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Peripheral nerve stimulation increases serotonin and dopamine metabolites in rat spinal cord
Brain Research Bulletin, Vol. 33, No. 6, pp. 625-632, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0361-9230/94 $6.00 + .OO
Pergamon 0361.9230(93)EOOO9-B
Peripheral Nerve Stimulation Increases Serotonin and Dopamine Metabolites in Rat Spinal Cord DI-SHENG MEN’ AND YOICHIRO
MATSUI
Department of Physiology, Showa University School of Dentistry, l-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan Received
19 July 1993; Accepted
4 October
1993
MEN, D.-S. AND Y. MATSUI. Peripheral nerve stimulation increases serotonin and dopamine metabolites in rat spinal cord. BRAIN RES BULL 33(6) 625-632, 1994.-Extracellular serotonin (5-HT), dopamine (DA), and their metabolites, 5-hydroxyinacid (HVA), were assessed in the rat doleacetic acid (5-H&4), 3,4_dihydroxyphenylacetic acid (DOPAC), and homovanillic lumbar spinal cord (W-4) by in vivo microdialysis with high performance liquid chromatography and electrochemical detection (HPLC-ECD). Under urethane-chloralose anesthesia, basal levels of 5-HT and DA in the dialysates were approximately 1.0-1.2 pg/22 ~1 sample, 5-HIAA, DOPAC, and HVA were constant at 322.6 2 14.9, 8.6 t 0.7, and 10.4 2 0.4 pg/22 ~1 sample (mean 2 SE), respectively. Local application of 100 mM KC1 via the dialysis probe increased the 5-HT and DA. Peripheral nerve stimulation that selectively excited the large (A-beta) or small (A-delta) myelinated fibers increased the metabolites. Excitation of the A-beta fibers increased the levels of 5-HIAA to 138%, DOPAC to 155%, and HVA to 143% of the controls. Stimulation of the A-delta fibers increased 5-HIAA to 121%, DOPAC to 120%, and HVA to 124% of the controls. The results suggest that normociceptive peripheral nerve stimulation may activate the descending 5-HT and DA systems in the spinal cord. Serotonin
Dopamine
Microdialysis
Peripheral
Spinal cord
nerve stimulation
large or small myelinated afferent fibers in the hindlimb suppressed the rat tail-flick reflex and nociceptive responses of dorsal horn neurons to noxious heating (20,30,31). In the present study, in vivo microdialysis was utilized in the rat lumbar spinal cord. This technique permitted examination of 5-HT or DA release in the rat lumbar spinal cord in response to selective stimulation of large and small myelinated afferents.
SEROTONIN (S-HT)and dopamine (DA)-containing neurons that originate in supraspinal structures innervate the spinal cord (6,21,22,28). The spinal 5-HT innervation originates mainly from the brain stem, particularly the nucleus raphe magnus (NRM). Descending 5-I-IT neurons have been shown physiologically and pharmacologically to be involved in stimulation-produced analgesia and modulation of nociceptive transmission at the spinal cord level (4,9,11,15). Histochemical studies have revealed DAcontaining nerve terminals in the spinal cord associated with DA neurons in the diencephalon (5,17,21,28). In comparison, knowledge of DA functions in the spinal cord is relatively limited. Some evidence suggests that a diencephalospinal DA projection participates in sensory processing at the spinal cord level (10,18). It is well known that spinal inhibitory phenomena produced by peripheral stimulation, such as transcutaneous electrical stimulation and acupuncture, are involved in endogenous monoaminergic functions (16,35). The presence of such intrinsic modulation systems in the central nervous system (CNS) raises the question of whether somatosensory inputs activate the systems and, if so, what peripheral nerve elements are involved in these inhibitory systems. Assay of cerebral spinal fluid (CSF) established that high intensity stimulation of sciatic nerve elicited 5I-IT and noradrenaline (NA) release from the spinal cord, and the evoked release was attenuated by cold block of the cervical spinal cord (32). This result indicates that somatic stimuli activate a descending monoaminergic system. Selective stimulation of
METHODS
Animals
and Surgical Operations
Female Wistar albino rats, weighing from 250 to 350 g, were anesthetized with a mixture of urethane (500 mg/kg) and chloralose (50 mg/kg) applied intraperitoneally. Supplemental urethane (5 mg/kg)-chloralose (0.5 mgkg) mixture was infused through a femoral vein as required to maintain light anesthesia. The lumbar spinal cord was exposed by laminectomy at the L34 level, and the vertebrae were rigidly mounted in a stereotaxic frame. The underlying dura and pia were carefully removed under a stereomicroscope. Hemorrhage was avoided. The anesthetic level and the condition of the animal were monitored by heart rate, respiration, and pupil size throughout the experiment. The body temperature was maintained between 37 and 38°C using a thermostatically controlled electric heating pad.
1To whom requests for reprints should be addressed. 625
626
MEN AND MATSlil
,
4
I
1
8
12
18 min
8
12
18 min
FIG. 1. HPLC chromatograms of (A) 22 ~1 standard mixture containing 2 pg/,ul 5-HT, DA, DOPAC, and HVA, and 20 pg/pl 5-HIAA; and (B) a 22 ~1 dialysis sample obtained from the spinal cord of a urethanechloralose anesthetized rat. The analytical conditions of HPLC-ECD are described in the text. Abscissas:
retention times. Full ordinate range: 2 n4.
Dialysis Procedure
Biochemical Analysis
Prior to the experiment, the recovery rate of a microdialysis probe was tested in vitro. A coaxial probe (2 mm long by 0.5 mm diameter, CMA/lO, Carnegie Medicin, Sweden) was immersed in a standard solution containing 8 pg/$ of S-hydroxytryptamine (5HT), 5hydroxyindoleacetic acid (5HIAA), dopamine (DA), 3,4_dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), and then perfused with physiological saline at a rate of 1 pl/min driven by a microinjection pump (CMA/lOO, Carnegie Medicin). The physiological saline consisted of 154 mM NaCl, 5.6 mM KCI, 2.2 mM CaC12, and 2.4 mM NaHC03. The average recovery of the probe was: 5-HT, 23.7%; 5-HIAA, 24.2%; DA, 23.1%; DOPAC, 23.4%, and HVA, 23.4%. The microdialysis probe was inserted into the lumbar spinal cord to a vertical depth of 1.5 mm ipsilateral to the stimulation side at an angle of 45-50 degrees to the spine by a micromanipulator. The in vivo dialysis was performed continuously with the physiological saline at a rate of 1 pl/min for more than 4 h. After the initial 1 h allowed for recovery from the injury induced-variation of collected substances, the dialysates were collected sequentially every 20 min into sample tubes with a microfraction collector (CMA/140, Carnegie Medicin). To prevent metabolism of the amines in the dialysates, each sample tube contained 5 ~1 of 1 M perchloric acid, and the entire sampling system was kept in an ice bath.
The dialysates were assayed immediately after collection. The concentrations of 5-HT, 5-H&4, DA, DOPAC, and HVA in the dialysates were determined by high performance liquid chromatography with electrochemical detection (HPLC-ECD, LC4BCC4, BAS). The reverse-phase column was Biophase ODS-4 (3 pm, 110 x 4 mm, BAS). A mobile phase composed of 0.1 M sodium acetate, 0.1 M tartaric acid, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 750 PM 1-octanesulfonic acid, 6% acetonitrile, and 0.5% tetrahydrofuran, adjusted to pH 3.2, was delivered at a flow rate of 0.7 ml/min by a high pressure pump (PM-60, BAS). Electrochemical detection was carried out with a glassy carbon working electrode setting of 0.75 V against a Ag/ AgCl reference electrode. Twenty-two ~1 of dialysate was injected directly into the HPLC-ECD. The detection limit of this system, estimated from measurement of the standard solution, was approximately 1 pg for all substances. The standard 5-HT, 5-HIAA, DA, DOPAC, and HVA were purchased from Sigma Chemical Co. Other reagents used in the experiment were analytical or HPLC grade. Administration
of Potassium
In a group of rats, a membrane depolarizing agent, was used to examine the neuronal release of substances.
KCl, The
627
SEROTONIN AND DOPAMINE IN SPINAL CORD TABLE 1
in vivo microdialysis in the spinal cord was performed as described above. When the substances in the dialysates maintained relatively constant levels, 100 mM KC1 saline instead of physiological saline was perfused through the probe for 20 min. Osmolarity was maintained by substituting the appropriate amount of sodium chloride in the saline. The dialysates were collected sequentially and measured for 80 min after the potassium administration. Selective Stimulation
BASAL
AND THEIR METABOLITES IN THE RAT SPINAL CORD Mean -+ SE (pgi22
n
5-HT
5-HLU DA DOPAC WA
of Large and Small Myelinated Fibers
For selective stimulation of large or small myelinated afferent fibers, two stainless steel needles were inserted into the skin of the hindlimb, and 0.1 ms rectangular, constant current pulses were delivered through the needles. The common peroneal nerve ipsilateral to the stimulation area was exposed and covered with warm paraffin oil. Compound action potentials evoked by the stimulation were recorded from the common peroneal nerve through a pair of platinum wires and monitored simultaneously on an oscilloscope. The selective stimulation of the large or small myelinated afferent fibers was confirmed from the conduction velocities of the compound action potentials. In the experiment, the electrical stimulation was applied at 3 times the threshold intensity for 40 min. The stimulus frequencies were 50 Hz for the large myelinated afferents and 2 I-Ix for the small myelinated afferents. Details of this stimulus manipulation have been described previously (20,31).
LEVELS OF SEROTONIN,DOPAMINE,
1.0 5
81 91 91 90 89
0.2
322.6 ” 14.9 1.2 + 0.2 8.6 2 0.7 10.4 I 0.4
n: Totai number of samples,
Data Analysis The concentrations of 5HT, S-HIAA, DA, DOPAC, and HVA in 22 ~1 dialysates are expressed in pica-grams per sample. Because the recovery rate of each probe is different, the values for statistics are calculated as the concentration corresponding to the average recovery rate. Results for basal levels and potassium action are expressed as mean 2 SE of the values of concentration of measured substance at individual time points. Results for peripheral nerve stimulation are expressed as mean -C SE of the percentage of the control value (100%). The control was the average value of three consecutive mea-
5-HIAA
120 Time in min
160
~1)
0 0
40
80
120
160
Time in min
FIG. 2. Basal levels of S-HT, DA, .5-HIAA, DOPAC, and HVA in dialysates. Zero time on abscissa: onset of co&&on, 60 min after start of dialysis. Each cobmm with bar represents the mean 2 SE for 12 rats. Sample size, 22 g.
628
MEN AND MA’I‘SUI KCI
5-HT
3oo
KCI
y.+
5-HIAA
1
HVA
0
40
Time
in mtn
-40
0
40
Time
in mnn
FIG. 3. Effects of local application of 100 mM K’ on the extracellular concentration of 5-HT and its metabolite, 5-HIAA (left); and DA and its metabolites, DOPAC, HVA (right). Zero time on abscissa: start of 100 mM K+ perfusion. Open columns with bars: mean k SE (n = 5-6) at indicated times before and after 100 mM K’ administration. Shaded columns with bars: mean 2 SE (n = 6) durine the hinh K’ oerfusion. * *p < 0.01; *I, < 0.05 vs. the values determined immediately before K+ pkfusion~ Sample size, 22 &.
surements immediately preceding the stimulation. Statistical comparisons between treatment and control were made by Student’s two-tailed t-test. RESULTS
Basal Levels of 5-HT, DA, and Their Metabolites Basal levels of 5-HT, 5-HIAA, DA, DOPAC, and HVA were measured in the lumbar spinal cord using in vivo microdialysis coupled with HPLC-ECD. Figure 1 shows analytical chromatograms of a standard mixture and a dialysate collected under a basal condition. The identity of a substance was determined by comparing its retention time with a standard mixture. The concentration of a substance in the dialysate was established by comparison with a standard concentration. After the initial 60 min, dialysate samples were collected continuously every 20 min over a total period of 180 min. The concentrations of S-HIAA, DOPAC, and HVA in the dialysates were high, but those of 5-HT and DA were barely above the detection limit of the assay system (Fig. 1B). Figure 2 shows the time courses of the basal levels of 5-HT’, .5-HJAA, DA, DOPAC, and HVA in the dialysates. The levels of 5-HIAA, DOPAC, and HVA remained relatively constant throughout the entire microdialysis. There were no statistically significant differences between sequential collections (p > 0.05) within 180 min. However, the
levels of 5-HT and DA declined progressively to values that were close to the detection limits under basal conditions. Table 1 summarizes the average basal levels of all substances. The values for 5-HT and DA are marginally reliable. It is noted that the basal level of 5-HIAA in the dialysates (322.6 -t 14.9 pg, n = 91) greatly exceeded that of 5-HT (1.0 5 0.2 pg, n = 81). Potassium-Induced
Release of 5-HT and DA
Figure 3 shows the time courses of the changes in 5-HT, DA, and their metabolites induced by administration of a high concentration of potassium. When saline containing 100 mM KC1 was perfused through the probe, the levels of 5-HT and DA in the dialysates increased remarkably, although their basal levels were very low. The 5-HT value rose from 0.8 -C 0.5 to 5.0 + 1.0 pg/sample 0, < 0.01) and DA value increased from 0.5 ? 0.3 to 3.4 -C 1.1 pg/sample (p < 0.05). After the high potassium saline was replaced by normal physiological saline, the levels of both substances subsided to their basal values. Induction of 5-HT and DA release by high potassium saline was accompanied by significant decreases in their metabolites. The level of the 5-HT metabolite, 5-HIAA, was reduced to 66.4% of the pretreatment level during the potassium administration @ < 0.01). The levels of DA metabolites, DOPAC and HVA, were
SEROTONIN
AND
DOPAMINE
IN
SPINAL
629
CORD
5-HIAA
A-beta
A-delta
%
L L
L
I
I
I
L
-
80
-40
lime
120
in min
80 lime
120
in min
FIG. 4. Effects on the extracellular concentration of 5-HIAA of selective stimulation of A-beta fibers (left) and A-delta fibers (right). Parameters of the stimulation are described in the text. Examples of evoked action potentials recorded from the common peroneal nerve arc shown in insets; black arrows: stimulus artifacts. Zero time on abscissas: start of stimulation. Each column with bar: mean 2 SE of changes in 17 rats (left) and 8 rats (right) during the stimulation (dotted columns), and before and after stimulation (open columns), compared to controls. **p < 0.01 vs. the controls.
reduced to 79.6% and 47.5%, respectively, by the high potassium saline (p < 0.05). Effects of Peripheral Nerve Stimulation on the Levels of 5-HT and DA Metabolites
When basal levels of S-HIAA, DOPAC, and HVA were stable for more than 1 h, large myelinated (A-beta) or small myelinated (A-delta) fibers in the hindlimb were selectively stimulated. The insets in Fig. 4 show the compound action potentials evoked by the stimuli. The mean threshold intensities were 219 PA for exciting the large myelinated and 413 PA for the small myelinated afferent fibers. The conduction velocities of large myelinated fibers were approximately 54 m/s, and those of small myelinated fibers were approximately 12 m/s. The S-HT metabolite, S-HIAA, in the dialysates was increased by the stimulation of either A-beta or A-delta afferents (Fig. 4). During the A-beta fiber stimulation (40 min), the increment in 5HIAA level appeared mostly in the initial 20 min. The average value of three consecutive measurements immediately prior to the stimulation was taken as the control (100%). The 5-H&.4 in the dialysate then reached 137.8 2 5.7% of the control during the initial stimulation period (p < 0.01). In the next 20 min period, the level of 5-HLAA diminished to about the control level. When the A-delta fiber was stimulated, the 5-HIAA level increased to 120.5 ? 5.8% of the control during the first 20 min of stimulation (p < 0.01). The level of 5-HIAA tended to remain high for more than 20 min. There were no statistically significant differences between these higher levels and the control level @ > 0.05), because of the high individual variation among the rats.
Figure 5 shows the time courses of changes of DA metabolites, DOPAC and HVA, in response to selective stimulation of A-beta or A-delta afferents. When A-beta fibers were stimulated (40 min), the levels of DOPAC increased to 155.0 ? 8.4% of the control and the levels of HVA increased to 142.6 2 6.1% of the control in the first 20 min of stimulation. When the A-delta fiber stimulation was applied, DOPAC and HVA levels increased to 120.3 ? 5.3% and 124.1 + 8.4% of the controls, respectively, in the initial 20 min of stimulation. No change in 5-I-R nor DA could be detected during either A-beta or A-delta fiber stimulation. DISCUSSION The present study used in vivo microdialysis to examine 5HT and DA activities in the spinal cord in response to selective stimulation of large or small myelinated afferent fibers. In previous studies using microdialysis in cerebral structures (2,2527), the dialysates driven from the tissue reflected the extracellular concentrations of substances released from nerve terminals and their metabolites around the microdialysis probe. In the present study, dialysates obtained sequentially from the rat spinal cord for a long period, over 3 h, showed low basal levels of 5-HT and DA, and relatively high and constant basal levels of their metabolites, 5-HIAA, DOPAC, and HVA. When a membrane depolarizing agent, KCl, was applied locally through the probe, extracellular 5-m and DA greatly increased. The results confirm innervation of regions around the probe insertion site by nerve terminals that contain 5-HT or DA. The low basal
630
MEN AND MATSUI
A-beta
,
A-delta
DOPAC
1
I
I
% 150
A-beta I-
-40
0
WA
A-delta
I
40
I
80
120
Time in min
-40
0 Time
1
40
a0
120
in min
FIG. 5. Effects of selective stimulation of A-beta (left) and A-delta (right) fibers on the extracellular concentrations of DOPAC (upper) and HVA (lower). Parameters of the stimulation are described in the text. Zero time: start of stimulation. Each column with bar: mean + SE of changes in 19 rats (left) and 9 rats (right) during stimulation (dotted columns), and before and after the stimulation (open columns), compared to control values.
**p < 0.01; *p < 0.05 vs. the controls.
levels of 5-HT and DA can be explained by the small amounts released from the nerve terminals, and their rapid metabolism immediately after the release and during the period of dialysate sampling. According to this explanation, any change in metabolite concentration could indicate 5-HT and DA release. However, it is noted that the concentration of 5-HIAA in the dialysates is far different from that of 5-HT. This might suggest another possible source of 5-HIAA, possibly arising directly from the nerve terminals. Studies of the mechanism of 5-HIAA release suggested that it depends on membrane potential of nerve terminals, which reduces 5-HIAA release by factors that induce membrane depolarization (24). This is consistent with our finding that 5-H&4 in dialysates was decreased by administration of high potassium. A similar phenomenon was also observed in the present study in which extracellular DOPAC and HVA were reduced by high potassium administration. The results led us to consider that the extracellular 5-HIAA, and probably DOPAC and HVA, include the results of 5-I-U and DA metabolism plus their release directly from the nerve terminals. When membrane depolarization was evoked by pharmacological or electrophysiological manipulations, 5-HT or DA was released from the nerve terminals and then turned to 5-HIAA, or DOPAC and HVA, respectively, under the action of monoamine oxidase in the extracellular space. Direct release of metabolites was suppressed, however, due to the membrane depolarization. Thus, it is possible
that either increase or decrease in metabolites may appear, depending on the membrane depolarization of the nerve terminal, whereas increase in the metabolites, 5-HIAA, DOPAC or HVA will reflect the release of 5-HT and DA from the nerve terminals. In the present study, it was found that selective stimulation of large or small myelinated afferent fibers (A-beta or A-delta) in the same conditions that suppressed nociceptive reflexes (20,30) and nociceptive responses in the dorsal horn neurons (31), increased 5-HIAA, DOPAC, and HVA in spinal dialysates. Based on the consideration that increment of the metabolites reflects increment of 5-HT or DA, it is suggested that the inhibition produced by peripheral nerve stimulation could be associated with activation of the endogenous 5-I-IT and DA systems. It is generally accepted that the endogenous 5-HT system, with cell bodies in the brain stem, projects to the spinal dorsal horn and is inhibitory at the spinal cord level. Direct stimulation of the descending 5-HT system, for instance, serotonin-rich raphe nuclei of the medulla, particularly the NRM, produced significant antinociceptive effects on nociceptive transmission at the spinal level (12,13,19). In addition, stimulation of the periaqueductal gray (PAG) that projects directly or indirectly to the NRM also elicited profound analgesia (3,33). However, studies of the activation of the descending 5-HT system by peripheral nerve stimulation are limited. Activation of small nerve fibers increased the concentration of 5-HT in the CSF of cat suggesting its release
631
SEROTONIN AND DOPAMINE IN SPINAL CORD
from the descending S-HT neurons, because evocation of release was abolished in spinalized animals (3236). Effects of tail stimulation on S-I-IT synthesis within the dorsal spinal cord suggested that peripheral nociceptive messages increased activity in some bulbo-spinal S-HT pathways (34). Because it is reported that NRM neurons respond to peripheral nerve stimulation (1,14,29), it is reasonable to consider that somatosensory inputs may trigger a complex loop that carries information from the spinal dorsal horn to the supraspinal level, then activates the descending 5-HT system directly or indirectly, and finally produces inhibitory effects at the spinal cord level. The present study, with direct data from the spinal cord, supports the conclusion that somatic information from small myelinated (A-delta) fibers activates the descending 5-HT system. In addition, it was demonstrated that inputs from large myelinated (A-beta) fibers also influence the intrinsic S-HT system. However, the different time courses of the A-beta and the A-delta fiber stimulation indicates different mechanisms or pathways in the CNS. Another interesting finding in the present study was that stimulation of A-beta or A-delta fibers increased DA metabolite concentrations in the spinal dialysates. Because it is clearly established that DA in the spinal cord is not only a precursor of NA, but also serves as an independent transmitter in the DA system (7,8), alternation of endogenous DA or its metabolites, DOPAC and HVA, may reflect activity of the DA system as well as that of the NA system. When combined with evidence that the same periphery nerve st~ulation produces no change in NA (23), the present results suggest that the endogenous DA system rather than the NA system was activated by peripheral nerve stimulation. The endogenous DA system innervating the spinal cord has received much attention in recent years. It has been demonstrated
by anatomical studies that DA neurons in the All cell group of the diencephalon give rise to descending DA projections to the rat spinal dorsal horn (21,28). Evidence that intrathecal injection of DA and the DA agonist, apomorphine, produced antinociceptive effects on thermally and chemically evoked noxious xesponses (18) suggests that spinal DA receptors are inhibitory to noxious inputs to the spinal cord. Furthermore, electrical stimulation in the AI 1 cell region in the diencephalon selectively sup pressed nociceptive responses of spinai m~tire~ptive neurons, and this was rapidly reversed by iontophoretic application of sulpiride, a D2 DA receptor antagonist, in the spinal dorsal horn (10). Such results suggest that the descending DA projection, as does the descending 5-HT system, inhibits the transmission of nociceptive info~ation in the spinal cord. Our fmding that peripheral nerve stimulation increased DOPAC and HVA in dialysates supports the hypothesis that spinal DA innervation participates in the processing of sensory information, and may suggest that inhibitory effects induced by selective stimulation of A-beta or A-delta fibers are related directly or indirectly to spinal DA activity. In summary, the present study suggests that the selective stimulation of large (A-beta) or small (A-delta) myelinated afferent fibers may activate the descending S-FIT and DA systems via supraspinal structures, and releases 5-HT and DA from the nerve terminals in the spinal cord. These neurotransmitters may be involved in inhibition of the processing of sensory transmission in the spinal cord.
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ACKNOWLEDGEMENTS
We thank Professor T. Sato and Dr. A. Matsui for their help and encouragement during this study.
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MEN
2Y. 30. 31.
32.
33. 34.
35.
36.
AND
.MATSIJI
neurones projecting to the spinal cord in the rat. Brain Rcs. 332:3411351; 1985. Toda, K. Response of raphe magnus neurons after acupuncture stim ulation in rat. Brain Res. 242:350-353; 1982. Tsuruokd, M. Suppression of the tail flick refex by Afl afferent nerve impulses. I. Showa Med. Assoc. 47:43-55; 1987. Tsuruoka, M.; Li, Q-J.; Matsui, A.; Matsui, Y. Inhibition of nociceptive responses of wide-dynamic-range neurons by peripheral nerve stimulation. Brain Res. Bull. 25:387-392; 1990. Tyce, G. M.: Yaksh, T. L. Monoamine release from cat spinal cord by somatic stimuli: An intrinsic modulatory system. J. Physiol. 314:513-529; 1981. Van Praag, H.; Frenk, H. The development of stimulation-produced analgesia (SPA) in the rat. Dev. Brain Res. 64:71-76: 1991. Weil-Fugazza, J.; Godefroy, F.; Le Bars, D. Increase in 5-H’I synthesis in the dorsal part of the spinal cord, induced by a nociceptive stimulus: Blockade by morphine. Brain Res. 297:247-264: 1984. Willis, W. D. Control of nociceptive transmission in the spinal cord. In: Autrum, H.; Ottoson, D.; Perl, E. R.; Schmidt, R. F., eds. Progress in sensory physiology. vol. 3. Berlin: Springer; 1982: I 159. Yaksh, T. L.; Hammond, D. L.: Tyce, G. M. Functional aspects of bulbospinal monoaminergic projections in modulating processing of somatosensory information. Fed. Proc. 40:2786-2794; 1981.
Pergamon 0361.9230(93)EOOO9-B
Peripheral Nerve Stimulation Increases Serotonin and Dopamine Metabolites in Rat Spinal Cord DI-SHENG MEN’ AND YOICHIRO
MATSUI
Department of Physiology, Showa University School of Dentistry, l-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan Received
19 July 1993; Accepted
4 October
1993
MEN, D.-S. AND Y. MATSUI. Peripheral nerve stimulation increases serotonin and dopamine metabolites in rat spinal cord. BRAIN RES BULL 33(6) 625-632, 1994.-Extracellular serotonin (5-HT), dopamine (DA), and their metabolites, 5-hydroxyinacid (HVA), were assessed in the rat doleacetic acid (5-H&4), 3,4_dihydroxyphenylacetic acid (DOPAC), and homovanillic lumbar spinal cord (W-4) by in vivo microdialysis with high performance liquid chromatography and electrochemical detection (HPLC-ECD). Under urethane-chloralose anesthesia, basal levels of 5-HT and DA in the dialysates were approximately 1.0-1.2 pg/22 ~1 sample, 5-HIAA, DOPAC, and HVA were constant at 322.6 2 14.9, 8.6 t 0.7, and 10.4 2 0.4 pg/22 ~1 sample (mean 2 SE), respectively. Local application of 100 mM KC1 via the dialysis probe increased the 5-HT and DA. Peripheral nerve stimulation that selectively excited the large (A-beta) or small (A-delta) myelinated fibers increased the metabolites. Excitation of the A-beta fibers increased the levels of 5-HIAA to 138%, DOPAC to 155%, and HVA to 143% of the controls. Stimulation of the A-delta fibers increased 5-HIAA to 121%, DOPAC to 120%, and HVA to 124% of the controls. The results suggest that normociceptive peripheral nerve stimulation may activate the descending 5-HT and DA systems in the spinal cord. Serotonin
Dopamine
Microdialysis
Peripheral
Spinal cord
nerve stimulation
large or small myelinated afferent fibers in the hindlimb suppressed the rat tail-flick reflex and nociceptive responses of dorsal horn neurons to noxious heating (20,30,31). In the present study, in vivo microdialysis was utilized in the rat lumbar spinal cord. This technique permitted examination of 5-HT or DA release in the rat lumbar spinal cord in response to selective stimulation of large and small myelinated afferents.
SEROTONIN (S-HT)and dopamine (DA)-containing neurons that originate in supraspinal structures innervate the spinal cord (6,21,22,28). The spinal 5-HT innervation originates mainly from the brain stem, particularly the nucleus raphe magnus (NRM). Descending 5-I-IT neurons have been shown physiologically and pharmacologically to be involved in stimulation-produced analgesia and modulation of nociceptive transmission at the spinal cord level (4,9,11,15). Histochemical studies have revealed DAcontaining nerve terminals in the spinal cord associated with DA neurons in the diencephalon (5,17,21,28). In comparison, knowledge of DA functions in the spinal cord is relatively limited. Some evidence suggests that a diencephalospinal DA projection participates in sensory processing at the spinal cord level (10,18). It is well known that spinal inhibitory phenomena produced by peripheral stimulation, such as transcutaneous electrical stimulation and acupuncture, are involved in endogenous monoaminergic functions (16,35). The presence of such intrinsic modulation systems in the central nervous system (CNS) raises the question of whether somatosensory inputs activate the systems and, if so, what peripheral nerve elements are involved in these inhibitory systems. Assay of cerebral spinal fluid (CSF) established that high intensity stimulation of sciatic nerve elicited 5I-IT and noradrenaline (NA) release from the spinal cord, and the evoked release was attenuated by cold block of the cervical spinal cord (32). This result indicates that somatic stimuli activate a descending monoaminergic system. Selective stimulation of
METHODS
Animals
and Surgical Operations
Female Wistar albino rats, weighing from 250 to 350 g, were anesthetized with a mixture of urethane (500 mg/kg) and chloralose (50 mg/kg) applied intraperitoneally. Supplemental urethane (5 mg/kg)-chloralose (0.5 mgkg) mixture was infused through a femoral vein as required to maintain light anesthesia. The lumbar spinal cord was exposed by laminectomy at the L34 level, and the vertebrae were rigidly mounted in a stereotaxic frame. The underlying dura and pia were carefully removed under a stereomicroscope. Hemorrhage was avoided. The anesthetic level and the condition of the animal were monitored by heart rate, respiration, and pupil size throughout the experiment. The body temperature was maintained between 37 and 38°C using a thermostatically controlled electric heating pad.
1To whom requests for reprints should be addressed. 625
626
MEN AND MATSlil
,
4
I
1
8
12
18 min
8
12
18 min
FIG. 1. HPLC chromatograms of (A) 22 ~1 standard mixture containing 2 pg/,ul 5-HT, DA, DOPAC, and HVA, and 20 pg/pl 5-HIAA; and (B) a 22 ~1 dialysis sample obtained from the spinal cord of a urethanechloralose anesthetized rat. The analytical conditions of HPLC-ECD are described in the text. Abscissas:
retention times. Full ordinate range: 2 n4.
Dialysis Procedure
Biochemical Analysis
Prior to the experiment, the recovery rate of a microdialysis probe was tested in vitro. A coaxial probe (2 mm long by 0.5 mm diameter, CMA/lO, Carnegie Medicin, Sweden) was immersed in a standard solution containing 8 pg/$ of S-hydroxytryptamine (5HT), 5hydroxyindoleacetic acid (5HIAA), dopamine (DA), 3,4_dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), and then perfused with physiological saline at a rate of 1 pl/min driven by a microinjection pump (CMA/lOO, Carnegie Medicin). The physiological saline consisted of 154 mM NaCl, 5.6 mM KCI, 2.2 mM CaC12, and 2.4 mM NaHC03. The average recovery of the probe was: 5-HT, 23.7%; 5-HIAA, 24.2%; DA, 23.1%; DOPAC, 23.4%, and HVA, 23.4%. The microdialysis probe was inserted into the lumbar spinal cord to a vertical depth of 1.5 mm ipsilateral to the stimulation side at an angle of 45-50 degrees to the spine by a micromanipulator. The in vivo dialysis was performed continuously with the physiological saline at a rate of 1 pl/min for more than 4 h. After the initial 1 h allowed for recovery from the injury induced-variation of collected substances, the dialysates were collected sequentially every 20 min into sample tubes with a microfraction collector (CMA/140, Carnegie Medicin). To prevent metabolism of the amines in the dialysates, each sample tube contained 5 ~1 of 1 M perchloric acid, and the entire sampling system was kept in an ice bath.
The dialysates were assayed immediately after collection. The concentrations of 5-HT, 5-H&4, DA, DOPAC, and HVA in the dialysates were determined by high performance liquid chromatography with electrochemical detection (HPLC-ECD, LC4BCC4, BAS). The reverse-phase column was Biophase ODS-4 (3 pm, 110 x 4 mm, BAS). A mobile phase composed of 0.1 M sodium acetate, 0.1 M tartaric acid, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 750 PM 1-octanesulfonic acid, 6% acetonitrile, and 0.5% tetrahydrofuran, adjusted to pH 3.2, was delivered at a flow rate of 0.7 ml/min by a high pressure pump (PM-60, BAS). Electrochemical detection was carried out with a glassy carbon working electrode setting of 0.75 V against a Ag/ AgCl reference electrode. Twenty-two ~1 of dialysate was injected directly into the HPLC-ECD. The detection limit of this system, estimated from measurement of the standard solution, was approximately 1 pg for all substances. The standard 5-HT, 5-HIAA, DA, DOPAC, and HVA were purchased from Sigma Chemical Co. Other reagents used in the experiment were analytical or HPLC grade. Administration
of Potassium
In a group of rats, a membrane depolarizing agent, was used to examine the neuronal release of substances.
KCl, The
627
SEROTONIN AND DOPAMINE IN SPINAL CORD TABLE 1
in vivo microdialysis in the spinal cord was performed as described above. When the substances in the dialysates maintained relatively constant levels, 100 mM KC1 saline instead of physiological saline was perfused through the probe for 20 min. Osmolarity was maintained by substituting the appropriate amount of sodium chloride in the saline. The dialysates were collected sequentially and measured for 80 min after the potassium administration. Selective Stimulation
BASAL
AND THEIR METABOLITES IN THE RAT SPINAL CORD Mean -+ SE (pgi22
n
5-HT
5-HLU DA DOPAC WA
of Large and Small Myelinated Fibers
For selective stimulation of large or small myelinated afferent fibers, two stainless steel needles were inserted into the skin of the hindlimb, and 0.1 ms rectangular, constant current pulses were delivered through the needles. The common peroneal nerve ipsilateral to the stimulation area was exposed and covered with warm paraffin oil. Compound action potentials evoked by the stimulation were recorded from the common peroneal nerve through a pair of platinum wires and monitored simultaneously on an oscilloscope. The selective stimulation of the large or small myelinated afferent fibers was confirmed from the conduction velocities of the compound action potentials. In the experiment, the electrical stimulation was applied at 3 times the threshold intensity for 40 min. The stimulus frequencies were 50 Hz for the large myelinated afferents and 2 I-Ix for the small myelinated afferents. Details of this stimulus manipulation have been described previously (20,31).
LEVELS OF SEROTONIN,DOPAMINE,
1.0 5
81 91 91 90 89
0.2
322.6 ” 14.9 1.2 + 0.2 8.6 2 0.7 10.4 I 0.4
n: Totai number of samples,
Data Analysis The concentrations of 5HT, S-HIAA, DA, DOPAC, and HVA in 22 ~1 dialysates are expressed in pica-grams per sample. Because the recovery rate of each probe is different, the values for statistics are calculated as the concentration corresponding to the average recovery rate. Results for basal levels and potassium action are expressed as mean 2 SE of the values of concentration of measured substance at individual time points. Results for peripheral nerve stimulation are expressed as mean -C SE of the percentage of the control value (100%). The control was the average value of three consecutive mea-
5-HIAA
120 Time in min
160
~1)
0 0
40
80
120
160
Time in min
FIG. 2. Basal levels of S-HT, DA, .5-HIAA, DOPAC, and HVA in dialysates. Zero time on abscissa: onset of co&&on, 60 min after start of dialysis. Each cobmm with bar represents the mean 2 SE for 12 rats. Sample size, 22 g.
628
MEN AND MA’I‘SUI KCI
5-HT
3oo
KCI
y.+
5-HIAA
1
HVA
0
40
Time
in mtn
-40
0
40
Time
in mnn
FIG. 3. Effects of local application of 100 mM K’ on the extracellular concentration of 5-HT and its metabolite, 5-HIAA (left); and DA and its metabolites, DOPAC, HVA (right). Zero time on abscissa: start of 100 mM K+ perfusion. Open columns with bars: mean k SE (n = 5-6) at indicated times before and after 100 mM K’ administration. Shaded columns with bars: mean 2 SE (n = 6) durine the hinh K’ oerfusion. * *p < 0.01; *I, < 0.05 vs. the values determined immediately before K+ pkfusion~ Sample size, 22 &.
surements immediately preceding the stimulation. Statistical comparisons between treatment and control were made by Student’s two-tailed t-test. RESULTS
Basal Levels of 5-HT, DA, and Their Metabolites Basal levels of 5-HT, 5-HIAA, DA, DOPAC, and HVA were measured in the lumbar spinal cord using in vivo microdialysis coupled with HPLC-ECD. Figure 1 shows analytical chromatograms of a standard mixture and a dialysate collected under a basal condition. The identity of a substance was determined by comparing its retention time with a standard mixture. The concentration of a substance in the dialysate was established by comparison with a standard concentration. After the initial 60 min, dialysate samples were collected continuously every 20 min over a total period of 180 min. The concentrations of S-HIAA, DOPAC, and HVA in the dialysates were high, but those of 5-HT and DA were barely above the detection limit of the assay system (Fig. 1B). Figure 2 shows the time courses of the basal levels of 5-HT’, .5-HJAA, DA, DOPAC, and HVA in the dialysates. The levels of 5-HIAA, DOPAC, and HVA remained relatively constant throughout the entire microdialysis. There were no statistically significant differences between sequential collections (p > 0.05) within 180 min. However, the
levels of 5-HT and DA declined progressively to values that were close to the detection limits under basal conditions. Table 1 summarizes the average basal levels of all substances. The values for 5-HT and DA are marginally reliable. It is noted that the basal level of 5-HIAA in the dialysates (322.6 -t 14.9 pg, n = 91) greatly exceeded that of 5-HT (1.0 5 0.2 pg, n = 81). Potassium-Induced
Release of 5-HT and DA
Figure 3 shows the time courses of the changes in 5-HT, DA, and their metabolites induced by administration of a high concentration of potassium. When saline containing 100 mM KC1 was perfused through the probe, the levels of 5-HT and DA in the dialysates increased remarkably, although their basal levels were very low. The 5-HT value rose from 0.8 -C 0.5 to 5.0 + 1.0 pg/sample 0, < 0.01) and DA value increased from 0.5 ? 0.3 to 3.4 -C 1.1 pg/sample (p < 0.05). After the high potassium saline was replaced by normal physiological saline, the levels of both substances subsided to their basal values. Induction of 5-HT and DA release by high potassium saline was accompanied by significant decreases in their metabolites. The level of the 5-HT metabolite, 5-HIAA, was reduced to 66.4% of the pretreatment level during the potassium administration @ < 0.01). The levels of DA metabolites, DOPAC and HVA, were
SEROTONIN
AND
DOPAMINE
IN
SPINAL
629
CORD
5-HIAA
A-beta
A-delta
%
L L
L
I
I
I
L
-
80
-40
lime
120
in min
80 lime
120
in min
FIG. 4. Effects on the extracellular concentration of 5-HIAA of selective stimulation of A-beta fibers (left) and A-delta fibers (right). Parameters of the stimulation are described in the text. Examples of evoked action potentials recorded from the common peroneal nerve arc shown in insets; black arrows: stimulus artifacts. Zero time on abscissas: start of stimulation. Each column with bar: mean 2 SE of changes in 17 rats (left) and 8 rats (right) during the stimulation (dotted columns), and before and after stimulation (open columns), compared to controls. **p < 0.01 vs. the controls.
reduced to 79.6% and 47.5%, respectively, by the high potassium saline (p < 0.05). Effects of Peripheral Nerve Stimulation on the Levels of 5-HT and DA Metabolites
When basal levels of S-HIAA, DOPAC, and HVA were stable for more than 1 h, large myelinated (A-beta) or small myelinated (A-delta) fibers in the hindlimb were selectively stimulated. The insets in Fig. 4 show the compound action potentials evoked by the stimuli. The mean threshold intensities were 219 PA for exciting the large myelinated and 413 PA for the small myelinated afferent fibers. The conduction velocities of large myelinated fibers were approximately 54 m/s, and those of small myelinated fibers were approximately 12 m/s. The S-HT metabolite, S-HIAA, in the dialysates was increased by the stimulation of either A-beta or A-delta afferents (Fig. 4). During the A-beta fiber stimulation (40 min), the increment in 5HIAA level appeared mostly in the initial 20 min. The average value of three consecutive measurements immediately prior to the stimulation was taken as the control (100%). The 5-H&.4 in the dialysate then reached 137.8 2 5.7% of the control during the initial stimulation period (p < 0.01). In the next 20 min period, the level of 5-HLAA diminished to about the control level. When the A-delta fiber was stimulated, the 5-HIAA level increased to 120.5 ? 5.8% of the control during the first 20 min of stimulation (p < 0.01). The level of 5-HIAA tended to remain high for more than 20 min. There were no statistically significant differences between these higher levels and the control level @ > 0.05), because of the high individual variation among the rats.
Figure 5 shows the time courses of changes of DA metabolites, DOPAC and HVA, in response to selective stimulation of A-beta or A-delta afferents. When A-beta fibers were stimulated (40 min), the levels of DOPAC increased to 155.0 ? 8.4% of the control and the levels of HVA increased to 142.6 2 6.1% of the control in the first 20 min of stimulation. When the A-delta fiber stimulation was applied, DOPAC and HVA levels increased to 120.3 ? 5.3% and 124.1 + 8.4% of the controls, respectively, in the initial 20 min of stimulation. No change in 5-I-R nor DA could be detected during either A-beta or A-delta fiber stimulation. DISCUSSION The present study used in vivo microdialysis to examine 5HT and DA activities in the spinal cord in response to selective stimulation of large or small myelinated afferent fibers. In previous studies using microdialysis in cerebral structures (2,2527), the dialysates driven from the tissue reflected the extracellular concentrations of substances released from nerve terminals and their metabolites around the microdialysis probe. In the present study, dialysates obtained sequentially from the rat spinal cord for a long period, over 3 h, showed low basal levels of 5-HT and DA, and relatively high and constant basal levels of their metabolites, 5-HIAA, DOPAC, and HVA. When a membrane depolarizing agent, KCl, was applied locally through the probe, extracellular 5-m and DA greatly increased. The results confirm innervation of regions around the probe insertion site by nerve terminals that contain 5-HT or DA. The low basal
630
MEN AND MATSUI
A-beta
,
A-delta
DOPAC
1
I
I
% 150
A-beta I-
-40
0
WA
A-delta
I
40
I
80
120
Time in min
-40
0 Time
1
40
a0
120
in min
FIG. 5. Effects of selective stimulation of A-beta (left) and A-delta (right) fibers on the extracellular concentrations of DOPAC (upper) and HVA (lower). Parameters of the stimulation are described in the text. Zero time: start of stimulation. Each column with bar: mean + SE of changes in 19 rats (left) and 9 rats (right) during stimulation (dotted columns), and before and after the stimulation (open columns), compared to control values.
**p < 0.01; *p < 0.05 vs. the controls.
levels of 5-HT and DA can be explained by the small amounts released from the nerve terminals, and their rapid metabolism immediately after the release and during the period of dialysate sampling. According to this explanation, any change in metabolite concentration could indicate 5-HT and DA release. However, it is noted that the concentration of 5-HIAA in the dialysates is far different from that of 5-HT. This might suggest another possible source of 5-HIAA, possibly arising directly from the nerve terminals. Studies of the mechanism of 5-HIAA release suggested that it depends on membrane potential of nerve terminals, which reduces 5-HIAA release by factors that induce membrane depolarization (24). This is consistent with our finding that 5-H&4 in dialysates was decreased by administration of high potassium. A similar phenomenon was also observed in the present study in which extracellular DOPAC and HVA were reduced by high potassium administration. The results led us to consider that the extracellular 5-HIAA, and probably DOPAC and HVA, include the results of 5-I-U and DA metabolism plus their release directly from the nerve terminals. When membrane depolarization was evoked by pharmacological or electrophysiological manipulations, 5-HT or DA was released from the nerve terminals and then turned to 5-HIAA, or DOPAC and HVA, respectively, under the action of monoamine oxidase in the extracellular space. Direct release of metabolites was suppressed, however, due to the membrane depolarization. Thus, it is possible
that either increase or decrease in metabolites may appear, depending on the membrane depolarization of the nerve terminal, whereas increase in the metabolites, 5-HIAA, DOPAC or HVA will reflect the release of 5-HT and DA from the nerve terminals. In the present study, it was found that selective stimulation of large or small myelinated afferent fibers (A-beta or A-delta) in the same conditions that suppressed nociceptive reflexes (20,30) and nociceptive responses in the dorsal horn neurons (31), increased 5-HIAA, DOPAC, and HVA in spinal dialysates. Based on the consideration that increment of the metabolites reflects increment of 5-HT or DA, it is suggested that the inhibition produced by peripheral nerve stimulation could be associated with activation of the endogenous 5-I-IT and DA systems. It is generally accepted that the endogenous 5-HT system, with cell bodies in the brain stem, projects to the spinal dorsal horn and is inhibitory at the spinal cord level. Direct stimulation of the descending 5-HT system, for instance, serotonin-rich raphe nuclei of the medulla, particularly the NRM, produced significant antinociceptive effects on nociceptive transmission at the spinal level (12,13,19). In addition, stimulation of the periaqueductal gray (PAG) that projects directly or indirectly to the NRM also elicited profound analgesia (3,33). However, studies of the activation of the descending 5-HT system by peripheral nerve stimulation are limited. Activation of small nerve fibers increased the concentration of 5-HT in the CSF of cat suggesting its release
631
SEROTONIN AND DOPAMINE IN SPINAL CORD
from the descending S-HT neurons, because evocation of release was abolished in spinalized animals (3236). Effects of tail stimulation on S-I-IT synthesis within the dorsal spinal cord suggested that peripheral nociceptive messages increased activity in some bulbo-spinal S-HT pathways (34). Because it is reported that NRM neurons respond to peripheral nerve stimulation (1,14,29), it is reasonable to consider that somatosensory inputs may trigger a complex loop that carries information from the spinal dorsal horn to the supraspinal level, then activates the descending 5-HT system directly or indirectly, and finally produces inhibitory effects at the spinal cord level. The present study, with direct data from the spinal cord, supports the conclusion that somatic information from small myelinated (A-delta) fibers activates the descending 5-HT system. In addition, it was demonstrated that inputs from large myelinated (A-beta) fibers also influence the intrinsic S-HT system. However, the different time courses of the A-beta and the A-delta fiber stimulation indicates different mechanisms or pathways in the CNS. Another interesting finding in the present study was that stimulation of A-beta or A-delta fibers increased DA metabolite concentrations in the spinal dialysates. Because it is clearly established that DA in the spinal cord is not only a precursor of NA, but also serves as an independent transmitter in the DA system (7,8), alternation of endogenous DA or its metabolites, DOPAC and HVA, may reflect activity of the DA system as well as that of the NA system. When combined with evidence that the same periphery nerve st~ulation produces no change in NA (23), the present results suggest that the endogenous DA system rather than the NA system was activated by peripheral nerve stimulation. The endogenous DA system innervating the spinal cord has received much attention in recent years. It has been demonstrated
by anatomical studies that DA neurons in the All cell group of the diencephalon give rise to descending DA projections to the rat spinal dorsal horn (21,28). Evidence that intrathecal injection of DA and the DA agonist, apomorphine, produced antinociceptive effects on thermally and chemically evoked noxious xesponses (18) suggests that spinal DA receptors are inhibitory to noxious inputs to the spinal cord. Furthermore, electrical stimulation in the AI 1 cell region in the diencephalon selectively sup pressed nociceptive responses of spinai m~tire~ptive neurons, and this was rapidly reversed by iontophoretic application of sulpiride, a D2 DA receptor antagonist, in the spinal dorsal horn (10). Such results suggest that the descending DA projection, as does the descending 5-HT system, inhibits the transmission of nociceptive info~ation in the spinal cord. Our fmding that peripheral nerve stimulation increased DOPAC and HVA in dialysates supports the hypothesis that spinal DA innervation participates in the processing of sensory information, and may suggest that inhibitory effects induced by selective stimulation of A-beta or A-delta fibers are related directly or indirectly to spinal DA activity. In summary, the present study suggests that the selective stimulation of large (A-beta) or small (A-delta) myelinated afferent fibers may activate the descending S-FIT and DA systems via supraspinal structures, and releases 5-HT and DA from the nerve terminals in the spinal cord. These neurotransmitters may be involved in inhibition of the processing of sensory transmission in the spinal cord.
1. Anderson, S. D.; Bar&aunt, A. I.; Fields, H. L. Response of medullary raphe neurons to peripheral stimulation and to systemic opiates. Brain Res. 123:363-368; 1977. 2. Auerbach, S. B.; Minxenberg, M. J.; Wilkinson, L. 0. Extracellular serotonin and 5-hyd~xy~doleacetic acid in hypothalamus of the unanes~e~ed rat measured by in vivo dialysis coupled to highperfomnmce liquid c~omato~aphy with electrochemical detection: Dialysate serotonin reflects neuronal release. Brain Res. 499:281290; 1989. 3. Behbehani, M. M.; Fields, H. L. Evidence that an excitatory connection between the periaqueductal gray and nucleus raphe magnus mediates stimulation produced analgesia. Brain Res. 17085-93; 1979. 4. Besson, J. M.; Chaouch, A. Peripheral and spinal mechanisms of nociception. Physiol. Rev. 67(1):67-185; 1987. 5. Bj&klund, A.; Skagerberg, G. Evidence for a major spinal cord projection from the diencephalic All dopamine cell group in the rat using transmitter-specitic fluorescent retrograde tracing. Brain Res. 177:170-175; 1979. 6. Bowker, R. M.; Westlund, K, N.; Coulter, .I. D. Origins of serotonergic projections to the spinal cord in rat: An imm~~yt~hemicalretrograde transport study. Brain Res. 226187-199; 1981. 7. Commissiong, J. W.; Galli, C. L.; Neff, N. H. Differentiation of dopaminergic and noradrenergic neurons in rat spinal cord. J. Neurochem. 30:1095-1099, 1978. 8. Commissiong, J. W.; Neff, N. H. Current status of dopamine in the mammalian spinalcord. Biochem. Pharmacol. 28:1569-1573; 1979. 9. Fields, H. L. Brainstem control of spinal pain-transmission neurons. Annu. Rev. Physiol. 40:217-248; 1978. 10. Fleetwood-Walker, S. M.; Hope, P. J.; Mitchell, R. Antinociceptive actions of descending dopaminergic tracts on cat and rat dorsal horn somatosensory neurones. J. Physiol. 399:335-348; 1988.
11. Gebhart, G. F. Modulatory effects of descending systems on spinal dorsal horn neurons. In: Yaksh, T. L., ed. Spinal afferent processing. New York: Plenum Press; 1986391-416. 12. Gerhart, K. D.; Wilcox, T. K.; Chung, I. M.; Willis, W. D. Inhibition of nociceptive and nonnociceptive responses of primate spinothalamic cells by stim~ation in medial brain stem. J. Neu~physiol. 45121-136; 1981. 13. Giesler, 0. J.; Gerhart, K. D.; Yexierski, R. P.; Wilcox, T. K.; Willis, W. D. Postsynaptic inhibition of primate spinothalamic neurons by stimulation in nucleus raphe maguus. Brain Res. 204:&t-188; 1981. 14. Guilbaud, G.; Peschanski, M.; Gautron, M.; Binder, D. Responses of neurons of the nucleus raphe magrms to noxious stimuli. Neurosci. I.&t. 12149-154; 1980. 15. Hammond, D. L. Control systems for nociceptive afferent processing In: Yaksh, T. L., ed. Spinal afferent processing. New York: Plenum Press; 1986:363-390. 16. Han, J. S.; Tang, J.; Ren, M. F.; Zhou, Z. F.; Fan, S. G.; Qiu, X. C. Central neurotransmitters and acupuncture analgesia. Am. J. Chin. Med. 8:331-348; 1980. 17. Hokfeit, T.; Philli~n, 0, Goldstein, M. Evidence for a dopaminergic pathway in the rat descending from the All cell group to the spinal cord. Acta Physiol. Stand. 107:393-395; 1979. 18. Jensen, T. S.; Yaksh, T. L. Effects of an intrathecal dopamine agonist, apomorpbine, on thermal and chemical evoked noxious responses in rats. Brain Res. 296285-293; 1984. 19. Jones, S. L.; Gebhart, G. F. Inhibition of spinal nociceptive transmission from the midbrain, pons and medulla in the rat: Activation of descending inhibition by morphine, glutamate and electrical stimulation. Brain Res. 46@281-296; 1988. 20. Kokubu, T.; Ishii, N.; Tsuruoka, M. Suppression of the tail flick reflex by selective stimulation of A6 afferent nerve fibers. J. Showa Univ. Dent. Sot. 7:37-&t; 1987.
ACKNOWLEDGEMENTS
We thank Professor T. Sato and Dr. A. Matsui for their help and encouragement during this study.
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