain slices treated with micromolar concentrations of SP were not used further. Even the lowest tested SP concentrations produced a significant inward shift in the holding current (Figure 2C, control vs. 0.1 μM SP, p = 0.0001, K-S test). In addition, the sIPSC frequency increased when 1 and 3 μM SP were administrated (Figure 2C, p = 0.0001, K-S test). In the 9 tested neurons, SP uniformly shifted the holding current inward, but the sIPSC frequency increased in only 8 of the 9 neurons. Across all neurons, 1 and 3 μM SP significantly increased the sIPSC frequency to 12.6 ± 4.9 and 14.6 ± 4.7 times the control frequency, respectively (Figure 2D, p < 0.016, vs. control, RM-ANOVA, n = 8). The SP concentrations had no effect on the amplitude of the sIPSC (P = 0.9, RM-ANOVA, n = 8). At all tested concentrations, the SP-induced holding current shifted inward (p < 0.032, vs. control, RM-ANOVA, n = 9).

The effect of SP on glutamatergic sEPSCs was recorded in the presence of the GABA_A receptor antagonist gabazine. In the representative neuron shown in Figure 3, the cumulative application of 0.03 to 3 μM SP induced an inward shift in the holding current and reduced the sEPSC frequency (Figures 3A,B). These responses did not fully recover to control levels even after a 20 min washout with ACSF. Statistical analysis revealed that all tested concentrations of SP reduced sEPSC frequency (Figure 3C, p < 0.03, K-S test) without affecting the amplitude (p > 0.24, K-S test). At the same time, SP induced a significant shift in the holding current at all tested concentrations (p < 0.0001, K-S test). Of the 7 tested neurons, SP induced an sEPSC frequency decrease in 6, but shifts in the holding current were observed in only 5 neurons. Summary aggregate data of the sEPSC events show that all tested concentrations of SP reduced the sEPSC frequency below control levels; however, a significant difference was observed only with the application of 1 and 3 μM SP (p < 0.03, n = 6, RM-ANOVA). SP concentrations had no effect on the amplitude of the sEPSCs (p > 0.1, RM-ANOVA, n = 7). At concentrations of 1 and 3 μM, SP treatment decreased the sEPSC frequency to 56.1 ± 24.7% and 58.1 ± 23.7% of the control frequency, respectively (Figure 3D, p < 0.001, RM-ANOVA, n = 6). In 5 of the SP-responsive neurons, all tested concentrations of SP induced an inward holding current (p < 0.01, RM-ANOVA, n = 5). After 5 min of treatment with 0.03 to 3 μM SP, the baseline holding current shifted inward from the control level at all tested concentrations. In summary, SP suppressed synaptic glutamate release without affect holding current.

In this experiment, SP induced an inward shift in the holding current at a resting potential of -50 mV. To examine this more closely, we measured the holding current with increasingly hyperpolarized steps up to -120 mV in the presence of tetrodotoxin (TTX), NBQX and picrotoxin. In all tested neurons (n = 5), 0.01 to 1 μM SP reduced the membrane current at all tested holding potentials (Figure 4A). The steady-state current-voltage (I-V) relationships for the control condition and 0.1 μM SP application cross were –89 mV (Figure 4B). The average value of -87.6 ± 0.8 mV (n = 5) was close to the potassium equilibrium potential (E_k) -86 mV. Figure 4C summarizes the baseline holding currents for the control conditions and during SP application. SP also increased membrane input resistance in a concentration-dependent manner (Figure 4D). Taken together, the results suggest that the inward shift in the holding current and the increase in input resistance were both driven by the inhibition of K⁺ conductance.

Substance P (SP) reduces the membrane current, but this finding raises the question of the current identity. The tandem pore domain K⁺ channel provides a prominent leak current in many central neurons, and its mRNA is present at high levels in the DMV (Duprat et al., 1997; Talley et al., 2000). Neurotransmitters, including SP, inhibit the tandem pore domain in a weak inwardly rectifying (TWIK)-related acid-sensitive potassium (TASK) channel, similar to the effect of acidification (Talley et al., 2000). Thus, we tested the pH sensitivity of the background-leak current in our mDMV neurons by measuring the steady state I-V relationships in the continuous presence of TTX, gabazine (GZ), and NBQX. Acidification of the extracellular solution (pH 6.8) from the control condition (pH 7.4) reduced the current responses at all voltage steps, in 78% of the tested neurons (n = 14/18, respectively; Figure 5A). The mean holding current at pH 6.8 was significantly less than that of the control condition at pH 7.4 (Figure 5C). The mean holding currents at pH 7.4 and 6.8 were 43.5 ± 67.3 and 1.1 ± 61.6 pA, respectively. The I-V relationship had a reversal potential of -86 ± 0.5 mV (Figure 5B).

The above results suggest that TASK channels participate in the SP-induced inward shift in the holding current and reduced input resistance. Indeed, high mRNA levels of TASK-1 and TASK-3 are found in the rodent DMV, while other two-pore domain K⁺ channel transcripts are expressed at lower levels (Talley et al., 2001). Hence, we characterized the background K⁺ current using selective inhibitors of TASK-1 and TASK-3. Application of a selective TASK-3 blocker, PK-THPP, induced similar responses as acidification (Figure 6). In all tested neurons (n = 5), the application of PK-THPP shifted the baseline holding current inward by approximately 9.6 ± 2.9 pA and reduced the membrane current. Meanwhile, application of a TASK-1 inhibitor, ML365, did not significantly affect the baseline holding current or membrane resistance (n = 5). Taken together, the results show that TASK-3 channels are likely involved in the regulation of excitability of mDMV neurons.

Substance P (SP) binds to all three NK receptor subtypes (NK1, NK2 and NK3), but NK1 has the highest affinity for SP. The expression of NK1 and NK3 receptors has been reported in rat DMV but not that of NK2 (Saffroy et al., 2003). Our lowest SP concentration (0.03 μM) preferentially induced a holding current shift without altering the sIPSC frequency. To isolate SP action, we tested the NK1 selective antagonist sendide, which affects SP-mediated responses (Lewis and Travagli, 2001). Sendide (1 μM) alone did not affect the holding current or frequency of the sIPSCs (Figure 7A). In the continuous presence of sendide, neither 0.1 nor 1 μM SP affected the holding current (RM-ANOVA, p > 0.45, n = 8). In contrast, 1 μM SP significantly increased the sIPSC frequency despite the presence of the NK1 antagonist in 6 of 8 tested neurons (RM-ANOVA, p = 0.03). The mean frequency of the sIPSCs measured before and after application of 1 μM SP was 2.9 ± 0.8 Hz and 8.9 ± 3.86 Hz, respectively (n = 6). In separate experiments, the effect of NK3R on the SP-mediated holding current and sIPSC frequency was tested in 6 different neurons. The NK3R antagonist, SB223412 (1 μM, SB), alone did not affect the holding current or frequency of the sIPSCs (Figure 7B). In the presence of SB (1 μM), perfusion with 0.1 and 1 μM SP shifted the holding current inward (RM-ANOVA, ** p < 0.003, * p < 0.012, n = 6). Neither 0.1 nor 1 μM SP induced any significant changes in sIPSC frequency (p > 0.4, n = 6).

In this study, micromolar concentrations of SP facilitated synaptic GABA release by activating NK3R and suppressed synaptic glutamate release via an unknown mechanism. In the neuronal circuitry of the brain stem, glutamatergic and GABAergic synaptic responses are often influenced by metabotropic receptor-mediated heterosynaptic modulation (Jin et al., 2004; Fernandes et al., 2011). To test whether a GABA_B receptor antagonist altered glutamate release, we tested the effect of SP in the presence of the selective GABA_B receptor antagonist CGP. The application of SP under control conditions decreased sEPSC rates, and the addition of the CGP blocked the SP-induced reduction in sEPSCs (Figure 8). The mean frequency of the sIPSC at control, SP, and SP + CGP was 2.1 ± 0.45, 1.14 ± 0.47 and 2.2 ± 0.47, respectively. Thus, our results imply that the SP-mediated increase in the release of GABA reduced synaptic glutamate release from glutamatergic neurons via GABA_B receptor activation. Perfusion of 1 μM SP reduced the sEPSC frequency to 60% of the control frequency (Figure 8, p = 0.045, RM -ANOVA, n = 5). In the same set of neurons, however, SP failed to reduce the sEPSC frequency in the presence of 5 μM CGP (p = 0.78, RM -ANOVA, n = 5). However, in the same experiments SP application shifted the holding current inward. The mean holding current at control, SP, and SP + CGP was 10.4 ± 15.8 pA, 31.6 ± 23.8 pA and 30.8 ± 21.3 pA, respectively.

These multiple effects on synaptic transmission as well as membrane conductance indicate that SP may alter neuronal excitability. To assess this in single neurons, we exposed neurons first to 0.1 μM SP under voltage-clamp and then followed that with current-clamp recordings of the responses. Application of 0.1 μM SP induced an inward current under voltage-clamp conditions (Figure 9A). During the current-clamp, 0.1μM SP both depolarized and elicited action potentials, as shown in the representative neuronal tracings in Figure 9. A high concentration of SP (1 μM) failed to increase the firing rate (Figure 9B). In twelve tested neurons, application of 0.1 μM SP shifted the holding current inward (Figure 10A). The mean holding current before and during 0.1 μM SP application was 32.3 ± 11.5 pA and -47.9 ± 21.3 pA, respectively. During the current-clamp, the neurons averaged a resting potential of -59.7 ± 2.6 mV in the control condition and 25% (n = 3/12) spontaneously fired action potentials. Application of 0.1 μM SP depolarized the mean resting membrane potential to -50.3 ± 2.6 mV; 75% of neurons fired action potentials in this condition (Figure 10B, n = 9/12). The application of 1 μM SP did not further affect the firing rate (p > 0.6, n = 12, RM ANOVA) or the percentage of firing neurons (p = 0.4, chi-square test). In the presence of 1 μM SP, the addition of GZ (p = 0.38, n = 11, paired t test) or the coapplication of GZ and NBQX (p = 0.65, n = 10, paired t test) failed to induce significant changes in action potential frequency (Figure 10C).