Multiple targets of μ-opioid receptor-mediated presynaptic inhibition at primary afferent Aδ- and C-fibers

Abstract

Agonists at μ-opioid receptors (MORs) represent the gold standard for the treatment of severe pain. A key element of opioid analgesia is the depression of nociceptive information at the first synaptic relay in spinal pain pathways. The underlying mechanisms are, however, largely unknown. In spinal cord slices with dorsal roots attached prepared from young rats, we determined the inhibitory effect of the selective MOR agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) on monosynaptic Aδ- and C-fiber-evoked EPSCs in lamina I neurons. DAMGO depressed presynaptically Aδ- and C-fiber-mediated responses, indicating that MORs are expressed on central terminals of both fiber types. We next addressed the mechanisms of presynaptic inhibition. The effect of DAMGO at both Aδ- and C-fiber terminals was mainly mediated by an inhibition of N-type voltage-dependent Ca(2+) channels (VDCCs), and to a lesser extent of P/Q-type VDCCs. Inhibition by DAMGO was not reduced by K(+) channel blockers. The rate of miniature EPSCs was reduced by DAMGO in a dose-dependent manner. The opioid also reduced Ca(2+)-dependent, ionomycin-induced EPSCs downstream of VDCCs. DAMGO had no effect on the kinetics of vesicle exocytosis in C-fiber terminals, but decreased the rate of unloading of Aδ-fiber boutons moderately, as revealed by two-photon imaging of styryl dye destaining. Together, these results suggest that binding of opioids to MORs reduces nociceptive signal transmission at central Aδ- and C-fiber synapses mainly by inhibition of presynaptic N-type VDCCs. P/Q-type VDCCs and the transmitter release machinery are targets of opioid action as well.

Figure 1.

Actions of MOR agonist DAMGO on presynaptic inhibition of monosynaptic Aδ- and C-fiber EPSCs. A, Time course of the reversible inhibition of DAMGO at different concentrations on evoked monosynaptic Aδ-fiber EPSCs. Right, Original traces taken from the indicated time points before (1), during (2), and after (3) the application of 1 μm DAMGO. B, Representative time course of the inhibitory effect of DAMGO (100 nm) on monosynaptic C-fiber EPSCs. Inset shows the corresponding original traces. C, Differential inhibition by DAMGO (100 nm) on Aδ- and C-fiber EPSCs recorded simultaneously from the same neuron. Inset, The corresponding original traces with Aδ- and C-fiber peaks. D, Dose–response curves of presynaptic DAMGO inhibition on monosynaptic C- (black circles) and Aδ-fiber (white circles)-evoked EPSCs. Numbers in parenthesis indicate the numbers of neurons tested. IC₅₀ = 157 nm for C-fiber EPSCs and IC₅₀ = 215 nm for Aδ-fiber EPSCs. Inset, The characterization of monosynaptic Aδ- and C-fiber-evoked EPSCs. Ten traces superimposed show a jitter of <10% of the response latency and no failures. Calibration: A, D (inset), 100 pA, 5 ms; B, C, 100 pA, 10 ms.

Figure 2.

Blockers of VDCCs reduced the inhibitory effect of MOR agonist DAMGO on monosynaptic C- and Aδ-fiber-evoked EPSCs, respectively. A, Representative time course of EPSC amplitudes (Aa) and corresponding bar graph (black bars, Ab) demonstrating the reduced inhibition of C-fiber-evoked EPSCs by DAMGO (100 nm) in the presence of the N-type-specific VDCC blocker ω-conotoxin GVIA (ω-CTX GVIA, 1 μm; n = 7). Ac, Bar graph (white bars) summarizes the effect of ω-CTX GVIA on the inhibition of Aδ-fiber-evoked EPSCs by 250 nm DAMGO (n = 8). B, Typical recording of EPSC amplitudes (Ba) and corresponding bar graph (black bars, Bb) showing the reduced inhibition of C-fiber-evoked EPSCs by 100 nm DAMGO in the presence of the P/Q-type-specific VDCC blocker ω-agatoxin (ω-AgTx, 300 nm; n = 5). Bc, Bar graph (white bars) summarizes the effect of ω-AgTx on the inhibition of Aδ-fiber-evoked EPSCs by 250 nm DAMGO (n = 6). C, Time course of EPSC amplitudes (Ca) and corresponding bar graph (black bars, Cb) demonstrating that the L-type-specific VDCC blocking toxin calciseptine (0.5 μm) has no effect on the inhibition of C-fiber-evoked EPSCs by 100 nm DAMGO (n = 5). Cc, Bar graph (white bars) shows the effect of calciseptine on the inhibition of Aδ-fiber-evoked EPSCs by 250 nm DAMGO (n = 5). *p < 0.05; **p < 0.01; n.s., not significant.

Figure 3.

MOR agonist DAMGO (10 μm) reduced the rate of ionomycin-induced mEPSCs in rat spinal lamina I neurons. A, Representative traces of mEPSC events (control), and mEPSCs, induced by 2 μm ionomycin, before, during, and after application of DAMGO, showing a reduction of ionomycin-induced mEPSCs (V_Hold = −70 mV). TTX (1 μm) and Cd²⁺ (200 μm) were included in the bath solution. B, Bar graph summarizes the mean mEPSC rates. C, Typical time course of the action of ionomycin (2 μm) and DAMGO on mEPSC rate. D, Bar graph shows the lacking effect of DAMGO on mEPSC amplitudes. Calibration: A, 50 pA, 1 s. Number of neurons tested: B, D, n = 6; **p < 0.01.

Figure 4.

MOR agonist DAMGO reduced the rate of mEPSCs in rat spinal lamina I neurons also under resting Ca²⁺ concentrations. A, Representative traces of mEPSCs recorded before (control), during, and after (wash) DAMGO (100 nm) application, demonstrating a reduction of mEPSC rates (V_Hold = −70 mV). TTX (1 μm) was included in the bath solution. B, Bar graphs show the mean rates and amplitudes of mEPSCs before, during, and after application of 100 nm DAMGO. C, Effect of 10 μm DAMGO on the mean rate and amplitude of mEPSCs. Calibration: A, 50 pA, 1 s. Number of neurons tested: B, n = 4; C, n = 7. *p < 0.05; **p < 0.01.

Figure 5.

Blockers of K⁺ conductances did not reduce the inhibitory effect of MOR agonist DAMGO (100 nm) on monosynaptic C-fiber-evoked EPSCs. Aa, Ba, Representative time courses of the amplitudes of evoked EPSCs showing no reduction of the inhibition by DAMGO in the presence of the inward rectifying K⁺ channel blockers Ba²⁺ (1 mm) and Cs⁺ (5 mm), respectively. Ab, Bb, Bar graphs illustrate no change in DAMGO action before and after application of Ba²⁺ and Cs⁺, respectively. Number of neurons tested: n = 5 for Ba²⁺, n = 3 for Cs⁺. n.s., not significant.

Figure 6.

MOR agonist DAMGO reduced the amount of vesicle exocytosis in A- and C-fiber boutons but did not alter the kinetics of styryl dye destaining in C-fiber terminals. A, Protocol for determining kinetics of styryl dye release from afferent C-fiber terminals. Terminals were labeled by exposure to Synaptogreen during 10 Hz stimulation of the dorsal root for 2 min (load). ADVASEP-7 (100 μm) was applied to remove bound extracellular dye. Subsequent electrical stimulation in dye-free solution then released dye (unload). B, Fluorescent images of the same field within the superficial lamina of the spinal dorsal horn before and after an unloading C-fiber stimulation. White arrows show some of the unloading boutons; gray arrows point to puncta not affected by the stimulation. C, Decay of average intensity of the fluorescent puncta during unloading C-fiber stimulation using 0.5 Hz (black squares), 2 Hz (black triangles), and 10 Hz (black circles) stimulation, respectively. Destaining of puncta induced by stimulation (10 Hz) with A-fiber intensity shows similar kinetics (white circles). Each point represents the average of a total of 99–535 boutons from 5–32 different slices. D, Average rates of puncta unloading (1/t_1/2) for the different stimulation frequencies applied with C-fiber (black bars) and A-fiber (white bar) intensity, respectively. E, Bath application of DAMGO (1 μm) reduced the proportion of slices reacting to dorsal root stimulation with C-fiber intensity at 2 Hz. Pretreatment with the specific MOR antagonist β-funaltrexamine (Funaltrex, 25 μm) abolished this effect. F, Box plot showing the decrease of the mean number of destaining C-fiber boutons per slice after DAMGO application and blockade of this effect by β-funaltrexamine. Frequency of unloading stimulation was 2 Hz. G, Bar chart showing average rates of puncta unloading (1/t_1/2) during stimulation (2 Hz) with C-fiber intensity for different DAMGO concentrations. H, Bar chart showing average rates of puncta unloading (1/t_1/2) during stimulation (10 Hz) with A-fiber intensity for increasing DAMGO concentrations. Number of slices tested: E, F, n = 32 for control, n = 11 for DAMGO, n = 25 for funaltrexamine, *p < 0.05; G, n = 25 for control, n = 4 for 50 nm, n = 6 for 200 nm, n = 4 for 1 μm DAMGO; H, n = 19 for control, n = 3 for 50 nm, n = 5 for 200 nm, n = 1 for 1 μm DAMGO.