Tonic GABAA receptor-mediated inhibition in the rat dorsal motor nucleus of the vagus

Abstract

Type A gamma-aminobutyric acid (GABA(A)) receptors expressed in the dorsal motor nucleus of vagus (DMV) critically regulate the activity of vagal motor neurons and, by inference, the gastrointestinal (GI) tract. Two types of GABA(A) receptor-mediated inhibition have been identified in the brain, represented by phasic (I(phasic)) and tonic (I(tonic)) inhibitory currents. The hypothesis that I(tonic) regulates neuron activity was tested in the DMV using whole cell patch-clamp recordings in transverse brain stem slices from rats. An I(tonic) was present in a subset of DMV neurons, which was determined to be mediated by different receptors than those mediating fast, synaptic currents. Preapplication of tetrodotoxin significantly decreased the resting I(tonic) amplitude in DMV neurons, suggesting that most of the current was due to action potential (AP)-dependent GABA release. Blocking GABA transport enhanced I(tonic) and multiple GABA transporters cooperated to regulate I(tonic). The I(tonic) was composed of both a gabazine-insensitive component that was nearly saturated under basal conditions and a gabazine-sensitive component that was activated when extracellular GABA concentration was elevated. Perfusion of THIP (10 muM) significantly increased I(tonic) amplitude without increasing I(phasic) amplitude. The I(tonic) played a major role in determining the overall excitability of DMV neurons by contributing to resting membrane potential and AP frequency. Our results indicate that I(tonic) contributes to DMV neuron membrane potential and activity and is thus an important regulator of vagally mediated GI function.

Fig. 1.

Relationship between type A γ-aminobutyric acid (GABA_A) receptor-mediated tonic (I_tonic) currents, synaptic currents, and action potential (AP)–dependent GABA release. A: the I_tonic under baseline conditions was revealed by addition of bicuculline (30 μM). B: plot showing the relationship between I_tonic amplitude and the mean I_phasic in 38 DMV (dorsal motor nucleus of vagus) neurons. There was little correlation (r² = 0.02), indicating that different receptor pools mediate I_tonic and I_phasic synaptic currents. C: nipecotic acid (1 mM) increased the I_tonic only slightly in the presence of tetrodotoxin (TTX, 1 μM); adding bicuculline (30 μM) revealed the total I_tonic. D: graph indicating that TTX produced a significant suppression of the I_tonic compared with control. Nipecotic acid enhanced the I_tonic in the presence of TTX, but the I_tonic was significantly smaller in amplitude than that in the absence of TTX. Asterisks indicate significant difference between groups indicated (unpaired t-test; P < 0.05); double asterisks indicate significant effect of TTX within cells (paired t-test; P < 0.05). Number of replicates is in parentheses above each bar.

Fig. 2.

The effects of GABA transporter blockers on I_tonic in DMV neurons. A: application of nipecotic acid (1 mM), a nonselective GABA transporter inhibitor, significantly increased the amplitude of I_tonic. B: graph showing the I_tonic amplitude, revealed by adding bicuculline (30 μM), under control conditions and in the presence of nipecotic acid. Asterisk indicates significant difference from control (P < 0.05; paired t-test). Number of replicates in parentheses. C: representative trace showing that coapplication of NO-711 and SNAP-5114 did not increase the I_tonic under normal conditions. D: comparison of the I_tonic amplitude changes induced by nipecotic acid (1 mM), GABA (5 μM), NO-711 (10 μM), SNAP-5114 (50 μM), NO-711 + SNAP-5114, and the combination of SKF89976A (30 or 90 μM) with β-alanine (100–200 μM; strychnine 10 μM was preapplied into the artificial cerebrospinal fluid [ACSF] to block glycine receptors). Asterisk indicates significant difference vs. I_tonic change in other groups [ANOVA; F_(5,37) = 7.94; P < 0.05]. Number of replicates is in parentheses above each bar. E: in the presence of GABA (5 μM), coapplication of NO-711 (10 μM) and SNAP-5114 (50 μM) increased the I_tonic. Adding bicuculline (30 μM) revealed the total I_tonic. F: graph summarizing the I_tonic in 5 μM GABA and the total I_tonic in the presence of NO-711 + SNAP-5114 and GABA. Asterisk indicates significant difference from GABA alone (P < 0.05). G: comparison of the I_tonic amplitude changes induced by NO-711 + SNAP-5114, NO-711 (10 μM) alone, and SNAP-5114 (50 μM) alone in the presence of GABA (5 μM). Asterisk indicates significant difference vs. other groups (P < 0.05). Number of replicates is in parentheses above each bar.

Fig. 3.

Sensitivity of I_tonic to GABA_A receptor antagonists. A: gabazine (2 or 25 μM) blocked spontaneous inhibitory postsynaptic currents (sIPSCs) but did not alter the I_tonic in normal ACSF. B: graph showing the I_tonic amplitude (revealed by addition of 30 μM bicuculline) under control conditions and in the presence of gabazine. There was no significant effect of gabazine on I_tonic in control ACSF (P > 0.05; paired t-test). Number of replicates in parentheses. C: in the presence of nipecotic acid (1 mM), gabazine (0.5 or 1 μM) decreased the I_tonic. Bicuculline (30 μM) completely blocked the I_tonic. D: in the presence of nipecotic acid, gabazine (0.5 or 1 μM) significantly decreased the tonic current. Raising gabazine concentration to 25 μM did not further alter the tonic current. Asterisks indicate significant differences between groups indicated [ANOVA; F_(3,33) = 3.64; P < 0.05]. E: gabazine (0.5 or 1 μM) almost completely blocked the sIPSCs (P < 0.05). Bic, bicuculline; GBZ, gabazine; Nip, nipecotic acid.

Fig. 4.

The effect of THIP on the I_tonic in PRV-152 labeled and unlabeled neurons. A1 and A2: fluorescence illumination (A1) and brightfield illumination (A2) during recording from a PRV-152–labeled DMV neuron. The arrows indicate the position of the recording pipette. A3 and A4: fluorescence illumination of the same neuron after fixation reveals the EGFP-labeled (A3) and biocytin-filled neuron (A4). B: a representative trace showing the increase caused by THIP (10 μM) in an unlabeled DMV neuron; adding bicuculline (30 μM) revealed the total I_tonic. C: a trace showing the I_tonic increase caused by THIP in the PRV-152–labeled DMV neuron in A. This neuron had little resting I_tonic. D: graph depicting the I_tonic amplitude in control ACSF and in THIP (10 μM). No difference was observed between labeled (n = 8; gray bar) and unlabeled (n = 8; black bar) cells in the response to THIP (P = 0.2). Asterisks indicate significant difference vs. control (P < 0.05). E: concentration–response relationship for the I_tonic change due to THIP. Replicates are in parentheses above each point. EGFP, enhanced green fluorescent protein; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol.

Fig. 5.

Effects of THIP and nipecotic acid on AP firing. A: current-clamp recording at resting membrane potential showing that THIP (10 μM) suppressed APs and hyperpolarized the membrane potential; bicuculline (30 μM) reversed the effect of THIP. B and C: bar graphs summarizing the effects of THIP and bicuculline on the resting membrane potential (B) and AP frequency (C). D and E: summary graphs indicating that nipecotic acid (1 mM) also suppressed APs and hyperpolarized the resting membrane potential; picrotoxin (100 μM) or bicuculline (30 μM) blocked the effects of nipecotic acid. Asterisks indicate significant differences between groups indicated (ANOVA; P < 0.05). Replicate number in parentheses.

Fig. 6.

Nipecotic acid and THIP decreased input resistance. A: current-clamp recording from a DMV neuron showing a decreased voltage response to current-step injection after nipecotic acid (1 mM) application. Bicuculline (30 μM) blocked the effect of nipecotic acid. The resting membrane potential was kept at −57 mV by injecting current through the recording electrode. B: bar graph illustrating the decrease in input resistance caused by nipecotic acid in 7 cells. C: bar graph indicating the decrease in input resistance caused by THIP (10 μM) and reversal of the effect by bicuculline. Asterisk indicates significant change vs. control (P < 0.05, ANOVA). Replicate number in parentheses.