Purinergic and vanilloid receptor activation releases glutamate from separate cranial afferent terminals in nucleus tractus solitarius

Abstract

Vanilloid (VR1) and purinergic (P2X) receptors are found in cranial afferent neurons in nodose ganglia and their central terminations within the solitary tract nucleus (NTS), but little is known about their function. We mechanically dissociated dorsomedial NTS neurons to preserve attached native synapses and tested for VR1 and P2X function primarily in spindle-shaped neurons resembling intact second-order neurons. All neurons (n = 95) exhibited spontaneous glutamate (EPSCs) and GABA (IPSCs)-mediated synaptic currents. VR1 agonist capsaicin (CAP; 100 nm) reversibly increased EPSC frequency, effects blocked by capsazepine. ATP (100 microm) increased EPSC frequency, actions blocked by P2X antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS; 20 microm). In all CAP-resistant neurons, P2X agonist alphabeta-methylene-ATP (alphabeta-m-ATP) increased EPSC frequency. Neither CAP nor alphabeta-m-ATP altered EPSC amplitudes, kinetics, or holding currents. Thus, activation of VR1 and P2X receptors selectively facilitated presynaptic glutamate release on different NTS neurons. PPADS and 2',3'-O-(2,4,6-trinitrophenyl)-ATP blocked alphabeta-m-ATP responses, but P2X1-selective antagonist NF023 (8,8'-[carbonylbis (imino-3,1-phenylene carbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid) did not. The pharmacological profile and transient kinetics of ATP responses are consistent with P2X3 homomeric receptors. TTX and Cd(2+) did not eliminate agonist-evoked EPSC frequency increases, suggesting that voltage-gated sodium and calcium channels are not required. In nodose ganglia, CAP but not alphabeta-m-ATP evoked inward currents in slow conducting neurons and the converse pattern in myelinated, rapidly conducting neurons (n = 14). Together, results are consistent with segregation of glutamatergic terminals into either P2X sensitive or VR1 sensitive that correspondingly identify myelinated and unmyelinated afferent pathways at the NTS.

Figure 1.

Synaptic responses recorded from a representative, acutely dispersed, medial NTS neuron. This neuron closely resembles second-order NTS neurons in its dimensions and spindle shape (inset). Spontaneous synaptic currents from this neuron were separated pharmacologically and kinetically into IPSCs and EPSCs. A, Pharmacological isolation of synaptic current subtypes within a single neuron. Control traces show large and small amplitude events. Bicuculline (100 μm) reversibly blocked large-amplitude, long-duration, synaptic currents (IPSCs) while preserving small-amplitude, brief synaptic currents (EPSCs). Wash returned to drug-free control solution. NBQX (20 μm) blocked EPSCs, but IPSCs remained. All synaptic currents were blocked by combined bicuculline and NBQX. B, Kinetic differences between bicuculline-sensitive IPSCs and NBQX-sensitive EPSCs from the same neuron as in A. Expanded traces illustrate that IPSCs (left) had prolonged time courses, whereas EPSCs (right) rapidly returned to baseline (amplitudes normalized to 100%). Average kinetic parameters for this neuron show that mean rise times (10–90%) and mean decay times were significantly shorter for EPSCs than IPSCs, and IPSC amplitudes (89 events) were larger than EPSCs (114 events). Bars represent mean ± SD; asterisks indicate significant differences (p < 0.05) by unpaired t test.

Figure 2.

CAP increased the frequency of spontaneous EPSCs in this representative CAP-sensitive NTS neuron. Counts of EPSC events were collected over time bins of 10 sec. CAP (100 nm) rapidly and reversibly increased the rate of EPSCs, and this CAP effect was reversibly blocked by VR1 antagonist CZ (500 nm). Wash of CZ rapidly restored sensitivity to CAP. The histogram is an unbroken record of this single neuron. The bottom left panel shows examples of original, expanded current traces from this experiment in each condition. The bottom right panel shows that the distribution of amplitudes was unaltered by CAP (K-S test; p > 0.05). Bicuculline was present in all experiments.

Figure 3.

ATP increased the frequency of spontaneous EPSCs in ATP-sensitive NTS neurons. A representative protocol is displayed in the top panel. Counts of EPSC events were collected over time bins of 10 sec. ATP (100 μm) rapidly and reversibly increased the rate of EPSCs, and this effect was reversibly blocked by P2X antagonist PPADS (20 μm). ATP increased mean EPSC frequency (distribution not shown) from 0.61 Hz in control to 9.42 Hz (K-S test; p = 0.0001). The bottom left panel shows examples of original, expanded current traces from this experiment in each condition. The bottom right panel shows that ATP did not alter the cumulative distribution of amplitudes in this neuron (K-S test; p > 0.05). All data came from same neuron, and bicuculline was present in all conditions.

Figure 4.

The nonmetabolized P2X agonistαβ-m-ATP (10 μm) quickly (Aa) and reversibly increased the rate of EPSCs in this ATP-sensitive NTS neuron, with the rate recovering quickly after agonist removal. Time bins were 10 sec. Analysis of the time course of the decrease in EPSC rate (points are EPSC events per 2 sec bin) during αβ-m-ATP (Ab) was best fit by a single exponential function depicted by the solid curve (Origin software). y = A1^–x/t1 + y0; Chi² = 0.99; R² = 0.89; y0 = 1.2 ± 0.7; A1 = 9.1 ± 0.8; t1 = 9.9 ± 2.5 sec. The P2X receptor antagonist PPADS (10 μm) reversibly blocked this effect (Aa). EPSC frequency increased from 0.5 to 2.4 Hz during αβ-m-ATP (K-S test; p = 0.0003). B displays examples of original, expanded current traces showing EPSCs in each condition for this purinergically sensitive neuron. C plots the distribution of EPSC amplitudes, and this was not altered by αβ-m-ATP (p > 0.05). Bicuculline was present in all experiments.

Figure 5.

VR1 are located on presynaptic terminals of different NTS neurons than neurons with P2X receptors. Tests of CAP (100 nm) andαβ-m-ATP (10 μm) were made on single neurons to test whether both receptors could modulate glutamate release. The bin size was 10 sec for both histograms. Bicuculline was present in all experiments. A, In a CAP-resistant NTS neuron, CAP failed to alter the rate of spontaneous EPSCs, but αβ-m-ATP increased the rate of glutamate release. No amplitude changes were found. B, Conversely in a CAP-sensitive NTS neuron, CAP triggered increased EPSCs, but αβ-m-ATP (10 μm) was without effect. No amplitude changes were found.

Figure 6.

Voltage-dependent Na⁺ and Ca²⁺ contribute to afferent glutamate release in VR1 and P2X responses. The bin size is 10 sec in histograms for each neuron. Bicuculline was present in all experiments. A, In a representative CAP-sensitive NTS neuron, application of CAP (100 nm) evoked a large transient increase in EPSCs (note broken y-axis). In the presence of both TTX (3 μm) and Cd²⁺ (200 μm), the CAP response was greatly reduced but not eliminated. Repeated CAP challenge in this same neuron with only TTX present evoked a similar peak response (amplitude of first 10 sec bin). B, In a representative CAP-resistant NTS neuron, αβ-m-ATP (10 μm) markedly increased the rate of spontaneous EPSCs. Such responses were greatly reduced but not eliminated by addition of TTX plus Cd²⁺ or TTX alone.

Figure 7.

A, B, Vanilloid and purinergic sensitivity of nodose sensory neurons with myelinated (A) or unmyelinated (B) CVs. Vagal trunk stimulation (arrow) evoked a single somatic action potential in neurons recorded in current clamp and CV calculated. In the same neurons, current responses (insets) to both CAP (100 nm) andαβ-m-ATP (10 μm) were measured in voltage clamp at –60 mV. Bars indicate periods of drug perfusion. Horizontal and vertical scale bars for inset traces are 125 sec and 0.5 nA, respectively. A, Representative myelinated nodose neuron in which vagal stimulation evoked a characteristically narrow action potential with an afferent fiber CV of 10.99 m/sec. Inset, Top, Voltage-clamp currents show that microperfusion of αβ-m-ATP produced a strong inward current from this same neuron. Inset, Bottom, In contrast, CAP evoked no current in the same neuron. B, Vagal stimulation evoked a characteristically broad action potential with an afferent fiber CV of 0.73 m/sec in a representative unmyelinated nodose neuron. In contrast to the myelinated neurons, CAP evoked large inward current (inset), and αβ-m-ATP evoked no current in this same neuron (top inset).