Direct GABAergic and glycinergic inhibition of the substantia gelatinosa from the rostral ventromedial medulla revealed by in vivo patch-clamp analysis in rats

Abstract

Stimulation of the rostral ventromedial medulla (RVM) is believed to exert analgesic effects through the activation of the serotonergic system descending to the spinal dorsal horn; however, how nociceptive transmission is modulated by the descending system has not been fully clarified. To investigate the inhibitory mechanisms affected by the RVM, an in vivo patch-clamp technique was used to record IPSCs from the substantia gelatinosa (SG) of the spinal cord evoked by chemical (glutamate injection) and electrical stimulation (ES) of the RVM in adult rats. In the voltage-clamp mode, the RVM glutamate injection and RVM-ES produced an increase in both the frequency and amplitude of IPSCs in SG neurons that was not blocked by glutamate receptor antagonists. Serotonin receptor antagonists were unexpectedly without effect, but a GABAA receptor antagonist, bicuculline, or a glycine receptor antagonist, strychnine, completely suppressed the RVM stimulation-induced increase in IPSCs. The RVM-ES-evoked IPSCs showed fixed latency and no failure at 20 Hz stimuli with a conduction velocity of >3 m/s (3.1-20.7 m/s), suggesting descending monosynaptic GABAergic and/or glycinergic inputs from the RVM to the SG through myelinated fibers. In the current-clamp mode, action potentials elicited by noxious mechanical stimuli applied to the receptive field of the ipsilateral hindlimb were suppressed by the RVM-ES in more than half of the neurons tested (63%; 10 of 16). These findings suggest that the RVM-mediated antinociceptive effects on noxious inputs to the SG may be exerted preferentially by the direct GABAergic and glycinergic pathways to the SG.

Figure 1.

Schematic diagram of the in vivo rat preparation and histochemical identification of SG neurons. A, While oxygen was supplied to a urethane-anesthetized rat from a nose cone, the lumbar spinal cord at the level from L1 to L6 was exposed by laminectomy. The surface of the spinal cord was perfused with 95% O₂–5% CO₂-equilibrated Krebs’ solution (15 ml/min) from inlet to outlet glass pipettes at 38 ± 1°C (small arrows). Ba, Representative SG neurons filled with neurobiotin from a patch pipette (red) that responded to the RVM-ES in a sagittal section of the spinal cord. Green fluorescence band shows IB4 binding, which indicates the inner layer of lamina II (SG). Bb, The same neuron at a higher magnification. Scale bars, 50 μm.

Figure 2.

RVM-GI facilitates sIPSCs in the SG. A, Typical example of the facilitation of sIPSCs induced by RVM-GI (30 nmol). Ba–Bc, Corresponding traces on an expanded time scale in Aa–Ac, respectively. C, Cumulative distributions of interevent intervals (left; p < 0.01) and current amplitudes (right; p < 0.05) for sIPSCs recorded from the same neuron shown in A (2281 and 5801 events in a 200 s period for sIPSCs before and after RVM-GI). Insets, Averages of sIPSC amplitude (amp.) and frequency (freq.), which are normalized to their controls (n = 9). *p < 0.05.

Figure 3.

RVM-ES facilitates sIPSCs in the SG. A, Typical example of the facilitation of sIPSCs induced by RMV-ES (100 μA; 30 Hz). B, Three sets of traces (a–c) demonstrate IPSCs on an expanded time scale recorded before, during, and after RVM-ES, corresponding to a–c in A. C, Cumulative distributions of interevent intervals (left; p < 0.05) and current amplitudes (right; p < 0.01) for sIPSCs recorded from the same neuron shown in A (292 and 359 events in a 5 s period for sIPSCs before and after RVM-ES). Insets, Amplitudes (amp.) and frequencies (freq.) of sIPSCs are normalized to their controls (n = 16). **p < 0.01.

Figure 4.

IPSCs evoked by RVM-ES are mediated by GABA or glycine. A, Time course of the relative peak amplitude of the RVM-ES-eIPSCs. Simultaneous application of the AMPA and NMDA receptor antagonists CNQX (10 μm) and APV (25 μm) was without effect, but the GABA_A receptor antagonist bicuculline (Bic; 20 μm) completely suppressed IPSCs in a reversible manner. Ba–Bd, Superimposed traces of the IPSCs at a–d in A. Note that the duration of the IPSCs was relatively long (>24 ms). C, RVM-ES-eIPSCs that were not abolished by glutamate antagonists and bicuculline but were blocked by a glycinergic receptor antagonist, strychnine (St; 2 μm). Da–Dd, Superimposed traces of the IPSCs with a short duration (<13 ms) at a–d in B. The intensity and frequency of the RVM-ES were 100 μA and 0.1 Hz, respectively. Holding potential: 0 mV.

Figure 5.

RVM-ES-mediated facilitation of sIPSCs was not blocked by a 5-HT₃ receptor blocker. A, Frequency and amplitude of sIPSCs were increased during RVM-ES in a reversible manner before drug application (left). The RVM-ES is always accompanied by IPSCs (dots). During application of a 5-HT₃ receptor blocker, ondansetron, the facilitations were again observed (right). The stimulus intensity and frequency were 100 μA and 30 Hz, respectively. B, Time courses of the frequency (filled circle) and amplitude (open circle). The enhancement of IPSCs induced by the RVM-ES was not affected by the application of ondansetron (20 μm). C, Average amplitude and frequency of sIPSCs that are normalized to their controls (n = 7). ns., Not significant; Ond, ondansetron. *p < 0.05.

Figure 6.

Monosynaptically evoked IPSCs by RVM-ES. A, The frequency of IPSCs was increased in a stimulus-frequency-dependent manner. The RVM-ES was always accompanied by IPSCs. B, Stimulus-frequency-dependent increase in relative frequency of IPSCs with the RVM-ES at 20, 50, and 100 Hz (100 μA). C, Superimposed traces of IPSCs evoked at 0.2 Hz (top) and 20 Hz (bottom) RVM-ES, respectively. Note that IPSCs were evoked at fixed latency and showed no failures. Holding potential: 0 mV.

Figure 7.

Stimulation sites of the RVM-ES. A, Example of a photomicrograph of a 50-μm-thick coronal section with a small electrolytic lesion marking a stimulation site in the RVM. B, Stimulation sites illustrated on representative coronal brain sections (Paxinos and Watson, 2005). Filled and open circles indicate the stimulation sites eliciting GABA_A- and glycine-dominant eIPSCs, respectively. X indicates the sites eliciting no significant responses. py, Pyramidal tract; 6, abducens nucleus; 7, facial nucleus; g7, genu of the facial nerve; NRO, nucleus raphe obscurus; Sp5, spinal trigeminal tract. The caudal distance of each section from bregma is indicated by values next to P (in millimeters). C, CV (left) and peak amplitudes (right) of GABAergic (n = 12) and glycinergic (n = 6) IPSCs.

Figure 8.

Suppression of the pinch-induced responses by the RVM-ES. A, Five sets of pinch stimuli for 5 s (black squares) were applied every 2 min to the receptive field of the ipsilateral hindlimb, and the RVM-ES was applied from 30 s before the third pinch stimulus for 1 min in current-clamp mode. Ba–Bd correspond to Aa–Ad on an expanded time scale. Ba, Pinch stimulation produced a depolarization accompanied by a barrage of action potentials under current-clamp condition. Resting membrane potential was −62 mV. Bb, RVM-ES (hatched square; 100 μA; 30 Hz) hyperpolarized the membrane potential, although 30 Hz artifacts overlapped. Bc, RVM-ES suppressed action potentials elicited by pinch stimulation. Bd, Action potentials were evoked again by pinch in the absence of RVM-ES