Pain, 49 (1992) 405-413 0 1992 Elsevier Science

405 Publishers

B.V. All rights reserved

0304-3959/92/$05.00

PAIN 02044

Differential influence of naloxone on the responses of nociceptive neurons in the superficial versus the deeper dorsal horn of the medulla in the rat S.S. Mokha Department of Physiology, Meharry Medical College, NashL+lle, TN 37208 (USA), and Dioision of Neurophysiogy and Neuropharmacology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA (UK) (Received

27 June 1991, revision

received

23 October

1991, accepted

9 December

1991)

Naloxone (200 pg/kg, i.v.) reduced the noxious thermal stimuli-evoked responses of 16/25 Summary nociceptive neurons in the superficial laminae whereas it enhanced the responses of 6/10 nociceptive neurons in the deeper dorsal horn. However, a different picture emerged when selectivity of neuronal responsivity (nocireceptive or multireceptive) was considered. In the superficial dorsal horn, naloxone reduced the responses of the majority of (15/18) selectively nocireceptive neurons. The reduction in responses became apparent within 60 set following naloxone administration and returned to control level within 48 min. In contrast, the responses of the majority of multireceptive neurons in the superficial (6/7), or the deeper (6/10) dorsal horn, were enhanced. The excitatory action in the superficial dorsal horn persisted for only 6-15 min, whereas it persisted for 40-70 min in the deeper dorsal horn. The firing of the majority of cold-receptive neurons (6/8) in the superficial dorsal horn was not altered. These effects were stereoselective since ( + )-naloxone, the inactive isomer of naloxone, did not affect the responses of 14/16 nociceptive neurons. It is concluded that naloxone differentially, and selectively, affects the firing of nociceptive neurons in the superficial versus the deeper dorsal horn, and the firing of selectively nocireceptive versus multireceptive neurons. The relevance of these findings to the behavioral effects of naloxone, hyperalgesia and analgesia, is discussed. Key words: Naloxone; Dorsal Horn; Trigeminal;

Nociception;

Introduction

Naloxone, an opiate antagonist, has been reported to alter the perception of pain in humans as well nociceptive thresholds in animal studies. Thus, naloxone has been reported to decrease the intensity of postoperative pain, i.e., analgesia (Levine and Gordon 19861, to increase the intensity of pain, i.e., hyperalgesia (Levine et al. 1978; Gracely et al. 1983), or to produce a biphasic effect, analgesia at low doses and hyperalgesia at high doses (Lasagna 1965; Buchsbaum et al. 1977; Levine et al. 1979). However, naloxone has

Correspondence to: Dr. S.S. Mokha, Meharry Medical College, 1005 D.B. 37208, USA. Tel.: (615) 327-6933/6288.

Department Todd Blvd.,

of Physiology, Nashville TN

Antinociception

also been reported to have no effect on experimental pain in humans (Grevert and Goldstein 1978). Data from animal studies have also been equivocal. Viz, naloxone, administered intravenously, has been reported to produce analgesia (Sewell and Spencer 1976; Rios and Jacob 19831, hyperalgesia (Frederickson et al. 1977; Jacob and Ramabadran 1978; Carmody et al. 19791, or no effect (reviewed in Duggan and North 1984). A biphasic effect has been observed in behavioral studies in arthritic (Kayser and Guilbaud 1981; Kayser et al. 1986) and mononeuropathic rats (Jazat and Guilbaud 1991). Intrathecal administration of naloxone has also been reported to produce a biphasic effect (Woolf 1980). Naloxone reduced behavioral analgesia, generated from the medial brain-stem in some studies, and altered segmental reflexes, evoked by activity in large and small diameter primary afferent

406

fibers in spinalized cats (Duggan and North 1984). Naloxone has also been reported to antagonize the inhibitory action of stimulation in the periacqueductal grey (PAG) and nucleus raphe magnus (NRM) on the digastric reflex and on some nociceptive neurons in laminae IV-V of the medullary dorsal horn (Sessle et al. 19811. Taken together these studies strongly support the suggestion of the existence of opioid peptide-mediated tonic and phasic control of nociceptive mechanisms. However, in other electrophysiological studies, naloxone failed to alter (Sinclair et al. 19801, or only partially reduced (Duggan and North 19841, the tonic descending inhibition of multireceptive neurons in the deeper dorsal horn and was reported not to alter excitability changes produced in terminals of tooth pulp afferent fibers by stimulation in PAG (Duggan and North 1984). There are, at least, 3 different opioid peptide gene families (pro-opiomelanocortin, pro-enkephalin and pro-dynorphin), and a heterogeneous population of opioid receptors (mu, delta and kappa) (reviewed in Goodman et al. 1980; Akil et al. 1984; Mansour et al. 1988; Romagnano et al. 1989; Zajac et al. 1989) and multiple receptor sub-types (mu,, mu,) (Pasternak et al. 1980; Moskowitz and Goodman 1985; Mansour et al. 1988). Multiple opioid receptors have been implicated in analgesia in animal studies (reviewed in Yaksh et al. 1988). Opioid peptides related to the pro-enkephalin family (enkephalins) (Cruz and Basbaum 1985; Ruda et al. 1986) and pro-dynorphin family (dynorphins) (Cruz and Basbaum 1985; Ruda et al. 1988; Weihi et al. 1989) are present in the dorsal horn, with a heavy concentration of opioid peptides and different opioid receptors in the superficial dorsal horn (Goodman et al. 1980; Akil et al. 1984; Mansour et al. 1988; Romagnano et al. 1989; Zajac et al. 1989). Therefore, opioid peptides acting on different opioid receptors may effectively modulate, tonically and/or phasically, various somatosensory inputs entering the dorsal horn. In previous electrophysiological investigations on deeper dorsal horn neurons, naloxone was reported to enhance responses evoked by electrical stimulation of C-fibers (Rivot et al. 1979; Fitzgerald and Woolf 1980) or by radiant heat (Henry 1979). The increase in the responsivity of dorsal horn neurons displayed a diurnal variation, naloxone being most effective during the day and early hours of the evening (Henry 1981). This is consistent with behavioral observations demonstrating that hyperalgesia produced by naloxone is more prominent in the late afternoon (Frederickson et al. 1977). Previous investigations, with the exception of a study by Fitzgerald and Woolf (19801, have focused mainly on neurons in the deeper dorsal horn. Naloxone was reported to influence differentially the firing of nociceptive neurons in the superficial versus the deeper laminae of the dorsal horn of the spinal cord of the rat

(Fitzgerald and Woolf 1980). The effects of naloxone were, however, investigated only on the responses evoked by electrical stimulation of primary afferent fibers. No previous reports examined the effects of naloxone on the responses of neurons in the superficial versus the deeper laminae of the dorsal horn of the medulla (trigeminal nucleus caudalis) in the rat. The present investigation was, therefore, designed to investigate the effects of naloxone on the noxious stimulievoked responses of physiologically characterized neurons in the superficial versus the deeper dorsal horn of the medulla. The effects of naloxone were also investigated on the responses of cold-receptive neurons in the superficial dorsal horn since very little is known about the modulation of these neurons. A preliminary report of some of this work has been published as an abstract (Mokha 1986).

Methods Experiments were performed on 42 male Sprague-Dawley rats weighing ZOO-400 g. Anesthesia was induced by 3% halothane in a mixture of 33% Oz /66% NzO. Following tracheal cannulation, anesthesia was maintained by l-1.5% halothane through the tracheal cannula. Additionally, cannulations were performed on carotid artery (for monitoring blood pressure) and jugular vein (for intravenous administration of naloxone). Anesthesia was monitored by observing the pupillary diameter and by detecting any sudden changes in arterial blood pressure. Body temperature was monitored continuously and maintained constant at 38°C by means of a feed back control unit (Harvard Apparatus Inc.). A small metallic brass plate was fiied to the skull and the head was ventroflexed in order to improve the stability for recording from the superficial and deeper dorsal horn of the medulla. The medulla was exposed by clearing the overlying musculature, cutting through and deflecting the dura and arachnoid mater. The exposed surface of the medulla was covered with agar (3-4% Agar in normal saline at about 40°C) in order to further improve the stability for recording from neurons in the superficial dorsal horn of the medulla. Extracellular, single-unit recordings were made with glass-coated, platinum-plated, tungsten microelectrodes. The electrode was advanced with a stepping motor driven micromanipulator. To limit the recordings from neurons in the superficial dorsal horn of the medulla, electrode penetrations were made caudal to obex and did not exceed 200 pm from the pial surface. Although recording sites were not reconstructed histologically, it is the experience of this and other laboratories that the depth of penetration below the surface of the spinal cord (Hylden et al. 1991) or medulla (Dickenson et al. 1979; Hubbard and Hellon 1980; Mokha et al. 1987) is a reliable guide of actual depth. For limiting recordings from neurons in the deeper dorsal horn (deeper magnocellular zone), penetrations did not exceed 500 pm below the pial surface. Action potentials were recorded and amplified using conventional means and were led to a spike processor which enabled construction of instantaneous frequency histograms. Binary coded decimal output from the spike processor was fed into the microcomputer to allow further analysis (on-line and off-line) of the data. Somatosensory stimuli (brush, pinch, radiant heat, etc.) were used to characterize neurons physiologically and map their receptive fields. The face was carefully shaved for adequate stimulation and mapping of receptive fields by thermal stimuli (Dickenson et al. 1979). Radiant heat from a quartz halogen lamp and gentle pinch were em-

407 ployed as neuronal search stimuli. Selectively nocireceptive (excited exclusively by noxious stimuli) and multireceptive neurons (excited by noxious as well as non-noxious stimuli) were activated by noxious thermal stimuli similar to those used in our previous studies (Mokha et al. 1987). Cold receptive neurons were initially identified by the abrupt cessation of their background activity by gentle warming of the face with a projector lamp. The firing rate of cold-receptive neurons increases as a function of decreasing temperature of the receptive field. The temperature of the receptive field of cold-receptive neurons was manipulated by the Peltier device as described previously (Davies et al. 1983; Mokha et al. 1987). Cutaneous receptive fields of cold-receptive and nociceptive neurons were small and located on the upper lip or in the area around the whisker pad. A Peltier thermal stimulator, described in detail in previous studies (Davies et al. 1983; Mokha et al. 19871,was used for applying noxious thermal stimuli and for activating cold-receptive neurons. The temperature of the stimulator-skin interface was measured by a thermocouple cemented in a groove on a shallow copper dome which is soldered on one corner of the Peltier semiconductor. Application of thermal stimuli for brief periods (lo-15 see) at a minimum inter-stimulus interval of 3 min and the slow rise of heat pulses ( < 2”C/sec) starting from a baseline of 40°C (or less) prevented the formation of edema. Although the noxious thermal stimuli were applied for lo-l.5 set, the peak temperature of 55°C is maintained for a small fraction of this time (Fig. 5). The reliable reproducibility of thermal stimuli is depicted in Fig. 5. The number of stimulus trials for each cell rarely exceeded 20, and only 1 stimulus was applied per trial at an inter-stimulus interval of minimum 3 min. Drugs (naloxone, (+ )-naloxone) were freshly dissolved in normai saline and administered intravenously (200 pg/kg). In a few experiments, a gradually increasing dose or a large dose (1 mg/kg) of naloxone was administered. Effects of naloxone were tested on the thermal stimuli-evoked responses (total number of action potentials minus the background activity if any) of nociceptive neurons in the superficial versus the deeper dorsal horn and on the activity of cold receptive neurons in the superficial dorsal horn. Several factors were taken into account for classifying a neuron excited or inhibited following naloxone administration. Some of the factors taken into account were a small variability in the control responses, a marked increase or a decrease in responses well above or below the control responses, and the return of responses to pre-naioxone control levels after a length of time as illustrated in Figs. 1 and 6. The effects of naloxone were generally tested only on the responses of 1 neuron per animal. However, when the effects were tested on more than 1 neuron per animal, the second dose of naloxone was administered at least 2-3 h after the first dose.

Results Responses of 59 neurons were recorded in the superficial and deeper dorsal horn of the medulla. Effects of naloxone were tested on the responses of 43/59 neurons. In order to asses that the actions of naloxone were stereoselective, the effects of (+ )-naloxone, the inactive isomer of naloxone, were examined on the responses of M/59 neurons in the superficial as well as deeper dorsal horn of the medulla. Effects of naloxone on the responses of nociceptive neurons in the superjkial dorsal horn

The effects of naloxone were tested on the thermal stimuli-evoked responses of 25 selectively nocireceptive

Minutes

Fig. 1. a: the effect of increasing doses of naloxone on the thermal stimuli-evoked responses of a selectively nocireceptive neuron in the superficial dorsal horn (40 pm below the pial surface). The thermal stimulus, starting from a baseline of 35°C was applied for 10 sec. b: the lack of effect of (+I-naloxone, the inactive stereoisomer of naloxone, on the thermal stimuli-evoked responses of another selectively nocireceptive neuron in the superficial dorsal horn. The thermal stimulus starting from a baseline of 40.8”C and peaking at 55°C was applied to the cutaneous receptive field for 10 sec.

and multireceptive neurons in the superficial dorsal horn. Naloxone reduced the responses of 16/25 neurons, enhanced the responses of 8/25 and produced a biphasic effect in l/25 neurons. (+ l-Naloxone did not alter the activity of 7/8 nociceptive neurons. ~tereose~ctiue eflects of na~o~one on the responses of selectively nocireceptiue neurons

The responses of 18 selectively nocireceptive neurons were recorded in the superficial dorsal horn of the medulla. Quantified noxious thermal stimuli were applied to the cutaneous receptive fields to activate these neurons. Naloxone, administered intravenously, reduced the thermal stimuli-evoked responses of the majority (15/l@ of these neurons (Figs. la and 2). The illustration (Fig. la> is from a neuron recorded 40 pm below the medullary surface and a noxious thermal stimulus (.55”C), rising from a baseline of 35°C was applied for 10 set to the cutaneous receptive field, located in the skin area around the whisker pad. The slow rise of thermal stimuli and starting from a baseline of less than 40°C prevented the formation of edema or sensitization of responses since the stimulus

40x

at the peak temperature stays only for a fraction of the total stimuius duration. Thermal stimuli-evoked reliable responses over the entire recording period. AIthough not shown in the examples (Figs. la, 21, the latency of reduction in responses following naloxonc administration (0.2 mg/kgl was less than 60 sec. Kesponses returned to control levels within 48 min in most of the neurons (Fig. 2). However, the duration of inhibition lasted longer than 48 min in some neurons. The peak inhibitory action developed within 15 min. In contrast to the above, an enhancement in the thermal stimuli-evoked responses (2/18) or a biphasic effect (1,031, reduction followed by an enhancement. was seen in the remaining neurons. The excitatory action occurred within 60 set, peaked within 3 min and persisted for 15 min. The inhibitory action of naloxone was stereoselective since (+ l-naloxone did not affect the thermal stimuli-evoked responses of 4/4 selectively nocireceptive neurons recorded in the superficial dorsal horn (Fig. lb). Fig. 3. a: excitatory action of naloxone f

Stereoselectitse ejrects of naloxone on the responses of multireceptil>e neurons

Thermal stimuli-evoked responses of 7 multireceptive neurons, activated by noxious as well as non-noxious stimuli, were recorded in the superficial dorsal horn. Although the responses of the majority (6/7) of these neurons were enhanced (Fig. 3a), reductions in the responses were also observed in one case. The excitatory action occurred within 40 set, peaked within 3 min and persisted for 6-15 min. The effects of naloxone were not investigated on the non-noxious stimuli-evoked responses in these neurons. Effects of

the thermal

stimuli-evoked

the superficial stimuli

tireceptive

mg/kg.

i.v.1 is illustrated

on

neuron

in

dorsal horn (30 wrn below the pial surface). Thermal

(36.S55°C)

f + )-naloxone

i

responses of a multireceptive

were

applied

on the thermal

neuron in the superficial

pial surface). Thermal

for

15 sec. h: lack of effect

stimuli-evoked

activity of another

of

mui-

dorsal horn (120 pm below the

stimuli (40-WCf,

were applied for IO sec.

naloxone were stereoselective since (+ 1 naloxone did not affect the responses of 3/4 multireceptive neurons (Fig. 3b). Stereoselectirle ejfects of nuloxone on the responses oj cold-receptirle neurons in the superficial darsal horn

The effects of naloxone were tested on the activity of 8 cold-receptive neurons in the superficial dorsal horn. After characterizing a cold-receptive neuron, the receptive field temperature was held constant during the entire period of testing with the Peltier device. The effects of naloxone were examined once the neuronal discharge had stabilized at a particular temperature. Naloxone did not affect the activity of 6/8 neurons (Fig. 4a1, reduced the activity in l/8 and enhanced the activity in l/8 neurons. ( + l-Naloxone did not alter the responses of 3/3 cold-receptive neurons in the superficial dorsal horn of he medulla (Fig. 4b). ~tereose~ect~t~eefjects of rIul~~xoneon the response.~ qf multireceptire neurons in the deeper dorsul horn Fig. 2. The figure illustrates of naloxone

(200

pg/ky,

sponses of selectively 170 ,um below initially repeated

the time course of the inhibitory i.v.) on the

nocireceptive

the pial surface).

thermal

neuron Thermal

stimuli-evoked

in the superficial stimuli

(3655°C)

at 3 min interval which were gradually prevent damage to the skin.

action re-

dorsal were

increased to

The effects of naloxone were tested on the thermal stimuli-evoked responses of 10 multireceptive neurons in the deeper dorsal horn of the medulla. The responses of the majority (6/10) of these neurons were enhanced (Figs. 5 and 6aL However, naloxone reduced the responses of 2/10 neurons; produced a biphasic

409

Fig. 4. a: lack of effect of naloxone (200 pg/kg, Lv.) on the activity of a cold-receptive neuron recorded in the superficial dorsal horn (50 pm below the pial surface). The receptive field temperature was held constant at 169°C. Each point represents the number of action ~tentials counted for 30 set at this temperature. b: lack of effect of (+ )-naloxone on the activity of another cold-receptive neuron in the superficial dorsal horn (70 grn below the pial surface). Receptive field temperature was held constant at 30°C. Each point represents number of action potentials over a period of 30 sec.

effect, enhancement followed by reduction, in l/10 neurons; and, did not alter the responses of l/10 neurons. The excitatory effect of naloxone is illustrated in Figs. 5 and 6a.

0-J

Fig. 6. a: the time course of the excitatory action of naloxone (200 pug/kg, i.v.) on the thermal stimuli-evoked responses of a multireceptive neuron in the deeper dorsal horn (370 pm below the pial surface). Thermal stimuli (36-55”Ck were applied for 15 sec. b: lack of effect of ( + rnaloxone on the thermal stimuli-evoked responses of another multireceptive neuron in the deeper dorsal horn (500 /*rn below the pial surface).

The reliable reproducibility of thermal stimuli is depicted in Fig. 5. The excitatory action occurred within 60 set, peaked within 15-21 min and persisted for 40-70 min. These effects were stereoselective since f + )-naloxone did not alter the responses of 3/5 multireceptive neurons (Fig. 6b).

Discussion

’

30s.

’

Fig. 5. Excitatory effect of naloxone (200 pg/kg,i.v.) on the thermal stimuli-evoked responses of a multireceptive neuron in the deeper dorsal horn (390 pm below the pial surface). Thermal stimuli (36.3SYC), were applied for 15 sec. Frequency histograms generated by the neuronal activity are represented in the top panel. The bottom panels show the reproducible thermal stimuli applied to the cutaneous receptive field.

The present investigation demonstrated a predominantly differential influence of naloxone on seIectively nocireceptive neurons in the superficial dorsal horn versus multireceptive neurons in the dorsal horn of the trigeminal nucleus caudalis. The responses of the majority of nociceptive neurons (16/25) in the superficial dorsal horn were reduced by naloxone, whereas the responses of multireceptive neurons in the deeper dorsal horn were enhanced. The differential effect on the firing of nociceptive neurons in the superficial versus the deeper dorsal horn corroborates and extends previ-

410

ous findings in the dorsal horn of the spinal cord (Fitzgerald and Woolf 1980). When examined in greater detail, a differential effect dependent upon stimulus selectivity in the superficial dorsal horn was revealed. The responses of selectively nocireceptive neurons were predominantly reduced, whereas the responses of multireceptive neurons were mainly enhanced. There appear to be some differences in our results and those obtained in a previous report. These small differences, however, may simply reflect different populations of neurons since the earlier report (Fitzgerald and Woolf 1980) concentrated on multireceptive neurons in the superficial dorsal horn of the spinal cord, whereas the present investigation focused on selectively nocireceptive neurons in the superficial dorsal horn of the medulla. Additionally, a decerebrate spinalized preparation was employed by Fitzgerald and Woolf (1980) whereas we used an intact preparation. Therefore, the possibility of supramedullary structures contributing to our results cannot be ruled out. The excitatory action of naloxone in the deeper dorsal horn, however, has previously been reported on multireceptive neurons in both spinalized (Henry 1979; Fitzgerald and Woolf 1980) and intact (Rivot et al. 1979) preparations. The excitatory actions of naloxone are suggested to be mediated at the level of the dorsal horn of the medulla. The methods used here do not provide exact information on the laminar location of neurons but do differentiate between recordings made in the superficial versus the deeper dorsal horn. Selectively nocireceptive neurons recorded in the superficial dorsal horn had small receptive fields and presumably, correspond to nociceptive specific (NS) neurons recorded in the marginal zone and the outer zone of the substantia gelatinosa by Yokota (1985). Selectively nocireceptive neurons were recorded in the close vicinity of cold-receptive neurons which are known to be present in the marginal zone (Dickenson et al. 1979; Dawson et al. 1983). The possibility that recordings in the superficial dorsal horn may have been made from dendrites of neurons located in the deeper dorsal horn can be ruled out because the spike shapes were generally triphasic which indicate that recordings were made closer to the cell bodies of neurons in the superficial dorsal horn (Millar and Armstrong-James 19821. Although there are reports suggesting the presence of selectively nocireceptive neurons in the deeper dorsal horn, these are reported to be concentrated in the superficial dorsal horn (Per1 1985). Multireceptive neurons recorded in the deeper dorsal horn also had small receptive fields and were possibly located in the magnocellular zone (lamina IV) and lamina V (Gobel et al. 1977). Some of these neurons may correspond to wide dynamic range (WDR) neurons recorded in lamina V in the cat (Yokota 1985). One can say with certainty that neurons recorded in the deeper dorsal horn were not

located in the subnucleus reticularis ventralis, since neurons located in this nucleus have rather large receptive fields (Yokota 1985). The differential influence of naloxone on neurons in the medullary dorsal horn appears to be mediated by its action on opiate receptors since (+ l-naloxone, the inactive isomer of naloxone, did not modify the responses. Compared to naloxone, ( + )-naloxone is 10,000 times less potent in opiate receptor binding studies (Iijima et al. 1978) and does not antagonize the actions of opioid peptides on neurons in the brain (Duggan and North 1984). While there are multiple opioid receptors (mu, delta and kappa) (Goodman et al. 1980; Akil et al. 1984; Mansour et al. 1988; Romagnano et al. 1989; Zajac et al. 1989) and receptor sub-types (mu,, mu,) (Pasternak et al. 1980; Moskowitz and Goodman 1985) in the dorsal horn, naloxone has the highest affinity at the mu receptor followed by its affinity at the delta and kappa receptors (Akil et al. 1984; Duggan and North 1984). Although low doses of naloxone (comparable to those used in the present study) have previously been found to antagonize the actions of selective agonists at mu receptors (Duggan et al. 1980; Oliveras et al. 1986; Mokha 1987, 1988) similar doses are inadequate in blocking the effects of selective agonists at delta- and kappa-opioid receptors in the medullary dorsal horn (Mokha 1988-90). The actions of selective agonists at kappa receptors were recently reported to be unaffected by doses of naloxone 5-50 times higher than used in the present investigation (Hylden et al. 1991). We suggest that the observed effects of naloxone are mediated, primarily, by blocking the action of endogenously released opioid peptides at mu receptors. Endogenous opioid peptides are present in abundance in the dorsal horn (Ruda et al. 1986). Basal levels of opioid peptides have been detected in the spinal cord (Yaksh and Elde 1981). Levels are increased by activation of A-delta and C-primary afferent fibers (Yaksh 1981; Hamon et al. 1988). Stress induced by surgery may cause the release of opioid peptides. Naloxone does not affect experimental pain in humans (Grevert and Goldstein 1978) whereas it does modulate post-surgical pain (Lasagna 1965; Levine et al. 1979; Gracely et al. 1983) and stress-induced analgesia in animals (Terman et al. 1984). However, the hyperalgesic action of naloxone has been demonstrated in animals with little or no surgical trauma (Frederickson et al. 1977; Jacob and Ramabadran 1978; Carmody et al. 1979) and it displays a diurnal rhythm (Frederickson et al. 1977). The present experiments involved very little surgery which was outside the area of the receptive fields of neurons. Based on our results, we predict that morphine, a mu-receptor agonist, would produce excitation in the superficial dorsal horn and inhibition in the deeper dorsal horn. Morphine does, indeed, excite some selec-

411

tively nocireceptive neurons in the superficial dorsal horn and inhibit all multireceptive neurons in the deeper dorsal horn of the medulla (Mokha 1987). It has previously been reported that enkephalins exert a selectively antinociceptive effect on multireceptive neurons in the medullary dorsal horn (Anderson et al. 1978). The actions of naloxone could be exerted at several sites; receptors on small diameter primary afferent fibers, on neurons in the dorsal horn, or on supramedullary neurons which, directly or indirectly, contribute to descending modulation of nociceptive mechanisms. Opioid receptors are present on small diameter primary afferent fibers (Fields et al. 1980; Zajac et al. 1989) and opioid analgesics inhibit the release of substance P (Jesse11 and Iversen 1977). Substance P is reported to excite nociceptive neurons in the trigeminal nucleus (Henry et al. 1980). Recent studies have demonstrated dose-dependent dual actions of opioid peptides on dorsal root ganglion cells in culture; low dose produced excitation whereas high dose produced inhibition (Crain and Shen 1990). In contrast, there is strong evidence that fails to support the hypothesis that peripheral opioid receptors are involved in mediating the actions of naloxone. Thus, excitatory action of naloxone on the spontaneous discharge of neurons in the deeper dorsal horn are not prevented by rhizotomy (Henry 1979) and, naloxone, per se, does not affect the activity of peripheral visceral nociceptors (Kumazawa et al. 1989). The time course of the excitatory action on multireceptive neurons in the deeper dorsal horn of the medulla is consistent with previous studies in the dorsal horn of the spinal cord (Henry 1979; Fitzgerald and Woolf 1980) and is comparable to the time course of the hyperalgesic action of naloxone in normal (Woolf 19801, arthritic (Kayser and Guilbaud 1981; Kayser et al. 1986) and mononeuropathic animals (Jazat and Guilbaud 1991). Multireceptive neurons contribute to the trigeminothalamic tract (Price et al. 1976) and have been suggested to play an important role in nociception (reviewed in Dubner 1985; Willis 1985). The excitatory action of naloxone on these neurons could, therefore, represent one of the neuronal mechanisms underlying the hyperalgesic action of naloxone reported in animal (Frederickson et al. 1977; Jacob and Ramabadran 1978; Carmody et al. 1979) and human studies (Levine et al. 1978; Gracely et al. 1983). Our observation that naloxone reduced the responses of selectively nocireceptive neurons in the superficial dorsal horn was unexpected since these neurons are believed to play a significant role in nociception (Dubner 1985; Per1 1985). Selectively nocireceptive neurons are abundantly present in the marginal zone (Per1 1985; Yokota 19851, a region which gives rise to projections to many areas of the brain involved in nociception, antinociception or affective mecha-

nisms of pain. These brains areas include the lateral thalamus (Hylden et al. 1979), the nucleus submedius thalamus (Craig and Burton 1981; Dado et al. 1990), the mesencephalon including the PAG (Swett et al. 1985) and the parabrachial area (Hylden et al. 1989). It is conceivable that selectively nocireceptive neurons may play a role in both nociceptive and antinociceptive mechanisms depending on their projections sites in the brain. Stimulation of the dorsolateral funiculus (DLF) which normally produces inhibition of nociceptive neurons in the deeper dorsal horn, has been reported to excite some projection neurons in the marginal zone (McMahon and Wall 1988). The inhibitory effect of naloxone on these neurons indicates the existence of a tonic excitatory control mechanism mediated by opiates. Opioid peptides can cause facilitation by disinhibition (Duggan and North 1984). Thus, opioid peptides are known to depress GABAergic transmission in the nervous system and can, therefore, produce facilitation due to a decrease in GABA-mediated inhibition. GABA-containing neurons are present in the superficial dorsal horn (Ruda et al. 1986). Alternatively, inhibitory interneurons in those brain areas that descend to the medullary dorsal horn could contribute to opioid peptide-mediated facilitation. Selectively nocireceptive neurons can, therefore, modulate nociceptive inputs in the deeper dorsal horn by their actions in the dorsal horn, or by activating descending pathways. In conclusion, we revealed a differential influence of naloxone dependent on: (a) location of nociceptive neurons (superficial vs. the deeper dorsal horn of the medulla) and (b) on stimulus selectivity (multireceptive vs. selectively nocireceptive neurons). We suggest that the modulation of nociceptive inputs by the tonic release of endogenous opioid peptides in the dorsal horn and/or in supraspinal structures contributes to descending modulation of nociception. The stereoselective effects of naloxone are suggested to be mediated, primarily, by its action at the mu receptors and may explain some of the behavioral effects observed in animal and human studies. Some selectively nocireceptive neurons may also have a role in antinociceptive mechanisms either in the dorsal horn or by activating descending pathways.

Acknowledgements I would like to thank Drs. Arthur Duggan, Jack Clark and Steven Fredman for their critical reading of the manuscript, Drs. R.F. Hellon and T.V.P. Bliss for their support during my stay at Mill Hill and Mr. Eric Archer for providing excellent technical assistance. ( + )-Naloxone was a gift from Dr. Richard L. Haws of the National Institute of Drug Abuse (NIDA). Sup-

ported by grants (RR-03032).

from

NSF (BNS-9109247)

and NIH

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