INTRODUCTION

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Opioids produce analgesia and other behavioral modifications by altering synaptic transmission at many sites in the CNS, including the spinal cord dorsal horn. Acting on a family of G protein-coupled receptors, including µ-, -, and -subtypes, opioids can reduce neuronal activity by a variety of pre- and postsynaptic mechanisms (reviewed by Law et al. 2000; North 1993; Yaksh 1997).

In the dorsal horn, µ-opioid receptor activation is well-known to inhibit excitatory transmission between primary afferent sensory fibers and their target neurons in the superficial laminae (Jeftinija 1988; Kohno et al. 1999). In contrast, a role for µ-opioid receptors in modulating inhibitory transmission between intrinsic dorsal horn neurons is less clear. In the hippocampus (Capogna et al. 1993), the nucleus raphe magnus (Pan et al. 1990), and the periaqueductal gray (Vaughan et al. 1997), µ-opioid receptor activation suppressed GABAergic transmission. A similar role for µ-opioid receptors has been demonstrated in the substantia gelatinosa of the rat spinal trigeminal ganglion (Grudt and Henderson 1998); however, in the corresponding area of the rat lumbar spinal cord, inhibitory transmission appeared unaffected by µ-opioid receptor agonists (Kohno et al. 1999).

In this study, we report that selective µ-opioid receptor activation did suppress inhibitory transmission between cultured spinal dorsal horn neurons. This action was presynaptic in origin and involved a mechanism independent of presynaptic Ca²⁺ entry and components downstream of presynaptic kainate (KA) receptors.

	METHODS

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Protocols for handling animals were approved by the Animal Studies Committee at Washington University. Dorsal horn neurons were taken from postnatal Sprague-Dawley rats (Harlan) and cultured as described previously (Kerchner et al. 2001a,b). Whole cell recordings were established using a pipette solution containing (in mM) 140 CsCH₃SO₃, 5 CsCl, 5 MgCl₂, 10 EGTA, 10 HEPES, 5 Mg-ATP, and 1 Li-GTP (pH 7.4 with CsOH) (Kerchner et al. 2001a,b). Neurons were voltage clamped at 0 mV with an Axopatch 200B amplifier (Axon Instruments, Union City, CA), and evoked inhibitory postsynaptic currents (eIPSCs) were triggered at 0.2 Hz by extracellular stimulation of a neuron close to the recorded cell with a bipolar glass stimulating electrode (Kerchner et al. 2001a,b).

During experiments, neurons were perfused constantly from a gravity-fed quartz glass pipette (ALA Scientific Instruments, Westbury, NY) with Tyrode's solution, containing (in mM) 150 NaCl, 4 KCl, 2 MgCl₂, 2 CaCl₂, 10 D-glucose, and 10 HEPES (pH 7.4 with NaOH) plus DL-2-amino-5-phosphono-pentanoic acid (AP-5; 25 µM) and the (S)--amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10-20 µM) or the AMPA receptor-selective antagonist (±)-4(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methyl- enedioxyphthalazine (SYM2206; 100 µM) (Wilding and Huettner 2001). All compounds were purchased from Sigma Chemical (St. Louis, MO), except SYM2206 and (+)-4-([R]--[{2S, 5R}-4-allyl-2,5-dimethyl-1-piperazinyl]-3-methoxybenzyl)-N,N-diethyl-benzamide (SNC80; Tocris Cookson, Ellisville, MO).

Data are presented as means ± SE. To detect significant differences between two means, a paired t-test or signed-rank test was used. For comparison of multiple groups, a one-way ANOVA was performed with Student-Newman-Keuls test for post hoc comparison. In all cases, P < 0.05 was considered significant.

	RESULTS

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In the presence of CNQX and AP-5 to block excitatory transmission, extracellular stimulation evoked bicuculline- and strychnine-sensitive IPSCs in cultured dorsal horn neurons (Kerchner et al. 2001a). The µ-opioid receptor-selective agonist Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGO; 1 µM) reduced the amplitude of eIPSCs (Fig. 1). This effect was blocked by the selective µ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP; 1 µM; Fig. 1). In contrast, activation of - or -opioid receptors with SNC80 (1 µM) or (5, 7, 8)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro [4.5]dec-8-yl)benzeneacetamide (U69593; 1 µM), respectively, caused little or no change in eIPSC amplitude (Fig. 1B). DAMGO reduced pure glycine and pure GABA receptor-mediated eIPSCs to a similar extent as the composite response (Fig. 1B); this is the expected result if DAMGO acted presynaptically, because GABA and glycine are co-released from individual dorsal horn interneurons (Kerchner et al. 2001a).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGO) inhibits evoked inhibitory postsynaptic currents (eIPSCs). A: averaged traces from a representative experiment show that 1 µM DAMGO reduced eIPSC amplitude in the absence but not the presence of the µ-opioid receptor-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP; 1 µM). B: pooled data indicate the percentage inhibition of eIPSCs by DAMGO alone (1 µM; n = 12); DAMGO in the presence of strychnine (1 µM; n = 4), bicuculline (10 µM; n = 2), or CTOP (1 µM; n = 4); U69593 (1 µM; n = 4); and SNC80 (1 µM; n = 4). * Significant difference from baseline.

Confirming that DAMGO acted presynaptically, it increased the paired-pulse ratio of eIPSCs (Fig. 2, A and B) and decreased miniature IPSC (mIPSC) frequency (Fig. 3, A and B). Indicating that there was no postsynaptic component to DAMGO action, the peptide had no effect on GABA-evoked currents in dorsal horn neurons (Fig. 2C), and it still reduced eIPSC amplitude when the postsynaptic cell was perfused with a pipette solution containing Li-GDP--S (2 mM) instead of Li-GTP, to inhibit G proteins (5 min after achieving whole cell mode, DAMGO reduced eIPSCs by 65 ± 10%; and after 10 min, by 65 ± 20%; n = 3; the magnitude of these effects did not differ significantly from the effect of DAMGO when cells were perfused with GTP; P = 0.36).

View larger version (19K): [in this window] [in a new window]   Fig. 2. DAMGO acts presynaptically. A: averaged traces from a representative experiment, with control (heavy line) and 1 µM DAMGO (light line) conditions superimposed. Scaling these traces to the amplitude of the 1st peak illustrates an increase in the paired-pulse ratio. B: pooled from experiments as in A (n = 6). * significant difference from control. C: DAMGO (1 µM) did not affect 500 µM GABA-evoked currents in dorsal horn neurons. GABA current amplitude in DAMGO was 97 ± 1% of control (n = 4; P = 0.12).

View larger version (25K): [in this window] [in a new window]   Fig. 3. DAMGO reduces GABA/glycine release by a mechanism not involving presynaptic Ca2+ entry or components downstream of presynaptic kainate (KA) receptors. A: mIPSC frequency, recorded in the presence of tetrodotoxin (0.5 µM), was reduced by 1 µM DAMGO. Data were normalized to miniature IPSC (mIPSC) frequency in the absence of DAMGO, averaged (n = 9), and plotted in 5-s bins. B: pooled data indicate the effect of 1-2 µM DAMGO on mIPSC frequency in different concentrations of KCl (n = 3-11 per condition). Whereas DAMGO caused a significant decrease in mIPSC frequency (P < 0.001), the magnitude of this decrease did not vary significantly with [KCl] (P = 0.70). C: DAMGO (1 µM) caused a significant decrease in eIPSC amplitude (*), and KA (3 µM) caused an additional significant decrease () in the same neurons (n = 3), comparable in magnitude to the effect of KA alone (see Kerchner et al. 2001a).

One way that µ-opioid receptors may reduce vesicle release is by inhibiting voltage-gated Ca²⁺ channels (reviewed by Law et al. 2000; North 1993; Yaksh 1997). If this is the case for dorsal horn interneurons, then the ability of DAMGO to reduce mIPSC frequency should be enhanced when a greater proportion of these events are Ca²⁺ dependent. To test this hypothesis, the effect of DAMGO on quantal GABA/glycine release was monitored in the context of elevated [KCl] (10 or 20 mM). In control conditions ([KCl] = 4 mM), mIPSCs were largely insensitive to Cd²⁺ (see Kerchner et al. 2001a), suggesting that they are Ca²⁺ independent. Elevating [KCl], which enhances mIPSC frequency (Kerchner et al. 2001a), presumably does so by activating terminal voltage-gated Ca²⁺ channels. The magnitude of DAMGO-induced reduction in mIPSC frequency was independent of [KCl] (Fig. 3B), suggesting that µ-opioid receptors are linked to processes independent of presynaptic Ca²⁺ entry.

Previously, we showed that presynaptic KA receptor activation inhibited action potential-dependent IPSCs between cultured dorsal horn neurons (Kerchner et al. 2001a). To test whether presynaptic µ-opioid receptors and presynaptic KA receptors share common mechanisms of eIPSC suppression, KA (3 µM) was applied in the presence of a saturating concentration of DAMGO (1 µM; 10 µM had no additional effect; data not shown) and SYM2206, instead of CNQX. Because KA still caused a significant reduction in eIPSC amplitude (Fig. 3C), µ-opioid receptors and KA receptors likely reduce GABA/glycine release in the dorsal horn by different mechanisms.

	DISCUSSION

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Presynaptic µ-opioid receptors inhibited action potential-dependent and -independent GABA and glycine release from cultured dorsal horn interneurons. In addition, the ability of DAMGO to reduce quantal GABA/glycine release was unaffected by the level of membrane depolarization, implying that this action was not related to the proportion of release events that were Ca²⁺ dependent. Besides modulating Ca²⁺ channels, µ-opioid receptors may suppress GABA/glycine release by direct inhibition of the exocytotic machinery (Capogna et al. 1993) or by activation of 4-aminopyridine (4-AP)- and dendrotoxin-sensitive K⁺ conductances (Vaughan et al. 1997). Finally, DAMGO did not occlude the ability of KA, and thus GABA_B receptors (see Kerchner et al. 2001a), to reduce eIPSC amplitude. µ-Opioid receptors and GABA_B receptors may couple to distinct downstream pathways either by using different subtypes of G proteins or by differential colocalization with substrates.

By demonstrating a clear effect of DAMGO on dorsal horn inhibitory transmission, the present report departs from past observations. It is not clear why these data differ from those of Kohno et al. (1999), who observed no effect of 1 µM DAMGO on eIPSCs in the substantia gelatinosa of rat spinal cord slices. Grudt and Henderson (1998) did report DAMGO-induced suppression of eIPSCs in the substantia gelatinosa of the rat spinal trigeminal nucleus, suggesting that there may be differential expression of µ-opioid receptors between the two regions. Using similar logic, differential receptor expression among spinal laminae could explain why an effect of DAMGO was apparent in cultures, where no distinction is made between superficial and deep dorsal horn neurons. Arguing against this notion, DAMGO reduced eIPSC amplitude in every recording reported here; i.e., there was apparently no unresponsive subpopulation that could have corresponded to neurons derived from lamina II.

Alternatively, developmental issues may account for the discrepancy. Kohno et al. (1999) observed no difference between 3- to 4- and 6- to 10-wk-old rats. However, cultures are prepared from newborn rats. Expression of µ-opioid receptors in the spinal cord peaked at postnatal day 7 and subsequently decreased to adult levels (Rahman et al. 1998), consistent with the possibility that very young rats may express a mechanism for µ-opioid receptor-dependent regulation of inhibitory transmission that is absent in adults.

In sum, the present report clearly establishes that there exist conditions in which dorsal horn inhibitory synapses are sensitive to µ-opioid receptor stimulation. Given that the net effect of opioid receptor activation is analgesia, spinal disinhibition may seem counterproductive, if the result is simply to enhance sensory transmission. However, µ-opioid receptor signaling in interneurons may play a more complex role, perhaps by disinhibiting other inhibitory pathways, or by providing a stabilizing counterpoise to the downregulation of primary afferent transmission. Further examination into the contribution of interneuronal µ-opioid receptors to dorsal horn sensory transmission, including the role of developmental changes, may extend our understanding of the mechanisms underlying opioid analgesia.