Opioids are important neuromodulators in the central nervous system and are widely used in clinical settings. Opioids primarily exert inhibitory effects that include postsynaptic hyperpolarization through the opening of several types of K+ channels, leading to a reduction in neuronal excitability and presynaptic inhibition of excitatory neurotransmitter release.1,2 Indeed, the powerful analgesic effect of μ opioid receptor (MOR) agonists is partially mediated by their suppressive actions in the spinal dorsal horn.3–5 The ventral horn of the spinal cord contains motoneuron pools, which are known as lamina IX,6 and although MORs are expressed in the spinal ventral horn,7–9 little is known about opioid effects on motoneurons in this region.
Traditionally, opioids are not thought to affect motor function. However, several lines of evidence suggest that opioids can affect spinal motoneuronal excitability. For example, multiple groups have reported that μ opioids decreased the amplitude of motor evoked potentials while increasing latency.10–12 Fernandez-Galinski et al.10 proposed that activation of MORs in the ventral horn could delay depolarization of spinal motoneurons and thereby prolong latency. In addition, they inferred that μ opioids inhibited the excitatory interneurons that depolarize spinal motoneurons. Conversely, it has also been reported that opioids have excitatory effects in some central nervous system regions.13,14 In this regard, MOR agonists were shown to cause spastic paraplegia in the setting of prior ischemic injury,15,16 and the MOR antagonist naloxone ameliorated motor deficits after spinal cord ischemia.17 Given that cell death after ischemic injury is thought to result from overexcitation of the neuronal membrane,18 the effects of MOR activation in this system may include depolarization of spinal motoneurons.
Despite these indications that μ opioids act as important neuromodulators in the spinal ventral horn, the cellular effects of MOR activation in this region have not been clarified. Therefore, the aim of the present study was to examine the neuromodulatory effects of μ opioids in spinal ventral horn neurons at the cellular level. Specifically, we examined the effects of a selective MOR agonist, [D-Ala2, -N-Me-Phe4, Gly5-ol]enkephalin (DAMGO), in spinal lamina IX neurons of rats, using the whole-cell patch-clamp technique. Our results provide new evidence that MOR activation modulates neurotransmission in the spinal ventral horn.
Preparation of Spinal Cord Slices
All experimental procedures involving the use of animals were approved by the Animal Care and Use Committee at Niigata University Graduate School of Medical and Dental Sciences (Niigata, Japan). Slices of rat spinal cord were prepared as previously described.19 In brief, neonatal Wistar rats (8–12 days old) were anesthetized with urethane (1.2–1.5 g · kg−1, intraperitoneally). After dorsal laminectomy, the lumbosacral segment of the spinal cord was removed, and the animal was immediately killed by exsanguination. The spinal cord was immersed in ice-cold artificial cerebrospinal fluid (ACSF; 117 mM NaCl, 3.6 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2orally4, 25 mM NaHCO3, and 11.5 mM D-glucose; pH 7.4) equilibrated with a gas mixture of 95% O2 and 5% CO2. The spinal cord was cut into 500-μm-thick transverse slices using a microslicer (DTK-1500; Dosaka, Kyoto, Japan). Slices were transferred to a recording chamber and placed on the stage of an upright microscope equipped with an infrared–differential interference contrast (IR-DIC) system (E600FN; Nikon, Tokyo, Japan). The slices were superfused at 5 mL · min−1 with ACSF solution and were maintained at 36°C using a temperature controller (TC-324B; Warner Instruments, Hamden, CT) for at least 1 hour before recordings (Fig. 1A).
Patch-Clamp Recordings from Spinal Lamina IX Neurons
Lamina regions were identified under low magnification (5× objective lens), and individual neurons were detected using a 40× objective lens under an IR-DIC microscope and monitored using a charge coupled device camera (C2400-79H; Hamamatsu Photonics, Hamamatsu, Japan) and a video monitor (Fig. 1A). The size of each neuron was calculated from the arithmetic mean diameter of the long and short axes of the soma intersecting at right angles. Whole-cell patch-clamp recordings were made from large lamina IX neurons (size, 15–25 μm), most frequently seen in the ventral lateral or ventral medial area (Fig. 1B).20,21 In a previous study, these neurons were identified as motoneurons by fluorescence labeling with Evans blue dye injected into the rat hindlimb the day before death.22 After the whole-cell configuration established, voltage-clamped neurons were held at −70 mV or 0 mV. Whole-cell patch pipettes were constructed from borosilicate glass capillaries (1.5 mm outer diameter; World Precision Instruments, Sarasota, FL). The resistance of a typical patch pipette was 4 to 8 MΩ when filled with internal solution. Two internal patch pipette solutions were used, with compositions as follows: 1 a potassium gluconate-based solution containing 135 mM potassium gluconate, 5 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 5 mM HEPES, and 5 mM ATP-Mg (pH 7.2); and 2 a cesium sulfate-based solution containing 110 mM Cs2SO4, 5 mM tetraethylammonium (TEA), 0.5 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 5 mM HEPES, 5 mM ATP-Mg, and 2 mM guanosine 5′-[β-thio]diphosphate trilithium salt (GDP-β-S) (pH 7.2). The latter solution was used to abolish the postsynaptic effects of DAMGO. Signals were amplified by an Axopatch 200B amplifier (Molecular Devices, Union City, CA) and filtered at 2 kHz and digitized at 5 kHz. Data were stored and analyzed using the pCLAMP 9.1 data acquisition program (Molecular Devices). Voltage ramps (duration: 400 milliseconds) from the −70 mV holding potential (approximately −110 to −50 mV) were used to examine current-voltage relationships.
DAMGO, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), and barium chloride dehydrate (Ba2+) were from Sigma-Aldrich (St. Louis, MO). Tetrodotoxin (TTX) was from Wako (Osaka, Japan). All drugs were first dissolved in distilled water at 1000 times the working concentration, then diluted to the working concentration in ACSF solution immediately before use. Drugs were applied by switching the perfusion solution via a 3-way stopcock without a change in the perfusion rate or temperature.
The continuous curve for the concentration-response relationship of DAMGO was drawn according to the Hill equation:
where x is the DAMGO concentration, y is the relative amplitude of DAMGO-induced current (%), and ymax is the maximal value of y. The term k is the 50% effective concentration (EC50) (μM), and n is the slope of the curve (Hill coefficient). Numerical data are expressed as mean ± SEM. Statistical significance was defined as P < 0.05. Student paired t test or the Kolmogorov-Smirnov test was used as indicated for statistical analyses. For electrophysiological data, n refers to the number of neurons studied.
Whole-cell recordings could be obtained from slices maintained in vitro for >12 hours, and stable recordings were made from single neurons for up to 4 hours. The data presented herein were obtained from 184 lamina IX neurons. All neurons recorded exhibited spontaneous excitatory postsynaptic currents (sEPSCs) at the holding potential of −70 mV. However, when the potential was held at 0 mV, the neurons exhibited spontaneous inhibitory postsynaptic currents (sIPSCs).
Postsynaptic Actions of DAMGO in Lamina IX Neurons
Bath-applied DAMGO (1 μM) induced an outward current in 34 (56%) of 61 neurons; the average peak amplitude was 35 ± 3 pA at −70 mV. After DAMGO washout, the outward current amplitude gradually returned to control level in approximately 6 minutes, and subsequent application of DAMGO produced the same response (Fig. 2A). Therefore, in this study, the time interval between DAMGO applications was at least 10 minutes. The DAMGO-induced outward current increased in peak amplitude with increasing concentrations (Fig. 2B). Figure 2C the dose-response curve for the DAMGO-induced currents. Analysis of the curve based on the Hill plot indicated that the EC50 was 0.10 μM with a Hill coefficient of 0.95.
To confirm that the DAMGO-induced currents were postsynaptic effects, we examined the currents in the presence of TTX to remove any possible influence of MOR on presynaptic neurons. TTX (0.5 μM) had no significant effect on the amplitude of DAMGO-induced currents (101% ± 3% of control, n = 6; P = 0.78; Fig. 3, A and C). However, DAMGO-induced currents were attenuated by the selective MOR antagonist CTAP (5% ± 3% of control, n = 5; P < 0.01; Fig. 3, B and C). These results suggested that the DAMGO-induced current was mediated by postsynaptic MOR.
We next investigated the nature of the channels that mediate the DAMGO-induced outward current by evaluating membrane currents in response to voltage pulses in the absence and presence of the DAMGO-induced current (Fig. 4A). Figure 4B the relationship between the step voltage and steady current at the end of the pulse obtained in the absence (open triangles) and presence (closed triangles) of the DAMGO-induced current. The net DAMGO-induced current (open circles), which was calculated from the difference between the 2 currents, exhibited a clear reversal. The average reversal potential was −86 ± 3 mV (n = 8), which was slightly different from the equilibrium potential for K+ (−97 mV) as calculated from the Nernst equation using the K+ concentrations ([K+]o, 3.6 mM; [K+]i, 140 mM) of the solutions. This slight difference might reflect a liquid junction potential (9–10 mV) between the ACSF and patch-pipette solutions.23 Furthermore, DAMGO-induced currents were suppressed by the application of the K+ channel blocker Ba2+ (1 mM) (48% ± 3% of control, n = 5; P < 0.01; Fig. 5, A and D). These Ba2+ effects were examined in the presence of TTX (0.5 μM), because Ba2+ could influence presynaptic neuronal activity. Next, we used a pipette solution containing Cs+ and TEA to inhibit the postsynaptic action of K+ channels. The DAMGO-induced outward current was recorded immediately after establishing the whole-cell configuration. However, this current was reduced after the second application of DAMGO (10% ± 4% of baseline, n = 4; P < 0.01; Fig. 5, B and D). Similar results were obtained when GDP-β-S (1 mM) was used in the K-gluconate pipette solution (11% ± 5% of baseline, n = 5; P < 0.01; Fig. 5, C and D). These results indicate that the DAMGO-induced current was produced by the activation of K+ channels through the activation of G proteins, thereby implicating G protein–coupled inwardly rectifying potassium channels (GIRKs).
Presynaptic Actions of DAMGO in Lamina IX Neurons
In this and subsequent experiments, a cesium sulfate-based pipette solution was used to inhibit any postsynaptic effect of DAMGO through activation of K+ channels, enabling us to focus on presynaptic actions of DAMGO. The sEPSCs had an average amplitude and frequency of 18 ± 1 pA and 23 ± 2 Hz, respectively, at a holding potential of −70 mV (n = 9). Superfusing DAMGO (1 μM) for 4 minutes resulted in a reversible reduction in the frequency of sEPSCs (Fig. 6A). Figure 6B the effects of DAMGO on the cumulative distribution of the amplitude and interevent interval of sEPSCs. DAMGO increased the proportion of sEPSCs with a significantly longer interevent interval (P < 0.01) compared with the control, but it had no effect on the cumulative distribution of the amplitude of sEPSCs (P = 0.70). Figure 6C the time course of the average changes in the amplitude and frequency of sEPSCs in response to DAMGO in comparison with those of the controls (n = 8). A maximal reduction in sEPSC frequency was seen just after the DAMGO washout (73% ± 5% of control; P < 0.01; Fig. 6C), but the suppressive effect was not seen in the presence of CTAP (1 μM) (data not shown). Conversely, sEPSC amplitude was unaffected by DAMGO (95% ± 4% of control; P = 0.25; Figs. 6C and 8A).
Next, we investigated the effect of DAMGO on inhibitory presynaptic input. The sIPSCs had an average amplitude and frequency of 89 ± 10 pA and 21 ± 2 Hz, respectively, at a holding potential of 0 mV (n = 6). DAMGO (1 μM) increased the proportion of sIPSCs with a significantly longer interevent interval (P < 0.01) without a significant change in the cumulative distribution of sIPSC amplitude (P = 0.13) (Fig. 7, A and B). A maximal reduction in sIPSC frequency was seen just after DAMGO washout (62% ± 6% of control, n = 6; P < 0.01; Fig. 7C), but the suppressive effect was not seen in the presence of CTAP (1 μM) (data not shown). In contrast, sIPSC amplitude was unaffected by DAMGO (102% ± 8% of control; P = 0.86; Figs. 7C and 8B).
To determine the site of the inhibitory action of DAMGO, the effects of DAMGO were examined in the presence of TTX (0.5 μM). Conduction of action potentials is blocked by TTX, so that the effects of DAMGO on the presynaptic terminal can be isolated. In the presence of TTX, postsynaptic responses to spontaneously released transmitter can be detected as miniature postsynaptic currents. TTX alone had no effect on the amplitude (98% ± 2% of control, n = 7; P = 0.34) or the frequency (101% ± 8% of control, n = 7; P = 0.94) of sEPSCs (Fig. 8A). In contrast, TTX decreased both the amplitude (70% ± 5% of control, n = 6; P < 0.01) and the frequency (30% ± 6% of control, n = 6; P < 0.01) of sIPSCs (Fig. 8B). As with the effect on spontaneous postsynaptic currents, DAMGO significantly decreased the frequency of the miniature excitatory postsynaptic currents (mEPSCs) (67% ± 5% of the TTX-treated value, n = 7; P < 0.01) and the miniature inhibitory postsynaptic currents (mIPSCs) (61% ± 7% of the TTX-treated value, n = 5; P < 0.01), but it had no effect on the amplitude of mEPSCs (P = 0.51) and mIPSCs (P = 0.33) compared with the baseline values recorded in the presence of TTX alone (Fig. 8, A and B).
MOR agonists hyperpolarize the membranes of central nervous system neurons via the activation of K+ channels caused by MOR activation.5,24 However, patch-clamp recordings examining μ opioid modulation of lumbar spinal motoneurons have not been reported and little is known of the effects of MOR activation in motoneurons. In this study, we examined the cellular effects of DAMGO in spinal lamina IX neurons of rat lumbar spinal cord. We demonstrated for the first time that MOR agonists produce an outward current and reduce both the excitatory and inhibitory neurotransmitter release in lamina IX neurons.
Bath application of DAMGO produced an outward current at −70 mV in 56% of spinal lamina IX neurons recorded. This percentage is similar to that seen in patch-clamp studies of spinal lamina II neurons.5 The EC50 value of 0.10 μM for activation of MORs in postsynaptic membranes of spinal lamina IX neurons is close to that of previous reports.25–27
Because the DAMGO-induced current was not affected by TTX, the production of the DAMGO-induced currents is a postsynaptic effect on lamina IX neurons. Moreover, the DAMGO-induced currents were suppressed by the simultaneous application of CTAP, indicating that MORs are expressed in the postsynaptic membranes of lamina IX neurons and that their activation directly hyperpolarizes the lamina IX neurons. The polarity of the current was reversed at a potential close to the equilibrium potential of K+ and was inhibited by perfusion with Ba2+ or a pipette solution containing Cs+ and TEA, indicating the involvement of K+ channels. The effect of Ba2+ was identical to that reported for currents produced by MOR activation in other central nervous system neurons.26,28 Moreover, the currents were blocked by the addition of G protein blocker, GDP-β-S, to the pipette solution. These results indicate that the DAMGO-induced current in lamina IX neurons was produced by the activation of K+ channels through the activation of G proteins, specifically implicating GIRKs in the effect.
The principally excitatory neurotransmitter glutamate depolarizes spinal motoneurons, whereas γ-aminobutyric acid or glycine (inhibitory neurotransmitters) hyperpolarize the motoneurons. After presynaptic release, these transmitters act on postsynaptic receptors and their integrated effect determines motoneuronal excitability.29 We show that TTX reduced both the amplitude and frequency of sIPSCs, in agreement with previous reports.30 These results indicate that sIPSCs depend largely on TTX-sensitive Na+ channels, and that ongoing spontaneous inhibition occurs in the spinal ventral horn.
In addition to producing an outward current via postsynaptic MOR activation, we showed that DAMGO decreased the frequency of sEPSC and sIPSC, without influencing amplitude. Furthermore, similar suppressive effects were observed when conduction of action potentials was blocked by TTX (yielding mEPSCs and mIPSCs). Because ongoing inhibition occurred in the spinal ventral horn, the DAMGO-induced changes in sEPSCs or sIPSCs could be secondary to alterations in the activity of presynaptic inhibitory neurons. Therefore, we measured the influences of DAMGO on spontaneous postsynaptic currents with or without TTX treatment. These results suggest that MORs are located on the axon terminals of both the excitatory and inhibitory neurons and that activation of MORs decreases neurotransmitter release by both types of neurons in spinal lamina IX.
Interestingly, a suppressive effect of opioids on mIPSC frequency was not observed in adult rat spinal lamina II neurons.4 In contrast, it has been reported that inhibitory transmission is depressed by DAMGO in substantia gelatinosa neurons of the spinal trigeminal nucleus in juvenile rat horizontal brain slices.31 This discrepancy may reflect age-related influences or regional differences in expression of opioid system components.
Consistent with our results, MORs have been detected in spinal lamina IX of adult rats using the selective MOR probe [125I]FK-33-824.8 Moreover, MOR mRNA was detected in spinal lamina IX of rats,7 as well as of humans,32 using in situ hybridization. Furthermore, GIRKs have been detected in the spinal cord gray matter33 and in brainstem motoneurons.34 Our data indicate that the MORs of the spinal ventral horn functioned through the activation of GIRK. However, these results should be interpreted cautiously for a combination of reasons. First, the MOR activation responses in spinal lamina IX neurons may vary throughout the lifetime of an organism. MOR expression has been shown to peak at postnatal day 4 and subsequently decrease in the gray matter, except in the superficial dorsal horn, in rats.9,35 To identify large neurons under IR-DIC microscopy in the present study, we excluded adult rats, in which spinal neurons are difficult to identify because of highly developed fibrous tissue. Second, because the spinal lamina IX neurons we studied may include propriospinal interneurons, our observations may not apply exclusively to spinal motoneurons. Further experiments that record from identified spinal motoneurons in the adult rat are required to address these caveats.
Our data indicate that MOR activation has 2 consequences in the ventral horn: 1 reduction in the release of neurotransmitters to spinal lamina IX neurons, and 2 direct hyperpolarization of spinal lamina IX neurons. Clinical significance of these opioidergic neuromodulatory effects in motor function are a matter for speculation. The MOR-mediated effects of postsynaptic hyperpolarization and the presynaptic inhibition of excitatory neurotransmitter release indicate that μ opioids can suppress excitability of spinal lamina IX neurons. This suggests that the reduction of volatile anesthetics' minimum alveolar concentration by μ opioid is attributable to direct suppression of spinal motoneuronal excitability by MOR activation. On the other hand, the presynaptic reduction of inhibitory neurotransmitter release represents the μ opioid–mediated disinhibition of the lamina IX neurons. In this respect, it is assumed that opioid-induced muscle rigidity36 or spasticity15,16 may be attributable to the spinal effects of μ opioids.
To assess the actual effects of MOR activation on motor pathways, several studies have analyzed transcranial motor evoked potentials in animals and humans. There are conflicting reports regarding μ opioid suppression of signaling to spinal motoneurons: some groups report suppression11,12 whereas others do not.37 These studies, however, used varying doses of μ opioids (fentanyl ∼15 μg · kg−1 IV versus fentanyl 3 μg · kg−1 IV). Studies in spinalized rats, which restricted the sites of action of the agonists to the spinal cord, demonstrated that nociceptive responses were significantly reduced by IV-administered fentanyl (4–16 μg · kg−1).38 The EC50 value of MORs of spinal lamina IX neurons is close to that of spinal lamina II neurons producing spinal antinociception.26 These results suggest that μ opioids used at relevant dosages for spinal antinociception are also able to activate MORs located at the spinal ventral horn, but high doses of MOR agonists are required to suppress signaling to spinal motoneurons. Our data provide a possible explanation for these observations. We suggest that MOR-mediated suppressive effects on inhibitory neurotransmission counteract the suppressive effects on excitatory signal transduction.
In the central nervous system, inhibitory interneurons can be suppressed by μ opioids, leading to the disinhibition of cells on which such neurons terminate.14,39,40 It has been reported that supraspinal opioid receptors solely mediate opioid-induced muscle rigidity.41,42 Although we also found that DAMGO can decrease inhibitory neurotransmitter release in the ventral horn, we have no direct evidence that DAMGO has excitatory effects on lamina IX neurons. In contrast, several studies have suggested that μ opioids suppressed inhibitory interneurons, which led to disinhibition of spinal motoneurons after transient spinal cord ischemia.15,16 Because transient ischemic insult possibly altered the response to MOR agonists,43 the increased sensitivity of the inhibitory interneurons to μ opioids may occur only after ischemic insult and cause spinal motoneuronal disinhibition.
In conclusion, our data demonstrate novel neuromodulatory effects of MOR stimulation in spinal lamina IX neurons. Our observations provide a new perspective on opioids and their receptors in motor systems.
Name: Hiroyuki Honda, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Hiroyuki Honda has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Yasuhiko Kawasaki, PhD.
Contribution: This author helped design the study, conduct the study, and analyze the data.
Attestation: Yasuhiko Kawasaki has seen the original study data and approved the final manuscript.
Name: Hiroshi Baba, MD, PhD.
Contribution: This author helped design the study and analyze the data.
Attestation: Hiroshi Baba has seen the original study data and approved the final manuscript.
Name: Tatsuro Kohno, MD, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Tatsuro Kohno has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Steven L. Shafer, MD.
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