Direct inhibition of substantia gelatinosa neurones in the rat spinal cord by activation of dopamine D2-like receptors
1 Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
2 Department of Physiology, Faculty of Medicine, Saga University, Saga 849-8501, Japan
Abstract
Dopaminergic innervation of the spinal cord is largely derived from the brain. To understand the cellular mechanisms of antinociception mediated by descending dopaminergic pathways, we examined the actions of dopamine (DA) on nociceptive transmission by using behavioural studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurones in the spinal cord. Intrathecal administration of DA increased the mechanical nociceptive threshold and this effect was mimicked by a D2-like receptor agonist, quinpirole, but not by a D1-like receptor agonist, SKF 38393. In current-clamp mode of patch-clamp recordings, bath application of DA hyperpolarized the membrane potential of SG neurones and suppressed action potentials evoked by electrical stimulation of a dorsal root. In voltage-clamp mode, DA induced an outward current that was resistant to TTX, was blocked by the addition of Cs+ or GDP--S in the pipette solution, and was inhibited in the presence of Ba+. The DA-induced current reversed its polarity at a potential close to the equilibrium potential of the K+ channel calculated from the Nernst equation. The DA-induced outward current was mimicked by quinpirole, but not by SKF 38393. The DA-induced outward current was suppressed by a D2-like receptor antagonist, sulpiride, but not by a D1-like receptor antagonist, SCH 23390. In contrast, DA did not cause any significant change in amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). These results indicate that DA mainly acts on postsynaptic SG neurones to induce an outward current via G-protein-mediated activation of K+ channels through D2-like receptors. This may be a possible mechanism for antinociception by the descending dopaminergic pathway.
, http://www.100md.com
Introduction
Dopamine (DA) is the most abundant catecholamine in the brain and exerts its action by binding to specific membrane regions of seven transmembrane domain G-protein-coupled receptors (for review see Missale et al. 1998). To date, five distinct types of DA receptors have been isolated and divided into two subfamilies, D1-like and D2-like receptors, on the basis of their biochemical and pharmacological properties. The D1-like subfamily includes D1 and D5 receptors, while the D2-like subfamily comprises D2, D3 and D4 receptors (Giros et al. 1989; Monsma et al. 1989; Sunahara et al. 1991). The important contribution of DA as a neurotransmitter and/or neuromodulator in the brain is well understood. DA controls a variety of functions including locomotor activity, cognition, emotion, positive reinforcement, food intake and endocrine regulation (for review see Missale et al. 1998). The involvement of dopaminergic systems in neurological and psychiatric disorders has been the focus of a large body of research. Compared with the enormous literature devoted to DA actions in the brain, little is known about the roles of DA in the spinal cord.
, 百拇医药
Superficial laminae of the spinal dorsal horn, particularly the substantia gelatinosa (SG), receive nociceptive information from the viscera, skin and other organs through fine myelinated A and unmyelinated C primary afferent fibres (Willis & Coggeshall, 2004). Nociceptive transmission in the spinal dorsal horn is modulated by a variety of endogenous systems and transferred to the higher centre. It has been well established that the descending noradrenergic and serotonergic pathways modulate nociceptive transmission in the spinal dorsal horn (for review see Sandkuhler, 1996). However, little is known about the roles of the descending dopaminergic pathway. DA-synthesizing neurones in the periventricular, posterior region of the hypothalamus (A11) also innervate the spinal cord (Commissiong et al. 1978; Swanson & Kuypers, 1980; Skagerberg et al. 1982). Focal electrical stimulation in the region of the A11 area suppresses the nociceptive responses of multireceptive neurones in the spinal dorsal horn (Fleetwood-Walker et al. 1988). Based on these findings, it seems highly likely that the descending dopaminergic pathway plays an important role in the process of antinociception in the spinal cord.
, 百拇医药
DA-containing fibres and terminals are widely distributed in the spinal dorsal horn (Holstege et al. 1996). The highest binding sites of D1, D2 and D3 receptors are found in the superficial layers of the spinal dorsal horn (Levant & McCarson, 2001). In human spinal cord, mRNA encoding D4 receptors is also detected at a high level (Matsumoto et al. 1996). In addition, behavioural studies have demonstrated that intrathecal administration of DA induces thermal antinociceptive effects through D2-like receptors when assessed by the tail flick test (Jensen & Yaksh, 1984; Barasi & Duggal, 1985). However, the cellular mechanisms of DA action on nociceptive transmission have never been documented. There are also no pharmacological studies using DA receptor agonists or antagonists in whole-cell patch-clamp recordings from spinal dorsal horn neurones. Therefore, the aim of this study was to evaluate spinal DA actions on synaptic transmission in SG neurones of spinal cord slices.
, 百拇医药
Methods
All the experimental procedures involving the use of animals were approved by the Ethics Committee on Animal Experiments, Kyushu University, and were in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines.
Intrathecal administration of drugs
Conscious male Sprague-Dawley rats (6–8 weeks of age, 200–300 g) were administered drugs intrathecally, as previously described (Hylden & Wilcox, 1980). A 28-gauge needle with a 25-μl Hamilton microsyringe was inserted into the intervertebral space between the L5 and L6 regions and drugs were administrated slowly in a total volume of 10 μl. The accurate placement of the needle was confirmed by a quick ‘flick’ of the rat's tail. Drugs were dissolved in Krebs solution. Control rats received Krebs solution alone.
, 百拇医药
Behavioural tests
Mechanical sensitivity was determined with von Frey filaments (semmes-weinstein monofilaments, Stoelting, IL, USA) with calibrated bending forces, as previously described (Koga et al. 2004). Briefly, rats were placed individually in a plastic cage with a wire mesh bottom. After rats had adapted to the testing environments for 60 min, the von Frey filaments were pressed perpendicularly against the mid-plantar surface of the hind paw from below the mesh floor and held for 3–5 s with it slightly buckled. Lifting of the paw was recorded as a positive response. Filaments were applied to the point of bending six times to the plantar surface of the left and right hind paw for a total of 12 times per rat at intervals of 5 s; the next lightest filament was chosen for each subsequent measurement. Paw withdrawal threshold was taken as the lowest force that caused 100% withdrawals, and was considered as the mechanical nociceptive threshold.
, 百拇医药
Spinal cord slice preparation
The methods used for obtaining adult rat spinal cord slice preparations have been previously described (Nakatsuka et al. 2000). In brief, male adult Sprague-Dawley rats were deeply anaesthetized with urethane (1.2 g kg–1, intraperitoneal), and then lumbosacral laminectomy was performed. The lumbosacral spinal cord (L1–S3) was removed and placed in pre-xygenated Krebs solution at 1–3°C. Immediately after the removal of the spinal cord, the rats were given an overdose of urethane and were then killed by exsanguination. The pia-arachnoid membrane was removed after cutting all the ventral and dorsal roots near the root entry zone, except for the L4 or L5 dorsal root on one side. The spinal cord was mounted on a vibratome and then a 600-μm thick transverse slice with a dorsal root was cut. The slice was placed on nylon mesh in the recording chamber, which had a volume of 0.5 ml, and then perfused at a rate of 15–20 ml min–1 with Krebs solution saturated with 95% O2–5% CO2, and maintained at 36 ± 1°C. The Krebs solution contained (mM): NaCl 117, KCl 3.6, CaCl2 2.5, MgCl2 1.2, NaH2PO4 1.2, NaHCO3 25 and glucose 11.
, 百拇医药
Patch-clamp recordings from SG neurones
Blind whole-cell patch-clamp recordings were made from SG neurones with patch-pipette electrodes having a resistance of 5–10 M (Nakatsuka et al. 2000). The composition of the patch-pipette solution was as follows (mM): potassium gluconate 135, KCl 5, CaCl2 0.5, MgCl2 2, EGTA 5, Hepes 5 and ATP-Mg 5 (pH 7.2). Guanosine-5'-O-(2-thiodiphosphate) (GDP--S) was added at a concentration of 2 mM to the patch-pipette solution when necessary. The patch-pipette solution was composed of (mM): Cs2SO4 110, tetraethylammonium (TEA) 5, CaCl2 0.5, MgCl2 2, EGTA 5, Hepes 5 and ATP-Mg 5 (pH 7.2), and was used for inhibiting a postsynaptic effect of K+ channels. Signals were acquired with a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Union City, CA, USA). Data were digitized with an A/D converter (Digidata 1200, Axon Instruments) and stored and analysed with a personal computer using the pCLAMP data acquisition program (Version 8.2, Axon Instruments). Stimuli to elicit excitatory postsynaptic potentials (EPSPs) or action potentials were given to the dorsal root at the frequency of 0.2 Hz via a suction electrode. To examine changes in membrane conductance of the DA-induced currents, voltage steps (duration, 320 ms) from a holding potential of –50 mV to voltages ranging from –50 to –120 mV in steps of 10 mV were given to SG neurones in the absence or presence of DA. SG neurones were viable for up to 24 h in slices perfused with pre-oxygenated Krebs solution. However, all the recordings described here were obtained within 12 h. Whole-cell patch-clamp recordings were stable for up to 4 h. A holding membrane potential of –50 mV was used unless otherwise mentioned. All of the neurones had membrane potentials more negative than –50 mV.
, http://www.100md.com
Application of drugs
Drugs were dissolved in Krebs solution and applied by perfusion via a three-way stopcock without any change in the perfusion rate or the temperature. The time necessary for the solution to flow from the stopcock to the surface of the spinal cord slice was approximately 20 s. The drugs used in this study were DA, SCH 23390, SKF 38393, sulpiride, TTX and yohimbine (Wako, Osaka, Japan), and quinpirole, barium chloride dihydrate and GDP--S (Sigma, St Louis, MO, USA).
, 百拇医药
Statistical analysis
All numerical data were expressed as mean ± S.E.M. Statistical significance was determined as P < 0.05 using the Mann-Whiney U test to compare monofilament-induced withdrawal thresholds, and the Student's paired t test to compare the amplitude of outward currents. In electrophysiological data, n refers to the number of neurones studied. The continuous theoretical curve for the concentration–response relationship of DA was drawn according to a modification of the Michaelis–Menten equation:
, http://www.100md.com
using a least-squares fitting routine (Newton-Raphson method) after normalizing the amplitude of the response, where I is the normalized value of the currents, Imax the maximal response, C the drug concentration, and n the apparent Hill coefficient. The membrane potentials were not corrected for the liquid junction potential between the Krebs solution and patch-pipette solution.
Results
Intrathecal administration of DA or DA receptor agonists
, 百拇医药
To investigate whether spinal DA has antinociceptive effects, we examined the effects of intrathecal DA administration on the mechanical withdrawal thresholds by using a set of von Frey filaments. As shown in Fig. 1A, the threshold was significantly increased 10 min after the DA (17.0 nmol (10 μl)–1) injection (100.8 ± 11.8 g; n = 7) compared with controls (61.0 ± 6.5 g; n = 6, P < 0.05), but it was not significantly increased 10 min after the DA (1.7 nmol (10 μl)–1) injection (69.4 ± 7.4 g; n = 11). The mechanical withdrawal threshold reached a peak at 10 min and had disappeared 60 min after the intrathecal DA administration. We subsequently tried to determine which subtype of DA receptors was involved in the DA-induced increase in the mechanical withdrawal threshold. Intrathecal administration of the D2-like receptor agonist quinpirole (30 nmol (10 μl)–1) significantly increased the mechanical withdrawal threshold (85.7 ± 5.3 g; n = 7, P < 0.05), but the D1-like receptor agonist SKF 38393 (30 nmol (10 μl)–1) did not significantly affect the threshold (64.3 ± 7.2 g; n = 6) at 10 min after administration (Fig. 1B).
, 百拇医药
A, the mechanical withdrawal thresholds were significantly increased 10 min after intrathecal DA (17.0 nmol (10 μl)–1) administration (n = 7) compared with controls (n = 6, P < 0.05). However, the threshold was not significantly increased 10 min after DA (1.7 nmol (10 μl)–1, n = 11) injection. B, intrathecal administration of a D2-like receptor agonist, quinpirole (30 nmol (10 μl)–1), significantly increased the threshold (n = 7, P < 0.05) 10 min after the injections, while a D1-like receptor agonist, SKF 38393 (30 nmol (10 μl)–1), did not affect the threshold (n = 6).
, 百拇医药
Effects of DA on action potentials evoked by dorsal root stimulation
As it was revealed that intrathecal administration of DA or a D2-like receptor agonist has an antinociceptive effect in our behavioural studies, we further investigated the effects of DA on the synaptic responses elicited in SG neurones by using whole-cell patch-clamp recordings. In the current-clamp mode, we continuously applied electrical stimuli to a dorsal root with a suction electrode at a frequency of 0.2 Hz. When the stimulus intensity was high enough to activate C-afferents as previously described (Nakatsuka et al. 2000), a single stimulation elicited an EPSP followed by an action potential (Fig. 2A and B, left- and right-hand traces). Perfusion with DA (100 μM) caused a clear hyperpolarization (14.2 ± 2.8 mV; n = 6) of the membrane potential and completely inhibited action potentials in a reversible manner (Fig. 2A and B, middle traces). The DA-induced hyperpolarization and inhibition of action potentials was observed in all six SG neurones tested.
, 百拇医药
A, electrical stimuli to elicit EPSPs or action potentials were given to a dorsal root at the frequency of 0.2 Hz with a suction electrode. EPSPs followed by action potentials were observed in a SG neurone (resting membrane potential, –63 mV) by stimulation of the dorsal root at the intensity of C primary afferents (left-hand trace). Perfusion with DA (100 μM) caused a clear hyperpolarization (8 mV) of the membrane potential and inhibited action potentials (middle trace) in a reversible manner (right-hand traces). B, any single stimulation of the dorsal root at the intensity of C primary afferents evoked an EPSP followed by an action potential on an expanded time scale (left-hand trace). Following the application of DA, action potentials disappeared (middle trace).
, 百拇医药
Postsynaptic DA action
In the voltage-clamp mode (holding potential, –50 mV), perfusion with DA (100 μM) induced a clear outward current in 53 out of 54 neurones recorded (Fig. 3A). The average amplitude of the DA-induced outward currents was 48.4 ± 3.4 pA (n = 53). When DA was applied repeatedly at 10-min intervals, it produced similar responses with almost the same amplitude (data not shown). Furthermore, an outward current did not show any accommodation during the continuous application of DA (100 μM) for 10 min (Fig. 3B; n = 3). In the presence of TTX (1 μM), DA (100 μM) also induced an outward current (Fig. 3C). The average amplitude of the DA-induced outward currents in the presence of TTX was 49.5 ± 11.5 pA (n = 4) and was not significantly different from that in the absence of TTX (52.3 ± 11.8 pA; n = 4). DA is the precursor of noradrenaline (NA). NA produces an outward current through 2-adrenoceptors in the majority of SG neurones (Sonohata et al. 2004). To determine whether the DA-induced outward currents are mediated by 2-adrenoceptors, the effects of DA were examined in the presence of an 2-adrenoceptor antagonist, yohimbine (4 μM). In the same neurones, the DA-induced outward current in the presence of yohimbine was similar to that in the absence of yohimbine (100.1 ± 7.7% of that in the control, see Fig. 6B). The average amplitude of the DA-induced outward currents in the presence of yohimbine was 60.0 ± 23.5 pA (n = 4) and was not significantly different from that in the absence of yohimbine (60.5 ± 23.9 pA, n = 4).
, 百拇医药
A, in voltage-clamp mode, bath application of DA (100 μM) produced an outward current in an SG neurone at a holding potential of –50 mV. B, DA (100 μM) application for 10 min induced an outward current without desensitization. C, in the presence of TTX (1 μM), DA also induced an outward current without any decrease in amplitude (left-hand trace, in the absence of TTX; right-hand trace, in the presence of TTX). D, the DA-induced outward currents were enhanced in amplitude with increasing concentrations (left-hand traces). Normalized amplitude of outward current induced by DA was plotted against the DA concentration (right graph). Vertical bar indicates S.E.M. (n = 3–6). The continuous curve was drawn according to a modified Michaelis–Menten equation with an EC50 value of 77.8 μM.
, 百拇医药
A, a D1-like receptor agonist, SKF 38393 (30 μM), did not cause any outward current, while a D2-like receptor agonist, quinpirole (30 μM), induced an outward current in the same neurone. B, an 2-adrenoceptor antagonist, yohimbine (4 μM), did not affect the DA-induced outward currents. C, a D1-like receptor antagonist, SCH 23390 (30 μM), did not affect the DA-induced outward current. On the other hand, in the presence of a D2-like receptor antagonist, sulpiride (30 μM), the DA-induced outward current was markedly attenuated (upper trace, DA alone; middle trace, in the presence of SCH 23390; lower trace, in the presence of sulpiride). D, the histogram shows the average amplitude of the outward currents induced by DA (100 μM, n = 53), SKF 38393 (30 μM, n = 9), SKF 38393 (100 μM, n = 4) and quinpirole (30 μM, n = 9). E, relative peak amplitude was calculated as the average amplitude of the DA-induced currents in the presence of SCH 23390 (n = 6) or sulpiride (n = 6) divided by the average amplitude of currents produced by DA alone (n = 6). The DA-induced outward currents in the presence of sulpiride were significantly smaller than those in the presence of DA alone or in the presence of SCH 23390 (P < 0.05).
, 百拇医药
When examined in the concentration range 1–300 μM, the DA-induced outward currents were enhanced in amplitude with increasing concentrations (Fig. 3D). The onset of the outward currents became rapid and the recovery was delayed with an increasing concentration of DA (Fig. 3D, left). The outward currents exhibited a clear concentration-dependency (Fig. 3D, right). An analysis of the curve based on the Hill plot gave 77.8 μM for the value of the effective concentration producing half-maximal response (EC50) with a Hill coefficient of 1.38.
, 百拇医药
Figure 4 demonstrates the relationships between the step voltage and the steady current at the end of its pulse in the absence () and presence () of DA (100 μM). The net DA-induced current (), estimated from a difference between the two currents, exhibited a clear reverse (Fig. 4B). This reversal potential averaged –92.0 ± 4.1 mV (n = 5). The equilibrium potential (–92 mV) of K+, as calculated from the Nernst equation using [K+]o of 3.6 and [K+]i of 140 mM, was approximately equal to the reversal potentials obtained from the patch-clamp experiment.
, http://www.100md.com
A, to examine a change in membrane conductance of the DA-induced currents, a voltage step (duration, 320 ms) from a holding potential of –50 mV to voltages ranging from –50 to –120 mV in steps of 10 mV was given to SG neurones in the absence (upper trace) and presence (lower trace) of DA (100 μM). B, amplitude of membrane currents in response to 320-ms duration voltage pulses holding potential of –50 mV was plotted against voltages in the absence () and presence () of DA (100 μM). The current–voltage relationship for net DA current was estimated from the difference between the current responses in the absence and presence of DA ().
, 百拇医药
Bath application of the K+ channel blocker Ba2+ (1 mM) alone induced a small inward current (Fig. 5A, lower trace), which seems to be due to an inhibition of K+ channels (Yoshimura & Jessell, 1989). The average of the DA-induced outward currents in the presence of Ba2+ was 14.0 ± 4.7 pA (n = 4) and it was significantly decreased to 21.1 ± 4.5% of that in the controls (Fig. 5A; P < 0.05). In addition, DA (100 μM) did not induce any outward current with the pipette solution containing Cs+ and TEA (Fig. 5B; n = 28). To examine the involvement of G-proteins in the DA-induced outward current, GDP--S (2 mM), a non-hydrolysable analogue of GDP that competitively inhibits G-proteins, was added to the pipette solution. When DA (100 μM) was applied just after establishing the whole-cell configuration with pipettes containing potassium gluconate and GDP--S, an outward current was clearly observed (Fig. 5C, upper trace, n = 4). When DA was again applied 30 min later, it was significantly suppressed (Fig. 5C, lower trace, n = 4, P < 0.05).
, 百拇医药
A, DA (100 μM) was administrated in the absence (upper trace) and presence (lower trace) of Ba2+ (1 mM). The outward current was significantly reduced in the presence of Ba2+ (P < 0.05). B, DA (100 μM) did not induce any outward current with the addition of Cs+ into the pipette solution. C, the DA-induced outward current was recorded with the pipette solution containing potassium gluconate and GDP--S. DA (100 μM) produced an outward current just after establishing whole-cell configuration (upper trace), but it was markedly suppressed when DA (100 μM) was again applied 30 min later (lower trace, P < 0.05).
, 百拇医药
Effects of DA receptor agonists and antagonists
We further examined which subtype of DA receptors is involved in the DA-induced outward currents by using DA receptor agonists and antagonists. SKF 38393 (30 μM), a D1-like receptor agonist, did not induce any outward current in any of the nine SG neurones tested, while quinpirole (30 μM), a D2-like receptor agonist, produced a clear outward current in the same neurones (37.6 ± 14.8 pA, n = 9, Fig. 6A and D). An even higher concentration of SKF 38393 (100 μM) induced a transient outward current in 2 out of 4 neurones recorded (2.5 ± 1.2 pA; Fig. 6D). The currents were significantly smaller than the current induced by quinpirole (30 μM). Thus, it appears that SKF 38393 (100 μM) activated dopamine receptors as a non-specific agonist. Moreover, DA receptor antagonists were administrated 5 min prior to the DA application. In the presence of a D1-like receptor antagonist, SCH 23390 (30 μM), the average amplitude of the DA-induced outward currents was 44.3 ± 8.0 pA (Fig. 6C, n = 6), and was not significantly different from that in the absence of SCH 23390 (Fig. 6C, 52.7 ± 9.1 pA, n = 6). On the other hand, in the presence of a D2-like receptor antagonist, sulpiride (30 μM), the average amplitude of the DA-induced outward currents was 8.7 ± 1.4 pA (Fig. 6C, n = 6), and was significantly decreased to 20.7 ± 5.8% of that in the absence of sulpiride (Fig. 6E, P < 0.05).
, 百拇医药
Presynaptic DA action
To investigate whether DA modulates glutamate release from presynaptic terminals, the effects of DA were examined on miniature excitatory postsynaptic currents (mEPSCs) in SG neurones. Patch-pipettes containing Cs2SO4 and TEA were used for inhibiting the postsynaptic effect of K+ channels. All of the neurones examined exhibited mEPSCs that were completely blocked by a non-NMDA receptor antagonist, CNQX (10 μM). When DA (100 μM) was perfused for 1 min, all of the neurones examined showed no noticeable change in mEPSC frequency and amplitude (Fig. 7A, n = 22). There was no significant difference in mEPSC frequency between control and after perfusion of DA (91.0 ± 8.0%, P > 0.05, Fig. 7B and C). As well as DA, the application SKF 38393 (100 μM, n = 8) or quinpirole (100 μM, n = 8) produced no significant change in mEPSC frequency (99.8 ± 12.6% or 108.5 ± 18.1% of control, respectively, P > 0.05, Fig. 7C).
, 百拇医药
A, continuous chart recording of mEPSCs before and during the application of DA (100 μM) (upper trace). Three consecutive traces of mEPSCs are shown on an expanded timescale (lower traces); these were obtained before (lower left-hand traces) and during the application of DA (lower right-hand traces). B, cumulative distributions of the inter-event interval (left) and the amplitude (right) of mEPSCs, before (continuous line) and during the application of DA (dashed line). DA had no significant effect on the inter-event interval and the amplitude distribution. C, the histogram shows relative mEPSC frequency in the control and in the presence of DA (100 μM, n = 22), SKF 38393 (100 μM, n = 8) and quinpirole (100 μM, n = 8).
, 百拇医药
Discussion
In the present study we have shown that spinal DA exerts mechanical antinociception by the activation of D2-like receptors. Furthermore, the patch-clamp experiments have provided a possible mechanism for antinociception by the descending dopaminergic pathway. DA directly hyperpolarized the membrane potentials of almost all SG neurones by G-protein-mediated activation of K+ channels through D2-like receptors. In addition, action potentials evoked by the electrical stimulation of a dorsal root were suppressed by the application of DA.
, 百拇医药
Several reports have described the direct actions of supraspinal DA on nociceptive responses (Magnusson & Fisher, 2000; Pertovaara et al. 2004), while others have provided evidence for an indirect role of supraspinal DA to alter nociceptive responses through its interaction with opioid systems in the brain (Takeshita & Yamaguchi, 1998; Rutledge et al. 2002). As supraspinal DA is involved in other important physiological functions, the spinal cord may be a better target for DA to modulate nociceptive transmission. Nociceptive information such as thermal or mechanical sensation is carried to the central nervous system by small-diameter fibres, which terminate in the superficial laminae of the spinal dorsal horn, particularly SG neurones (Willis & Coggeshall, 2004). The nociceptive transmission can be modulated by a variety of endogenous systems in the spinal dorsal horn. Therefore, intrathecal drug delivery has been established to have an important role in the treatment of various pain sensations, such as postoperative, neuropathic, chronic and cancer pain. Previous studies have demonstrated that the intrathecal administration of DA induced thermal antinociceptive effects (Jensen & Yaksh, 1984; Barasi & Duggal, 1985). This antinociceptive action of DA agonists is selectively antagonized by D2-like receptors, but not D1-like receptors (Barasi & Duggal, 1985). However, there is no information about spinal DA actions on acute mechanical sensations. In the present study, we first demonstrated that the intrathecal administration of DA also produced mechanical antinociceptive effects and that it was mimicked by the intrathecal administration of a D2-like receptor agonist, quinpirole, but not by a D1-like receptor agonist, SKF 38393. These results suggested that the intrathecal administration of DA exerts not only thermal antinociception, but also mechanical antinociception through D2-like receptors. Of more importance, it was recently reported that intrathecal administration of DA or a D2-like receptor agonist markedly reduced the development of thermal hyperalgesia in response to chronic inflammation induced by carrageenan (Gao et al. 2001). Thus, D2-like receptors within the spinal dorsal horn may play a significant regulatory role in a variety of pain sensations.
, 百拇医药
Although early studies have suggested that the descending dopaminergic pathway plays an important role in spinal antinociception (Commissiong et al. 1978; Swanson & Kuypers, 1980; Skagerberg et al. 1982), the effects of DA in the spinal dorsal horn neurones have never been examined to elucidate the cellular mechanisms of nociceptive transmission. In the present study, action potentials evoked by electrical stimulation of a dorsal root were substantially suppressed by the application of DA, probably due to the hyperpolarization of the membrane potentials. Bath application of DA induced an outward current in almost all SG neurones, while DA and its receptor agonists did not modulate glutamatergic mEPSCs. The DA-induced outward currents exhibited a clear concentration-dependency. The EC50 value was very high in the present study, compared with that reported in dissociated cells (Einhorn et al. 1991). The enzymatic degradation of DA could greatly affect the EC50 in the spinal cord slice experiments. The finding of no effect of TTX on the DA-induced outward currents in the present study suggests that DA acts directly on SG neurones and not through an activation of interneurones. However, it may be possible that DA affects the release of other inhibitory neurotransmitters such as NA, serotonin, somatostatin, enkephalin or neuropeptide Y in a TTX-independent manner, resulting in an outward current in the SG neurones. In fact, it has been reported that DA receptors control NA release from sympathetic nerve endings in other preparations (Artalejo et al. 1990). NA has a similar chemical structure to DA (Missale et al. 1998) and induces an outward current through 2-adrenoceptors in the majority of SG neurones (Sonohata et al. 2004). However, the DA-induced outward currents were not suppressed by an 2-adrenoceptor antagonist, yohimbine (4 μM), in the present study. In addition, the DA-induced outward currents were mimicked by a selective D2-like receptor agonist and attenuated by a selective D2-like receptor antagonist. Thus, DA directly acts on D2-like receptors on the postsynaptic membrane of SG neurones. The DA-induced outward currents showed a reversal potential of –92 mV and were completely blocked by Cs+ and GDP--S in the pipette solution. The perfusion of a non-selective K+ channel blocker, Ba2+, also inhibited the outward currents induced by DA. These findings suggest that the DA-induced outward currents in SG neurones are mediated by G-protein-activated K+ channels through D2-like receptors. In other neural tissues, the role of D2-like receptors in modulating K+ channels has been studied extensively. Consistent with the present study of SG neurones in the spinal cord, D2-like receptor-mediated hyperpolarization mediated by G-protein-activated K+ conductance was first reported in DA neurones in the substantia nigra pars compacta (Lacey et al. 1987). In addition, D2-like receptors increase K+ outward currents, leading to cell hyperpolarization in striatal neurones, mesencephalic neurones and pituitary cells (Castelletti et al. 1989; Einhorn et al. 1991; Greif et al. 1995). These activations of K+ channels are also modulated by a G-protein-dependent mechanism. It has been demonstrated that D2-like receptors are coupled with inward rectified K+ channels in other neural tissues (Einhorn et al. 1991; Kim et al. 1995; Uchida et al. 2000). In contrast, the DA-induced outward currents in SG neurones of the spinal cord did not show a clear rectification in the inward direction, although we have not fully characterized the conductance underlying the DA-induced outward currents. This discrepancy may be the result of a distinct G-protein subunit coupled with D2-like receptors or it may reflect the modulation of a different K+ conductance by D2-like receptors in SG neurones of the spinal cord.
, http://www.100md.com
It has been demonstrated that there is no dopaminergic cell body in the rat, cat and monkey spinal cord and only fibres and terminals are immunoreactive for DA (Holstege et al. 1996). DA-containing fibres and terminals are widely distributed in the spinal cord (Skagerberg et al. 1982; Yoshida & Tanaka, 1988; Ridet et al. 1992). Dopaminergic innervation of the spinal cord is largely derived from the brain. The periventricular, posterior region of the hypothalamus (A11) is the principle source of descending dopaminergic pathways to the spinal cord (Commissiong et al. 1978; Skagerberg et al. 1982; Yoshida & Tanaka, 1988; Ridet et al. 1992). The highest density of descending dopaminergic fibres from the region of A11 is seen in the dorsal horn and lamina X of the spinal cord (Skagerberg et al. 1982; Yoshida & Tanaka, 1988; Ridet et al. 1992). Focal electrical stimulation in the region of A11 suppresses the nociceptive responses of the multireceptive neurones in the spinal dorsal horn, and the suppression of nociceptive responses is reversed by a D2-like receptor antagonist (Fleetwood-Walker et al. 1988). Therefore, the potential origin of endogenous DA appears to be from dopaminergic neurones in the region of A11. On the other hand, it has been reported that there is a small population of DRG neurones that exhibits a clear DA-immunoreactivity (Weil-Fugazza et al. 1993). Another possibility remains that DA is released from the peripheral nerve terminals; however, there is no reported evidence for this. In addition, our previous study showed that stimulating a dorsal root either singly or repetitively does not evoke a slow current in SG neurones (Nakatsuka et al. 2000). Therefore it is possible that DA is not released from the central terminal of primary afferents innervated onto SG neurones. Although the possibility cannot be completely excluded that DA is released from the peripheral nerve terminals, the principal source of endogenous DA in the spinal dorsal horn seems to be from descending dopaminergic fibres from cerebral structures. An autoradiographic study has demonstrated that both D1-like and D2-like receptors are densely localized in superficial laminae of the spinal dorsal horn (Levant & McCarson, 2001). Consistent with this, the present study has demonstrated that D2-like receptors are expressed in SG neurones and mediate the DA-induced outward currents. Because a selective agonist for D2, D3 or D4 receptors was not commercially available at this time, the subtype of DA receptors involving the DA-induced outward currents was not further examined. D1-like receptor-mediated postsynaptic excitation by inhibiting K+ conductances or activating cation conductances has been reported in striatal cholinergic interneurones (Aosaki et al. 1998). Although D1-like receptors also exist in the superficial laminae of the spinal cord (Levant & McCarson, 2001), the effect of D1-like receptors on membrane currents was not detected in the present study. Thus, further investigations will be required to clarify the role of D1-like receptors in the spinal dorsal horn.
, 百拇医药
It has been reported that D2-like receptor agonists markedly reduce acute and inflammatory pain in mammals (Barasi & Duggal, 1985; Gao et al. 2001). In addition, the precursor of DA, L-dihydroxyphenylalamine (L-DOPA), is used for the relief of cancer pain (Dickey & Minton, 1972; Nixon, 1975) and neuropathic pain in humans (Kernbaum & Hauchecorne, 1981; Ertas et al. 1998). However, the clinical use of DA agonists or L-DOPA for the treatment of pain sensations is not established yet, because the mechanism of the DA-induced antinociception is unclear. For the first time, the present study has provided a cellular mechanism of the spinal DA-induced antinociception. DA directly hyperpolarizes the membrane potentials of spinal dorsal horn neurones by G-protein-mediated activation of K+ channels through D2-like receptors. The activation of D2-like receptors in the spinal dorsal horn may be capable of inhibiting nociceptive signalling in a variety of pathological conditions.
, 百拇医药
References
Aosaki T, Kiuchi K & Kawaguchi Y (1998). Dopamine D1-like receptor activation excites rat striatal large aspiny neurons in vitro. J Neurosci 18, 5180–5190.
Artalejo CR, Ariano MA, Perlman RL & Fox AP (1990). Activation of facilitation calcium channels in chromaffin cells by D1 dopamine receptors through a cAMP/protein kinase A-dependent mechanism. Nature 348, 239–242.
Barasi S & Duggal KN (1985). The effect of local and systemic application of dopaminergic agents on tail flick latency in the rat. Eur J Pharmacol 117, 287–294.
, 百拇医药
Castelletti L, Memorandum M, Missale C, Spano PF & Valerio A (1989). Potassium channels involved in the transduction mechanism of dopamine D2 receptors in rat lactotrophs. J Physiol 410, 251–265.
Commissiong JW, Galli CL & Neff NH (1978). Differentiation of dopaminergic and noradrenergic neurons in rat spinal cord. J Neurochem 30, 1095–1099.
Dickey RP & Minton JP (1972). Levodopa relief of bone pain from breast cancer. N Engl J Med 286, 843.
, http://www.100md.com
Einhorn LC, Gregerson KA & Oxford GS (1991). D2 dopamine receptor activation of potassium channels in identified rat lactotrophs: whole-cell and single-channel recording. J Neurosci 11, 3727–3737.
Ertas M, Sagduyu A, Arac N, Uludag B & Ertekin C (1998). Use of levodopa to relieve pain from painful symmetrical diabetic polyneuropathy. Pain 75, 257–259.
Fleetwood-Walker SM, Hope PJ & Mitchell R (1988). Antinociceptive actions of descending dopaminergic tracts on cat and rat dorsal horn somatosensory neurones. J Physiol 399, 335–348.
, 百拇医药
Gao X, Zhang Y & Wu G (2001). Effects of dopaminergic agents on carrageenan hyperalgesia in rats. Eur J Pharmacol 406, 53–58.
Giros B, Sokoloff P, Martres MP, Riou JF, Emorine LJ & Schwartz JC (1989). Alternative splicing directs the expression of two D2 dopamine receptor isoforms. Nature 342, 923–926.
Greif GJ, Lin YJ, Liu JC & Freedman JE (1995). Dopamine-modulated potassium channels on rat striatal neurons: specific activation and cellular expression. J Neurosci 15, 4533–4544.
, 百拇医药
Holstege JC, Van Dijken H, Buijs RM, Goedknegt H, Gosens T & Bongers CM (1996). Distribution of dopamine immunoreactivity in the rat, cat and monkey spinal cord. J Comp Neurol 376, 631–652.
Hylden JLK & Wilcox GL (1980). Intrathecal morphine in mice: a new technique. Eur J Pharamacol 67, 313–316.
Jensen TS & Yaksh TL (1984). Effects of an intrathecal dopamine agonist, apomorphine, on thermal and chemical evoked noxious responses in rats. Brain Res 296, 285–293.
, 百拇医药
Kernbaum S & Hauchecorne J (1981). Administration of levodopa for relief of herpes zoster pain. JAMA 246, 132–134.
Kim KM, Nakajima Y & Nakajima S (1995). G protein-coupled inward rectifier modulated by dopamine agonists in cultured substantia nigra neurons. Neuroscience 69, 1145–1158.
Koga K, Honda K, Ando S, Harasawa I, Kamiya H & Takano Y (2004). Intrathecal clonidine inhibits mechanical allodynia via activation of the spinal muscarinic M1 receptor in streptozotocin-induced diabetic mice. Eur J Pharmacol 505, 75–82.
, http://www.100md.com
Lacey MG, Mercuri NB & North RA (1987). Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J Physiol 392, 397–416.
Levant B & McCarson KE (2001). D3 dopamine receptors in rat spinal cord: implications for sensory and motor function. Neurosci Lett 303, 9–12.
Magnusson JE & Fisher K (2000). The involvement of dopamine in nociception: the role of D1 and D2 receptors in the dorsolateral striatum. Brain Res 855, 260–266.
, 百拇医药
Matsumoto M, Hidaka K, Akiho H, Tada S, Okada M & Yamaguchi T (1996). Low stringency hybridization study of the dopamine D4 receptor revealed D4-like mRNA distribution of the orphan seven-transmembrane receptor, APJ, in human brain. Neurosci Lett 219, 119–122.
Missale C, Nash SR, Robinson SW, Jaber M & Caron MG (1998). Dopamine receptors: from structure to function. Physiol Rev 78, 189–225.
Monsma FJ Jr, McVittie LD, Gerfen CR, Mahan LC & Sibley DR (1989). Multiple D2 dopamine receptors produced by alternative RNA splicing. Nature 342, 926–929.
, 百拇医药
Nakatsuka T, Ataka T, Kumamoto E, Tamaki T & Yoshimura M (2000). Alteration in synaptic inputs through C-afferent fibers to substantia gelatinosa neurons of the rat spinal dorsal horn during postnatal development. Neuroscience 99, 549–556.
Nixon DW (1975). Letter: Use of L-dopa to relieve pain from bone metastases. N Engl J Med 292, 647.
Pertovaara A, Martikainen IK, Hagelberg N, Mansikka H, Nagren K, Hietala J & Scheinin H (2004). Striatal dopamine D2/D3 receptor availability correlates with individual response characteristics to pain. Eur J Neurosci 20, 1587–1592.
, 百拇医药
Ridet JL, Sandillon F, Rajaofetra N, Geffard M & Privat A (1992). Spinal dopaminergic system of the rat: light and electron microscopic study using an antiserum against dopamine, with particular emphasis on synaptic incidence. Brain Res 598, 233–241.
Rutledge LP, Ngong JM, Kuperberg JM, Samaan SS, Soliman KF & Kolta MG (2002). Dopaminergic system modulation of nociceptive response in long-term diabetic rats. Pharmacol Biochem Behav 74, 1–9.
, 百拇医药
Sandkuhler J (1996). The organization and function of endogenous antinociceptive systems. Prog Neurobiol 50, 49–81.
Skagerberg G, Bjorklund A, Lindvall O & Schmidt RH (1982). Origin and termination of the diencephalo-spinal dopamine system in the rat. Brain Res Bull 9, 237–244.
Sonohata M, Furue H, Katafuchi T, Yasaka T, Doi A, Kumamoto E & Yoshimura M (2004). Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording. J Physiol 555, 515–526.
, 百拇医药
Sunahara RK, Guan HC, O'Dowd BF, Seeman P, Laurier LG, Ng G, George SR, Torchia J, Van Tol HH & Niznik HB (1991). Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature 350, 614–619.
Swanson LW & Kuypers HG (1980). The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol 194, 555–570.
, 百拇医药
Takeshita N & Yamaguchi I (1998). Antinociceptive effects of morphine were different between experimental and genetic diabetes. Pharmacol Biochem Behav 60, 889–897.
Uchida S, Akaike N & Nabekura J (2000). Dopamine activates inward rectifier K+ channel in acutely dissociated rat substantia nigra neurones. Neuropharmacology 39, 191–201.
Weil-Fugazza J, Onteniente B, Audet G & Philippe E (1993). Dopamine as trace amine in the dorsal root ganglia. Neurochem Res 18, 965–969.
, http://www.100md.com
Willis WD & Coggeshall RE (2004). Sensory Mechanisms of the Spinal Cord, 3rd edn. Plenum, New York.
Yoshida M & Tanaka M (1988). Existence of new dopaminergic terminal plexus in the rat spinal cord: assessment by immunohistochemistry using anti-dopamine serum. Neurosci Lett 94, 5–9.
Yoshimura M & Jessell TM (1989). Membrane properties of rat substantia gelatinosa neurons in vitro. J Neurophysiol 62, 109–118., 百拇医药(Akihiro Tamae, Terumasa N)
2 Department of Physiology, Faculty of Medicine, Saga University, Saga 849-8501, Japan
Abstract
Dopaminergic innervation of the spinal cord is largely derived from the brain. To understand the cellular mechanisms of antinociception mediated by descending dopaminergic pathways, we examined the actions of dopamine (DA) on nociceptive transmission by using behavioural studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurones in the spinal cord. Intrathecal administration of DA increased the mechanical nociceptive threshold and this effect was mimicked by a D2-like receptor agonist, quinpirole, but not by a D1-like receptor agonist, SKF 38393. In current-clamp mode of patch-clamp recordings, bath application of DA hyperpolarized the membrane potential of SG neurones and suppressed action potentials evoked by electrical stimulation of a dorsal root. In voltage-clamp mode, DA induced an outward current that was resistant to TTX, was blocked by the addition of Cs+ or GDP--S in the pipette solution, and was inhibited in the presence of Ba+. The DA-induced current reversed its polarity at a potential close to the equilibrium potential of the K+ channel calculated from the Nernst equation. The DA-induced outward current was mimicked by quinpirole, but not by SKF 38393. The DA-induced outward current was suppressed by a D2-like receptor antagonist, sulpiride, but not by a D1-like receptor antagonist, SCH 23390. In contrast, DA did not cause any significant change in amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). These results indicate that DA mainly acts on postsynaptic SG neurones to induce an outward current via G-protein-mediated activation of K+ channels through D2-like receptors. This may be a possible mechanism for antinociception by the descending dopaminergic pathway.
, http://www.100md.com
Introduction
Dopamine (DA) is the most abundant catecholamine in the brain and exerts its action by binding to specific membrane regions of seven transmembrane domain G-protein-coupled receptors (for review see Missale et al. 1998). To date, five distinct types of DA receptors have been isolated and divided into two subfamilies, D1-like and D2-like receptors, on the basis of their biochemical and pharmacological properties. The D1-like subfamily includes D1 and D5 receptors, while the D2-like subfamily comprises D2, D3 and D4 receptors (Giros et al. 1989; Monsma et al. 1989; Sunahara et al. 1991). The important contribution of DA as a neurotransmitter and/or neuromodulator in the brain is well understood. DA controls a variety of functions including locomotor activity, cognition, emotion, positive reinforcement, food intake and endocrine regulation (for review see Missale et al. 1998). The involvement of dopaminergic systems in neurological and psychiatric disorders has been the focus of a large body of research. Compared with the enormous literature devoted to DA actions in the brain, little is known about the roles of DA in the spinal cord.
, 百拇医药
Superficial laminae of the spinal dorsal horn, particularly the substantia gelatinosa (SG), receive nociceptive information from the viscera, skin and other organs through fine myelinated A and unmyelinated C primary afferent fibres (Willis & Coggeshall, 2004). Nociceptive transmission in the spinal dorsal horn is modulated by a variety of endogenous systems and transferred to the higher centre. It has been well established that the descending noradrenergic and serotonergic pathways modulate nociceptive transmission in the spinal dorsal horn (for review see Sandkuhler, 1996). However, little is known about the roles of the descending dopaminergic pathway. DA-synthesizing neurones in the periventricular, posterior region of the hypothalamus (A11) also innervate the spinal cord (Commissiong et al. 1978; Swanson & Kuypers, 1980; Skagerberg et al. 1982). Focal electrical stimulation in the region of the A11 area suppresses the nociceptive responses of multireceptive neurones in the spinal dorsal horn (Fleetwood-Walker et al. 1988). Based on these findings, it seems highly likely that the descending dopaminergic pathway plays an important role in the process of antinociception in the spinal cord.
, 百拇医药
DA-containing fibres and terminals are widely distributed in the spinal dorsal horn (Holstege et al. 1996). The highest binding sites of D1, D2 and D3 receptors are found in the superficial layers of the spinal dorsal horn (Levant & McCarson, 2001). In human spinal cord, mRNA encoding D4 receptors is also detected at a high level (Matsumoto et al. 1996). In addition, behavioural studies have demonstrated that intrathecal administration of DA induces thermal antinociceptive effects through D2-like receptors when assessed by the tail flick test (Jensen & Yaksh, 1984; Barasi & Duggal, 1985). However, the cellular mechanisms of DA action on nociceptive transmission have never been documented. There are also no pharmacological studies using DA receptor agonists or antagonists in whole-cell patch-clamp recordings from spinal dorsal horn neurones. Therefore, the aim of this study was to evaluate spinal DA actions on synaptic transmission in SG neurones of spinal cord slices.
, 百拇医药
Methods
All the experimental procedures involving the use of animals were approved by the Ethics Committee on Animal Experiments, Kyushu University, and were in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines.
Intrathecal administration of drugs
Conscious male Sprague-Dawley rats (6–8 weeks of age, 200–300 g) were administered drugs intrathecally, as previously described (Hylden & Wilcox, 1980). A 28-gauge needle with a 25-μl Hamilton microsyringe was inserted into the intervertebral space between the L5 and L6 regions and drugs were administrated slowly in a total volume of 10 μl. The accurate placement of the needle was confirmed by a quick ‘flick’ of the rat's tail. Drugs were dissolved in Krebs solution. Control rats received Krebs solution alone.
, 百拇医药
Behavioural tests
Mechanical sensitivity was determined with von Frey filaments (semmes-weinstein monofilaments, Stoelting, IL, USA) with calibrated bending forces, as previously described (Koga et al. 2004). Briefly, rats were placed individually in a plastic cage with a wire mesh bottom. After rats had adapted to the testing environments for 60 min, the von Frey filaments were pressed perpendicularly against the mid-plantar surface of the hind paw from below the mesh floor and held for 3–5 s with it slightly buckled. Lifting of the paw was recorded as a positive response. Filaments were applied to the point of bending six times to the plantar surface of the left and right hind paw for a total of 12 times per rat at intervals of 5 s; the next lightest filament was chosen for each subsequent measurement. Paw withdrawal threshold was taken as the lowest force that caused 100% withdrawals, and was considered as the mechanical nociceptive threshold.
, 百拇医药
Spinal cord slice preparation
The methods used for obtaining adult rat spinal cord slice preparations have been previously described (Nakatsuka et al. 2000). In brief, male adult Sprague-Dawley rats were deeply anaesthetized with urethane (1.2 g kg–1, intraperitoneal), and then lumbosacral laminectomy was performed. The lumbosacral spinal cord (L1–S3) was removed and placed in pre-xygenated Krebs solution at 1–3°C. Immediately after the removal of the spinal cord, the rats were given an overdose of urethane and were then killed by exsanguination. The pia-arachnoid membrane was removed after cutting all the ventral and dorsal roots near the root entry zone, except for the L4 or L5 dorsal root on one side. The spinal cord was mounted on a vibratome and then a 600-μm thick transverse slice with a dorsal root was cut. The slice was placed on nylon mesh in the recording chamber, which had a volume of 0.5 ml, and then perfused at a rate of 15–20 ml min–1 with Krebs solution saturated with 95% O2–5% CO2, and maintained at 36 ± 1°C. The Krebs solution contained (mM): NaCl 117, KCl 3.6, CaCl2 2.5, MgCl2 1.2, NaH2PO4 1.2, NaHCO3 25 and glucose 11.
, 百拇医药
Patch-clamp recordings from SG neurones
Blind whole-cell patch-clamp recordings were made from SG neurones with patch-pipette electrodes having a resistance of 5–10 M (Nakatsuka et al. 2000). The composition of the patch-pipette solution was as follows (mM): potassium gluconate 135, KCl 5, CaCl2 0.5, MgCl2 2, EGTA 5, Hepes 5 and ATP-Mg 5 (pH 7.2). Guanosine-5'-O-(2-thiodiphosphate) (GDP--S) was added at a concentration of 2 mM to the patch-pipette solution when necessary. The patch-pipette solution was composed of (mM): Cs2SO4 110, tetraethylammonium (TEA) 5, CaCl2 0.5, MgCl2 2, EGTA 5, Hepes 5 and ATP-Mg 5 (pH 7.2), and was used for inhibiting a postsynaptic effect of K+ channels. Signals were acquired with a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Union City, CA, USA). Data were digitized with an A/D converter (Digidata 1200, Axon Instruments) and stored and analysed with a personal computer using the pCLAMP data acquisition program (Version 8.2, Axon Instruments). Stimuli to elicit excitatory postsynaptic potentials (EPSPs) or action potentials were given to the dorsal root at the frequency of 0.2 Hz via a suction electrode. To examine changes in membrane conductance of the DA-induced currents, voltage steps (duration, 320 ms) from a holding potential of –50 mV to voltages ranging from –50 to –120 mV in steps of 10 mV were given to SG neurones in the absence or presence of DA. SG neurones were viable for up to 24 h in slices perfused with pre-oxygenated Krebs solution. However, all the recordings described here were obtained within 12 h. Whole-cell patch-clamp recordings were stable for up to 4 h. A holding membrane potential of –50 mV was used unless otherwise mentioned. All of the neurones had membrane potentials more negative than –50 mV.
, http://www.100md.com
Application of drugs
Drugs were dissolved in Krebs solution and applied by perfusion via a three-way stopcock without any change in the perfusion rate or the temperature. The time necessary for the solution to flow from the stopcock to the surface of the spinal cord slice was approximately 20 s. The drugs used in this study were DA, SCH 23390, SKF 38393, sulpiride, TTX and yohimbine (Wako, Osaka, Japan), and quinpirole, barium chloride dihydrate and GDP--S (Sigma, St Louis, MO, USA).
, 百拇医药
Statistical analysis
All numerical data were expressed as mean ± S.E.M. Statistical significance was determined as P < 0.05 using the Mann-Whiney U test to compare monofilament-induced withdrawal thresholds, and the Student's paired t test to compare the amplitude of outward currents. In electrophysiological data, n refers to the number of neurones studied. The continuous theoretical curve for the concentration–response relationship of DA was drawn according to a modification of the Michaelis–Menten equation:
, http://www.100md.com
using a least-squares fitting routine (Newton-Raphson method) after normalizing the amplitude of the response, where I is the normalized value of the currents, Imax the maximal response, C the drug concentration, and n the apparent Hill coefficient. The membrane potentials were not corrected for the liquid junction potential between the Krebs solution and patch-pipette solution.
Results
Intrathecal administration of DA or DA receptor agonists
, 百拇医药
To investigate whether spinal DA has antinociceptive effects, we examined the effects of intrathecal DA administration on the mechanical withdrawal thresholds by using a set of von Frey filaments. As shown in Fig. 1A, the threshold was significantly increased 10 min after the DA (17.0 nmol (10 μl)–1) injection (100.8 ± 11.8 g; n = 7) compared with controls (61.0 ± 6.5 g; n = 6, P < 0.05), but it was not significantly increased 10 min after the DA (1.7 nmol (10 μl)–1) injection (69.4 ± 7.4 g; n = 11). The mechanical withdrawal threshold reached a peak at 10 min and had disappeared 60 min after the intrathecal DA administration. We subsequently tried to determine which subtype of DA receptors was involved in the DA-induced increase in the mechanical withdrawal threshold. Intrathecal administration of the D2-like receptor agonist quinpirole (30 nmol (10 μl)–1) significantly increased the mechanical withdrawal threshold (85.7 ± 5.3 g; n = 7, P < 0.05), but the D1-like receptor agonist SKF 38393 (30 nmol (10 μl)–1) did not significantly affect the threshold (64.3 ± 7.2 g; n = 6) at 10 min after administration (Fig. 1B).
, 百拇医药
A, the mechanical withdrawal thresholds were significantly increased 10 min after intrathecal DA (17.0 nmol (10 μl)–1) administration (n = 7) compared with controls (n = 6, P < 0.05). However, the threshold was not significantly increased 10 min after DA (1.7 nmol (10 μl)–1, n = 11) injection. B, intrathecal administration of a D2-like receptor agonist, quinpirole (30 nmol (10 μl)–1), significantly increased the threshold (n = 7, P < 0.05) 10 min after the injections, while a D1-like receptor agonist, SKF 38393 (30 nmol (10 μl)–1), did not affect the threshold (n = 6).
, 百拇医药
Effects of DA on action potentials evoked by dorsal root stimulation
As it was revealed that intrathecal administration of DA or a D2-like receptor agonist has an antinociceptive effect in our behavioural studies, we further investigated the effects of DA on the synaptic responses elicited in SG neurones by using whole-cell patch-clamp recordings. In the current-clamp mode, we continuously applied electrical stimuli to a dorsal root with a suction electrode at a frequency of 0.2 Hz. When the stimulus intensity was high enough to activate C-afferents as previously described (Nakatsuka et al. 2000), a single stimulation elicited an EPSP followed by an action potential (Fig. 2A and B, left- and right-hand traces). Perfusion with DA (100 μM) caused a clear hyperpolarization (14.2 ± 2.8 mV; n = 6) of the membrane potential and completely inhibited action potentials in a reversible manner (Fig. 2A and B, middle traces). The DA-induced hyperpolarization and inhibition of action potentials was observed in all six SG neurones tested.
, 百拇医药
A, electrical stimuli to elicit EPSPs or action potentials were given to a dorsal root at the frequency of 0.2 Hz with a suction electrode. EPSPs followed by action potentials were observed in a SG neurone (resting membrane potential, –63 mV) by stimulation of the dorsal root at the intensity of C primary afferents (left-hand trace). Perfusion with DA (100 μM) caused a clear hyperpolarization (8 mV) of the membrane potential and inhibited action potentials (middle trace) in a reversible manner (right-hand traces). B, any single stimulation of the dorsal root at the intensity of C primary afferents evoked an EPSP followed by an action potential on an expanded time scale (left-hand trace). Following the application of DA, action potentials disappeared (middle trace).
, 百拇医药
Postsynaptic DA action
In the voltage-clamp mode (holding potential, –50 mV), perfusion with DA (100 μM) induced a clear outward current in 53 out of 54 neurones recorded (Fig. 3A). The average amplitude of the DA-induced outward currents was 48.4 ± 3.4 pA (n = 53). When DA was applied repeatedly at 10-min intervals, it produced similar responses with almost the same amplitude (data not shown). Furthermore, an outward current did not show any accommodation during the continuous application of DA (100 μM) for 10 min (Fig. 3B; n = 3). In the presence of TTX (1 μM), DA (100 μM) also induced an outward current (Fig. 3C). The average amplitude of the DA-induced outward currents in the presence of TTX was 49.5 ± 11.5 pA (n = 4) and was not significantly different from that in the absence of TTX (52.3 ± 11.8 pA; n = 4). DA is the precursor of noradrenaline (NA). NA produces an outward current through 2-adrenoceptors in the majority of SG neurones (Sonohata et al. 2004). To determine whether the DA-induced outward currents are mediated by 2-adrenoceptors, the effects of DA were examined in the presence of an 2-adrenoceptor antagonist, yohimbine (4 μM). In the same neurones, the DA-induced outward current in the presence of yohimbine was similar to that in the absence of yohimbine (100.1 ± 7.7% of that in the control, see Fig. 6B). The average amplitude of the DA-induced outward currents in the presence of yohimbine was 60.0 ± 23.5 pA (n = 4) and was not significantly different from that in the absence of yohimbine (60.5 ± 23.9 pA, n = 4).
, 百拇医药
A, in voltage-clamp mode, bath application of DA (100 μM) produced an outward current in an SG neurone at a holding potential of –50 mV. B, DA (100 μM) application for 10 min induced an outward current without desensitization. C, in the presence of TTX (1 μM), DA also induced an outward current without any decrease in amplitude (left-hand trace, in the absence of TTX; right-hand trace, in the presence of TTX). D, the DA-induced outward currents were enhanced in amplitude with increasing concentrations (left-hand traces). Normalized amplitude of outward current induced by DA was plotted against the DA concentration (right graph). Vertical bar indicates S.E.M. (n = 3–6). The continuous curve was drawn according to a modified Michaelis–Menten equation with an EC50 value of 77.8 μM.
, 百拇医药
A, a D1-like receptor agonist, SKF 38393 (30 μM), did not cause any outward current, while a D2-like receptor agonist, quinpirole (30 μM), induced an outward current in the same neurone. B, an 2-adrenoceptor antagonist, yohimbine (4 μM), did not affect the DA-induced outward currents. C, a D1-like receptor antagonist, SCH 23390 (30 μM), did not affect the DA-induced outward current. On the other hand, in the presence of a D2-like receptor antagonist, sulpiride (30 μM), the DA-induced outward current was markedly attenuated (upper trace, DA alone; middle trace, in the presence of SCH 23390; lower trace, in the presence of sulpiride). D, the histogram shows the average amplitude of the outward currents induced by DA (100 μM, n = 53), SKF 38393 (30 μM, n = 9), SKF 38393 (100 μM, n = 4) and quinpirole (30 μM, n = 9). E, relative peak amplitude was calculated as the average amplitude of the DA-induced currents in the presence of SCH 23390 (n = 6) or sulpiride (n = 6) divided by the average amplitude of currents produced by DA alone (n = 6). The DA-induced outward currents in the presence of sulpiride were significantly smaller than those in the presence of DA alone or in the presence of SCH 23390 (P < 0.05).
, 百拇医药
When examined in the concentration range 1–300 μM, the DA-induced outward currents were enhanced in amplitude with increasing concentrations (Fig. 3D). The onset of the outward currents became rapid and the recovery was delayed with an increasing concentration of DA (Fig. 3D, left). The outward currents exhibited a clear concentration-dependency (Fig. 3D, right). An analysis of the curve based on the Hill plot gave 77.8 μM for the value of the effective concentration producing half-maximal response (EC50) with a Hill coefficient of 1.38.
, 百拇医药
Figure 4 demonstrates the relationships between the step voltage and the steady current at the end of its pulse in the absence () and presence () of DA (100 μM). The net DA-induced current (), estimated from a difference between the two currents, exhibited a clear reverse (Fig. 4B). This reversal potential averaged –92.0 ± 4.1 mV (n = 5). The equilibrium potential (–92 mV) of K+, as calculated from the Nernst equation using [K+]o of 3.6 and [K+]i of 140 mM, was approximately equal to the reversal potentials obtained from the patch-clamp experiment.
, http://www.100md.com
A, to examine a change in membrane conductance of the DA-induced currents, a voltage step (duration, 320 ms) from a holding potential of –50 mV to voltages ranging from –50 to –120 mV in steps of 10 mV was given to SG neurones in the absence (upper trace) and presence (lower trace) of DA (100 μM). B, amplitude of membrane currents in response to 320-ms duration voltage pulses holding potential of –50 mV was plotted against voltages in the absence () and presence () of DA (100 μM). The current–voltage relationship for net DA current was estimated from the difference between the current responses in the absence and presence of DA ().
, 百拇医药
Bath application of the K+ channel blocker Ba2+ (1 mM) alone induced a small inward current (Fig. 5A, lower trace), which seems to be due to an inhibition of K+ channels (Yoshimura & Jessell, 1989). The average of the DA-induced outward currents in the presence of Ba2+ was 14.0 ± 4.7 pA (n = 4) and it was significantly decreased to 21.1 ± 4.5% of that in the controls (Fig. 5A; P < 0.05). In addition, DA (100 μM) did not induce any outward current with the pipette solution containing Cs+ and TEA (Fig. 5B; n = 28). To examine the involvement of G-proteins in the DA-induced outward current, GDP--S (2 mM), a non-hydrolysable analogue of GDP that competitively inhibits G-proteins, was added to the pipette solution. When DA (100 μM) was applied just after establishing the whole-cell configuration with pipettes containing potassium gluconate and GDP--S, an outward current was clearly observed (Fig. 5C, upper trace, n = 4). When DA was again applied 30 min later, it was significantly suppressed (Fig. 5C, lower trace, n = 4, P < 0.05).
, 百拇医药
A, DA (100 μM) was administrated in the absence (upper trace) and presence (lower trace) of Ba2+ (1 mM). The outward current was significantly reduced in the presence of Ba2+ (P < 0.05). B, DA (100 μM) did not induce any outward current with the addition of Cs+ into the pipette solution. C, the DA-induced outward current was recorded with the pipette solution containing potassium gluconate and GDP--S. DA (100 μM) produced an outward current just after establishing whole-cell configuration (upper trace), but it was markedly suppressed when DA (100 μM) was again applied 30 min later (lower trace, P < 0.05).
, 百拇医药
Effects of DA receptor agonists and antagonists
We further examined which subtype of DA receptors is involved in the DA-induced outward currents by using DA receptor agonists and antagonists. SKF 38393 (30 μM), a D1-like receptor agonist, did not induce any outward current in any of the nine SG neurones tested, while quinpirole (30 μM), a D2-like receptor agonist, produced a clear outward current in the same neurones (37.6 ± 14.8 pA, n = 9, Fig. 6A and D). An even higher concentration of SKF 38393 (100 μM) induced a transient outward current in 2 out of 4 neurones recorded (2.5 ± 1.2 pA; Fig. 6D). The currents were significantly smaller than the current induced by quinpirole (30 μM). Thus, it appears that SKF 38393 (100 μM) activated dopamine receptors as a non-specific agonist. Moreover, DA receptor antagonists were administrated 5 min prior to the DA application. In the presence of a D1-like receptor antagonist, SCH 23390 (30 μM), the average amplitude of the DA-induced outward currents was 44.3 ± 8.0 pA (Fig. 6C, n = 6), and was not significantly different from that in the absence of SCH 23390 (Fig. 6C, 52.7 ± 9.1 pA, n = 6). On the other hand, in the presence of a D2-like receptor antagonist, sulpiride (30 μM), the average amplitude of the DA-induced outward currents was 8.7 ± 1.4 pA (Fig. 6C, n = 6), and was significantly decreased to 20.7 ± 5.8% of that in the absence of sulpiride (Fig. 6E, P < 0.05).
, 百拇医药
Presynaptic DA action
To investigate whether DA modulates glutamate release from presynaptic terminals, the effects of DA were examined on miniature excitatory postsynaptic currents (mEPSCs) in SG neurones. Patch-pipettes containing Cs2SO4 and TEA were used for inhibiting the postsynaptic effect of K+ channels. All of the neurones examined exhibited mEPSCs that were completely blocked by a non-NMDA receptor antagonist, CNQX (10 μM). When DA (100 μM) was perfused for 1 min, all of the neurones examined showed no noticeable change in mEPSC frequency and amplitude (Fig. 7A, n = 22). There was no significant difference in mEPSC frequency between control and after perfusion of DA (91.0 ± 8.0%, P > 0.05, Fig. 7B and C). As well as DA, the application SKF 38393 (100 μM, n = 8) or quinpirole (100 μM, n = 8) produced no significant change in mEPSC frequency (99.8 ± 12.6% or 108.5 ± 18.1% of control, respectively, P > 0.05, Fig. 7C).
, 百拇医药
A, continuous chart recording of mEPSCs before and during the application of DA (100 μM) (upper trace). Three consecutive traces of mEPSCs are shown on an expanded timescale (lower traces); these were obtained before (lower left-hand traces) and during the application of DA (lower right-hand traces). B, cumulative distributions of the inter-event interval (left) and the amplitude (right) of mEPSCs, before (continuous line) and during the application of DA (dashed line). DA had no significant effect on the inter-event interval and the amplitude distribution. C, the histogram shows relative mEPSC frequency in the control and in the presence of DA (100 μM, n = 22), SKF 38393 (100 μM, n = 8) and quinpirole (100 μM, n = 8).
, 百拇医药
Discussion
In the present study we have shown that spinal DA exerts mechanical antinociception by the activation of D2-like receptors. Furthermore, the patch-clamp experiments have provided a possible mechanism for antinociception by the descending dopaminergic pathway. DA directly hyperpolarized the membrane potentials of almost all SG neurones by G-protein-mediated activation of K+ channels through D2-like receptors. In addition, action potentials evoked by the electrical stimulation of a dorsal root were suppressed by the application of DA.
, 百拇医药
Several reports have described the direct actions of supraspinal DA on nociceptive responses (Magnusson & Fisher, 2000; Pertovaara et al. 2004), while others have provided evidence for an indirect role of supraspinal DA to alter nociceptive responses through its interaction with opioid systems in the brain (Takeshita & Yamaguchi, 1998; Rutledge et al. 2002). As supraspinal DA is involved in other important physiological functions, the spinal cord may be a better target for DA to modulate nociceptive transmission. Nociceptive information such as thermal or mechanical sensation is carried to the central nervous system by small-diameter fibres, which terminate in the superficial laminae of the spinal dorsal horn, particularly SG neurones (Willis & Coggeshall, 2004). The nociceptive transmission can be modulated by a variety of endogenous systems in the spinal dorsal horn. Therefore, intrathecal drug delivery has been established to have an important role in the treatment of various pain sensations, such as postoperative, neuropathic, chronic and cancer pain. Previous studies have demonstrated that the intrathecal administration of DA induced thermal antinociceptive effects (Jensen & Yaksh, 1984; Barasi & Duggal, 1985). This antinociceptive action of DA agonists is selectively antagonized by D2-like receptors, but not D1-like receptors (Barasi & Duggal, 1985). However, there is no information about spinal DA actions on acute mechanical sensations. In the present study, we first demonstrated that the intrathecal administration of DA also produced mechanical antinociceptive effects and that it was mimicked by the intrathecal administration of a D2-like receptor agonist, quinpirole, but not by a D1-like receptor agonist, SKF 38393. These results suggested that the intrathecal administration of DA exerts not only thermal antinociception, but also mechanical antinociception through D2-like receptors. Of more importance, it was recently reported that intrathecal administration of DA or a D2-like receptor agonist markedly reduced the development of thermal hyperalgesia in response to chronic inflammation induced by carrageenan (Gao et al. 2001). Thus, D2-like receptors within the spinal dorsal horn may play a significant regulatory role in a variety of pain sensations.
, 百拇医药
Although early studies have suggested that the descending dopaminergic pathway plays an important role in spinal antinociception (Commissiong et al. 1978; Swanson & Kuypers, 1980; Skagerberg et al. 1982), the effects of DA in the spinal dorsal horn neurones have never been examined to elucidate the cellular mechanisms of nociceptive transmission. In the present study, action potentials evoked by electrical stimulation of a dorsal root were substantially suppressed by the application of DA, probably due to the hyperpolarization of the membrane potentials. Bath application of DA induced an outward current in almost all SG neurones, while DA and its receptor agonists did not modulate glutamatergic mEPSCs. The DA-induced outward currents exhibited a clear concentration-dependency. The EC50 value was very high in the present study, compared with that reported in dissociated cells (Einhorn et al. 1991). The enzymatic degradation of DA could greatly affect the EC50 in the spinal cord slice experiments. The finding of no effect of TTX on the DA-induced outward currents in the present study suggests that DA acts directly on SG neurones and not through an activation of interneurones. However, it may be possible that DA affects the release of other inhibitory neurotransmitters such as NA, serotonin, somatostatin, enkephalin or neuropeptide Y in a TTX-independent manner, resulting in an outward current in the SG neurones. In fact, it has been reported that DA receptors control NA release from sympathetic nerve endings in other preparations (Artalejo et al. 1990). NA has a similar chemical structure to DA (Missale et al. 1998) and induces an outward current through 2-adrenoceptors in the majority of SG neurones (Sonohata et al. 2004). However, the DA-induced outward currents were not suppressed by an 2-adrenoceptor antagonist, yohimbine (4 μM), in the present study. In addition, the DA-induced outward currents were mimicked by a selective D2-like receptor agonist and attenuated by a selective D2-like receptor antagonist. Thus, DA directly acts on D2-like receptors on the postsynaptic membrane of SG neurones. The DA-induced outward currents showed a reversal potential of –92 mV and were completely blocked by Cs+ and GDP--S in the pipette solution. The perfusion of a non-selective K+ channel blocker, Ba2+, also inhibited the outward currents induced by DA. These findings suggest that the DA-induced outward currents in SG neurones are mediated by G-protein-activated K+ channels through D2-like receptors. In other neural tissues, the role of D2-like receptors in modulating K+ channels has been studied extensively. Consistent with the present study of SG neurones in the spinal cord, D2-like receptor-mediated hyperpolarization mediated by G-protein-activated K+ conductance was first reported in DA neurones in the substantia nigra pars compacta (Lacey et al. 1987). In addition, D2-like receptors increase K+ outward currents, leading to cell hyperpolarization in striatal neurones, mesencephalic neurones and pituitary cells (Castelletti et al. 1989; Einhorn et al. 1991; Greif et al. 1995). These activations of K+ channels are also modulated by a G-protein-dependent mechanism. It has been demonstrated that D2-like receptors are coupled with inward rectified K+ channels in other neural tissues (Einhorn et al. 1991; Kim et al. 1995; Uchida et al. 2000). In contrast, the DA-induced outward currents in SG neurones of the spinal cord did not show a clear rectification in the inward direction, although we have not fully characterized the conductance underlying the DA-induced outward currents. This discrepancy may be the result of a distinct G-protein subunit coupled with D2-like receptors or it may reflect the modulation of a different K+ conductance by D2-like receptors in SG neurones of the spinal cord.
, http://www.100md.com
It has been demonstrated that there is no dopaminergic cell body in the rat, cat and monkey spinal cord and only fibres and terminals are immunoreactive for DA (Holstege et al. 1996). DA-containing fibres and terminals are widely distributed in the spinal cord (Skagerberg et al. 1982; Yoshida & Tanaka, 1988; Ridet et al. 1992). Dopaminergic innervation of the spinal cord is largely derived from the brain. The periventricular, posterior region of the hypothalamus (A11) is the principle source of descending dopaminergic pathways to the spinal cord (Commissiong et al. 1978; Skagerberg et al. 1982; Yoshida & Tanaka, 1988; Ridet et al. 1992). The highest density of descending dopaminergic fibres from the region of A11 is seen in the dorsal horn and lamina X of the spinal cord (Skagerberg et al. 1982; Yoshida & Tanaka, 1988; Ridet et al. 1992). Focal electrical stimulation in the region of A11 suppresses the nociceptive responses of the multireceptive neurones in the spinal dorsal horn, and the suppression of nociceptive responses is reversed by a D2-like receptor antagonist (Fleetwood-Walker et al. 1988). Therefore, the potential origin of endogenous DA appears to be from dopaminergic neurones in the region of A11. On the other hand, it has been reported that there is a small population of DRG neurones that exhibits a clear DA-immunoreactivity (Weil-Fugazza et al. 1993). Another possibility remains that DA is released from the peripheral nerve terminals; however, there is no reported evidence for this. In addition, our previous study showed that stimulating a dorsal root either singly or repetitively does not evoke a slow current in SG neurones (Nakatsuka et al. 2000). Therefore it is possible that DA is not released from the central terminal of primary afferents innervated onto SG neurones. Although the possibility cannot be completely excluded that DA is released from the peripheral nerve terminals, the principal source of endogenous DA in the spinal dorsal horn seems to be from descending dopaminergic fibres from cerebral structures. An autoradiographic study has demonstrated that both D1-like and D2-like receptors are densely localized in superficial laminae of the spinal dorsal horn (Levant & McCarson, 2001). Consistent with this, the present study has demonstrated that D2-like receptors are expressed in SG neurones and mediate the DA-induced outward currents. Because a selective agonist for D2, D3 or D4 receptors was not commercially available at this time, the subtype of DA receptors involving the DA-induced outward currents was not further examined. D1-like receptor-mediated postsynaptic excitation by inhibiting K+ conductances or activating cation conductances has been reported in striatal cholinergic interneurones (Aosaki et al. 1998). Although D1-like receptors also exist in the superficial laminae of the spinal cord (Levant & McCarson, 2001), the effect of D1-like receptors on membrane currents was not detected in the present study. Thus, further investigations will be required to clarify the role of D1-like receptors in the spinal dorsal horn.
, 百拇医药
It has been reported that D2-like receptor agonists markedly reduce acute and inflammatory pain in mammals (Barasi & Duggal, 1985; Gao et al. 2001). In addition, the precursor of DA, L-dihydroxyphenylalamine (L-DOPA), is used for the relief of cancer pain (Dickey & Minton, 1972; Nixon, 1975) and neuropathic pain in humans (Kernbaum & Hauchecorne, 1981; Ertas et al. 1998). However, the clinical use of DA agonists or L-DOPA for the treatment of pain sensations is not established yet, because the mechanism of the DA-induced antinociception is unclear. For the first time, the present study has provided a cellular mechanism of the spinal DA-induced antinociception. DA directly hyperpolarizes the membrane potentials of spinal dorsal horn neurones by G-protein-mediated activation of K+ channels through D2-like receptors. The activation of D2-like receptors in the spinal dorsal horn may be capable of inhibiting nociceptive signalling in a variety of pathological conditions.
, 百拇医药
References
Aosaki T, Kiuchi K & Kawaguchi Y (1998). Dopamine D1-like receptor activation excites rat striatal large aspiny neurons in vitro. J Neurosci 18, 5180–5190.
Artalejo CR, Ariano MA, Perlman RL & Fox AP (1990). Activation of facilitation calcium channels in chromaffin cells by D1 dopamine receptors through a cAMP/protein kinase A-dependent mechanism. Nature 348, 239–242.
Barasi S & Duggal KN (1985). The effect of local and systemic application of dopaminergic agents on tail flick latency in the rat. Eur J Pharmacol 117, 287–294.
, 百拇医药
Castelletti L, Memorandum M, Missale C, Spano PF & Valerio A (1989). Potassium channels involved in the transduction mechanism of dopamine D2 receptors in rat lactotrophs. J Physiol 410, 251–265.
Commissiong JW, Galli CL & Neff NH (1978). Differentiation of dopaminergic and noradrenergic neurons in rat spinal cord. J Neurochem 30, 1095–1099.
Dickey RP & Minton JP (1972). Levodopa relief of bone pain from breast cancer. N Engl J Med 286, 843.
, http://www.100md.com
Einhorn LC, Gregerson KA & Oxford GS (1991). D2 dopamine receptor activation of potassium channels in identified rat lactotrophs: whole-cell and single-channel recording. J Neurosci 11, 3727–3737.
Ertas M, Sagduyu A, Arac N, Uludag B & Ertekin C (1998). Use of levodopa to relieve pain from painful symmetrical diabetic polyneuropathy. Pain 75, 257–259.
Fleetwood-Walker SM, Hope PJ & Mitchell R (1988). Antinociceptive actions of descending dopaminergic tracts on cat and rat dorsal horn somatosensory neurones. J Physiol 399, 335–348.
, 百拇医药
Gao X, Zhang Y & Wu G (2001). Effects of dopaminergic agents on carrageenan hyperalgesia in rats. Eur J Pharmacol 406, 53–58.
Giros B, Sokoloff P, Martres MP, Riou JF, Emorine LJ & Schwartz JC (1989). Alternative splicing directs the expression of two D2 dopamine receptor isoforms. Nature 342, 923–926.
Greif GJ, Lin YJ, Liu JC & Freedman JE (1995). Dopamine-modulated potassium channels on rat striatal neurons: specific activation and cellular expression. J Neurosci 15, 4533–4544.
, 百拇医药
Holstege JC, Van Dijken H, Buijs RM, Goedknegt H, Gosens T & Bongers CM (1996). Distribution of dopamine immunoreactivity in the rat, cat and monkey spinal cord. J Comp Neurol 376, 631–652.
Hylden JLK & Wilcox GL (1980). Intrathecal morphine in mice: a new technique. Eur J Pharamacol 67, 313–316.
Jensen TS & Yaksh TL (1984). Effects of an intrathecal dopamine agonist, apomorphine, on thermal and chemical evoked noxious responses in rats. Brain Res 296, 285–293.
, 百拇医药
Kernbaum S & Hauchecorne J (1981). Administration of levodopa for relief of herpes zoster pain. JAMA 246, 132–134.
Kim KM, Nakajima Y & Nakajima S (1995). G protein-coupled inward rectifier modulated by dopamine agonists in cultured substantia nigra neurons. Neuroscience 69, 1145–1158.
Koga K, Honda K, Ando S, Harasawa I, Kamiya H & Takano Y (2004). Intrathecal clonidine inhibits mechanical allodynia via activation of the spinal muscarinic M1 receptor in streptozotocin-induced diabetic mice. Eur J Pharmacol 505, 75–82.
, http://www.100md.com
Lacey MG, Mercuri NB & North RA (1987). Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J Physiol 392, 397–416.
Levant B & McCarson KE (2001). D3 dopamine receptors in rat spinal cord: implications for sensory and motor function. Neurosci Lett 303, 9–12.
Magnusson JE & Fisher K (2000). The involvement of dopamine in nociception: the role of D1 and D2 receptors in the dorsolateral striatum. Brain Res 855, 260–266.
, 百拇医药
Matsumoto M, Hidaka K, Akiho H, Tada S, Okada M & Yamaguchi T (1996). Low stringency hybridization study of the dopamine D4 receptor revealed D4-like mRNA distribution of the orphan seven-transmembrane receptor, APJ, in human brain. Neurosci Lett 219, 119–122.
Missale C, Nash SR, Robinson SW, Jaber M & Caron MG (1998). Dopamine receptors: from structure to function. Physiol Rev 78, 189–225.
Monsma FJ Jr, McVittie LD, Gerfen CR, Mahan LC & Sibley DR (1989). Multiple D2 dopamine receptors produced by alternative RNA splicing. Nature 342, 926–929.
, 百拇医药
Nakatsuka T, Ataka T, Kumamoto E, Tamaki T & Yoshimura M (2000). Alteration in synaptic inputs through C-afferent fibers to substantia gelatinosa neurons of the rat spinal dorsal horn during postnatal development. Neuroscience 99, 549–556.
Nixon DW (1975). Letter: Use of L-dopa to relieve pain from bone metastases. N Engl J Med 292, 647.
Pertovaara A, Martikainen IK, Hagelberg N, Mansikka H, Nagren K, Hietala J & Scheinin H (2004). Striatal dopamine D2/D3 receptor availability correlates with individual response characteristics to pain. Eur J Neurosci 20, 1587–1592.
, 百拇医药
Ridet JL, Sandillon F, Rajaofetra N, Geffard M & Privat A (1992). Spinal dopaminergic system of the rat: light and electron microscopic study using an antiserum against dopamine, with particular emphasis on synaptic incidence. Brain Res 598, 233–241.
Rutledge LP, Ngong JM, Kuperberg JM, Samaan SS, Soliman KF & Kolta MG (2002). Dopaminergic system modulation of nociceptive response in long-term diabetic rats. Pharmacol Biochem Behav 74, 1–9.
, 百拇医药
Sandkuhler J (1996). The organization and function of endogenous antinociceptive systems. Prog Neurobiol 50, 49–81.
Skagerberg G, Bjorklund A, Lindvall O & Schmidt RH (1982). Origin and termination of the diencephalo-spinal dopamine system in the rat. Brain Res Bull 9, 237–244.
Sonohata M, Furue H, Katafuchi T, Yasaka T, Doi A, Kumamoto E & Yoshimura M (2004). Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording. J Physiol 555, 515–526.
, 百拇医药
Sunahara RK, Guan HC, O'Dowd BF, Seeman P, Laurier LG, Ng G, George SR, Torchia J, Van Tol HH & Niznik HB (1991). Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature 350, 614–619.
Swanson LW & Kuypers HG (1980). The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol 194, 555–570.
, 百拇医药
Takeshita N & Yamaguchi I (1998). Antinociceptive effects of morphine were different between experimental and genetic diabetes. Pharmacol Biochem Behav 60, 889–897.
Uchida S, Akaike N & Nabekura J (2000). Dopamine activates inward rectifier K+ channel in acutely dissociated rat substantia nigra neurones. Neuropharmacology 39, 191–201.
Weil-Fugazza J, Onteniente B, Audet G & Philippe E (1993). Dopamine as trace amine in the dorsal root ganglia. Neurochem Res 18, 965–969.
, http://www.100md.com
Willis WD & Coggeshall RE (2004). Sensory Mechanisms of the Spinal Cord, 3rd edn. Plenum, New York.
Yoshida M & Tanaka M (1988). Existence of new dopaminergic terminal plexus in the rat spinal cord: assessment by immunohistochemistry using anti-dopamine serum. Neurosci Lett 94, 5–9.
Yoshimura M & Jessell TM (1989). Membrane properties of rat substantia gelatinosa neurons in vitro. J Neurophysiol 62, 109–118., 百拇医药(Akihiro Tamae, Terumasa N)