Effect of systemic morphine on the responses of convergent neurons to noxious heat stimuli applied over graded surface areas.

BACKGROUND: Stimulus intensity is a major determinant of the antinociceptive activity of opiates. This study focused on the influence of the spatial characteristics of nociceptive stimuli, on opiate-induced depressions of nociceptive transmission at the level of the spinal cord. METHODS: Anesthetized rats were prepared to allow extracellular recordings to be made from convergent neurons in the lumbar dorsal horn. The effects of systemic morphine (1 and 10 mg/kg) were compared with those of saline for thermal stimuli of constant intensity, applied to the area of skin surrounding the excitatory receptive field (1.9 cm2) or to a much larger adjacent area (18 cm2). RESULTS: The responses (mean +/- SD) elicited by the 1.9-cm2 stimulus were not modified by 1 mg/kg intravenous morphine, although they were decreased by the 10-mg/kg dose (to 11+/-4% of control values compared with saline; P < 0.05). In contrast, when the 18-cm2 stimulus was applied, 1 mg/kg intravenous morphine produced a paradoxical facilitation of the neuronal responses (159+/-36% of control values; P < 0.05) and 10 mg/kg intravenous morphine resulted in a weaker depression of the responses (to 42+/-24% of control values; P < 0.05) than was observed with the smaller stimulus. CONCLUSIONS: Doses of systemic morphine in the analgesic range for rats had dual effects on nociceptive transmission at the level of the spinal cord, depending on the surface area that was stimulated. Such effects are difficult to explain in terms of accepted pharmacodynamic concepts and may reflect an opioid-induced depression of descending inhibitory influences triggered by spatial summation.

Involvement of the caudal medulla in negative feedback mechanisms triggered by spatial summation of nociceptive inputs.

In the rat, applying noxious heat stimuli to the excitatory receptive fields and simultaneously to adjacent, much larger, areas of the body results in a surface-related reduction in the responses of lumbar dorsal horn convergent neurons. These inhibitory effects induced by spatial summation of nociceptive inputs have been shown to involve a supraspinally mediated negative feedback loop. The aim of the present study was to determine the anatomic level of integration of these controls and hence to ascertain what relationships they might share with other descending controls modulating the transmission of nociceptive signals. The responses of lumbar convergent neurons to noxious stimulation (15-s immersion in a 48 degrees C water bath) applied to increasing areas of the ipsilateral hindlimb were examined in several anesthetized preparations: sham-operated rats, rats with acute transections performed at various levels of the brain stem, and spinal rats. The effects of heterotopic noxious heat stimulation (tail immersion in a 52 degrees C water bath) on the C-fiber responses of these neurons also were analyzed. The electrophysiological properties of dorsal horn convergent neurons, including their responses to increasing stimulus surface areas, were not different in sham-operated animals and in animals the brain stems of which had been transected completely rostral to a plane -2. 8 mm remote from interaural line (200 micron caudal to the caudal end of the rostral ventromedial medulla). In these animals, increasing the stimulated area size from 4.8 to 18 cm2 resulted in a 35-45% reduction in the responses. In contrast, relative to responses elicited by 4.8 cm2 stimuli, responses to 18 cm2 were unchanged or even increased in animals with transections at more caudal level and in spinal animals. Inhibitions of the C-fiber responses elicited by heterotopic noxious heat stimulation were in the 70-80% range during conditioning in sham-operated animals and in animals with rostral brain stem transections. Such effects were reduced significantly (residual inhibitions in the 10-20% range) in animals with transections >500 micron caudal to the caudal end of the rostral ventromedial medulla and in spinal animals. It is concluded that the caudal medulla constitutes a key region for the expression of negative feed-back mechanisms triggered by both spatial summation of noxious inputs and heterotopic noxious inputs.

The spinal transmission of nociceptive information: modulation by the caudal medulla.

Multiple descending systems for pain control originate from the rostral medulla and midbrain. These systems are involved in the antinociceptive action produced by opioids. One category of descending inhibitory controls is activated specifically by noxious stimuli and has been termed diffuse noxious inhibitory controls. These controls have been described in both animal and man, but their supraspinal circuitry has not been fully localized. To determine the supraspinal level of integration of nociceptor activated controls and hence their potential relationships with previously described descending controls, we studied in halothane-anesthetized rats the effects of transections performed at various levels in the brainstem. The physiological properties of dorsal horn convergent neurons, including supraspinally-mediated inhibitory processes elicited by heterotopic noxious stimuli, i.e. diffuse noxious inhibitory controls, were not altered in rats in which the brainstem had been completely transected up to 200 microns caudal to the caudal end of the rostral ventromedial medulla. In contrast, the spontaneous activity of these neurons was significantly enhanced and the inhibitory phenomena significantly reduced in animals with transections more than 500 microns caudal to the caudal end of the rostral ventromedial medulla. These effects were not related to cardiovascular changes induced by the transections. These data indicate that some tonic descending inhibitory controls and diffuse noxious inhibitory controls depend upon connections in the caudal medulla. It is proposed that this area constitutes another level from which the transmission of nociceptive information can be modulated and that it acts co-operatively with previously described modulatory systems in the spinal cord and at more rostral levels of the brainstem.

Morphine and diffuse noxious inhibitory controls in the rat: effects of lesions of the rostral ventromedial medulla.

The aim of this study was to investigate whether the rostral ventromedial medulla (RVM) participates in the lifting of diffuse noxious inhibitory controls (DNIC) by systemic morphine. The effects of morphine (1 mg/kg i.v.) on DNIC were compared in sham-operated rats and animals with electrolytic lesions of the RVM performed one or three weeks earlier. The C-fibre-evoked responses of spinal dorsal horn convergent neurones were similar in the sham-operated and lesioned animals. DNIC acting on these responses were also similar in these groups. DNIC were similarly reduced naloxone reversibly following morphine injections in sham-operated animals and animals tested one week after lesioning of the RVM. In contrast, DNIC were not significantly altered by morphine in animals tested three weeks after lesioning. The lesions were similar in both groups of animals. This time-dependent attenuation of the effects of morphine indicates that the RVM is not directly involved in the reduction of DNIC induced by systemic morphine. However, it is suggested that lesions in this region can induce a reorganization of brainstem opioidergic systems.

Involvement of the dorsolateral funiculus in the descending spinal projections responsible for diffuse noxious inhibitory controls in the rat.

Recordings were made from convergent neurons in the lumbar dorsal horn of the spinal cord of the rat. These neurons were activated by both innocuous and noxious mechanical stimuli applied to their excitatory receptive fields located on the extremity of the hindpaw. Transcutaneous application of suprathreshold 2-ms square-wave electrical stimuli to the center of the excitatory field, resulted in responses to C-fiber activation being observed. This type of response was inhibited by applying a noxious thermal conditioning stimulus on the muzzle. The immersion of the muzzle in a 52 degrees C waterbath resulted in a strong reduction of the response during the application of the noxious conditioning stimulus and this was followed by long lasting poststimulus effects. Such inhibitory processes have been termed diffuse noxious inhibitory controls (DNIC). The effects on these inhibitions of lesions including the dorsolateral funiculus (DLF) were investigated in acute experiments: tests were performed before and at least 30 min after the DLF lesion. A lesion including the DLF ipsilateral to the neuron under study completely abolished the inhibitory processes triggered from the muzzle. Concomitantly, a facilitation of C-fiber responses was observed. Nevertheless, DNIC was still impaired even using a juxtathreshold current to elicit a weak C-fiber response. To ascertain further the main, if not entire, participation of the ipsilateral DLF in the descending projections responsible for the heterotopic inhibitory processes, the effects of a lesion of the contralateral DLF were investigated. Neither the inhibitory processes nor the unconditioned C-fiber responses were altered by this procedure. Again, a second lesion including the ipsilateral DLF induced a blockade of DNIC. It is concluded that the descending projections involved in the triggering of DNIC are mainly, if not entirely, confined to the DLF ipsilateral to the neuron under study. The contralateral DLF did not appear to play a role in these processes.

Lesions of dorsolateral funiculi (DLF) do not affect the depressive effects of systemic morphine upon dorsal horn convergent neuronal activities related to pain in the rat.

It has been shown that morphine could interact with supraspinal inhibitory controls which modulate the transmission of nociceptive messages at the spinal level. However, the way in which such interactions occur is still a subject of controversy. Based on behavioural experiments, it has been proposed that systemic morphine increases descending inhibitory controls travelling via the dorsolateral funiculus (DLF). To directly test this hypothesis from an electrophysiological standpoint, we have investigated the effects of morphine upon C-fibre responses of dorsal horn convergent neurones in rats with bilateral lesions of the DLF. Previous studies by our group have shown that 6 mg/kg morphine chlorhydrate was the intravenous mean effective dose for depressing by 50% (ED50) the C-fibre evoked responses of convergent neurones recorded at the lumbar level. In the present work, the effects of 6 mg/kg morphine were investigated under identical experimental conditions, except that a bilateral destruction of the dorsolateral funiculus (DLF) was performed previously. These lesions did not change the mean C-fibre evoked responses. Following morphine administration, the C-fibre evoked responses were depressed by 47.1 +/- 10.1% (n = 13) and reversal of these effects by naloxone was always observed. The A-fibre evoked responses, concurrently recorded, were not modified by the drug. As the depressive effects observed with this dose of morphine appear to be essentially of the same magnitude as those previously found in the intact or spinal preparations, we conclude that the DLF is not involved in the depressive effects of systemic morphine on dorsal horn convergent neurones.

Failure of ES 52, a highly potent enkephalinase inhibitor, to affect nociceptive transmission by rat dorsal horn convergent neurones.

The effects of ES 52, a highly potent derivative of the enkephalinase (enkephalin-dipeptidylcarboxypeptidase) inhibitor thiorphan, were studied on nociceptive activities of dorsal horn convergent neurones in the anaesthetized rat. Neither the C-fibre component of the responses elicited by supramaximal electrical stimulation of the hindpaw excitatory receptive fields nor diffuse noxious inhibitory controls triggered by immersion of the tail in 46-48 degrees C waterbaths, were affected by ES 52. Thus we conclude that, in our experimental conditions, modulations of the transmission of nociceptive messages at the spinal level are not greatly modified by specifically blocking the degradation of enkephalins. If a major role for enkephalinase (vs aminopeptidase) in the catabolism of enkephalins at the spinal level can be confirmed, then comparison of the present data with our previous results obtained using the opioid antagonist naloxone, might suggest a predominant role for proenkephalin B products (i.e. dynorphins and/or alpha-neo-endorphin) in modulating nociceptive transmission in the spinal cord.

Depression of activities of dorsal horn convergent neurones by propriospinal mechanisms triggered by noxious inputs; comparison with diffuse noxious inhibitory controls (DNIC).

The ability of heterotopic noxious stimuli to inhibit the activity of dorsal horn convergent neurones was investigated in both intact anesthetized, and spinal unanesthetized rats. Forty-four convergent neurones in lumbar dorsal horn were recognized by their ability to respond to both noxious and non-noxious natural stimuli and by their characteristic responses corresponding to A- and C-fibre activity following electrical stimulation of their cutaneous excitatory receptive fields on the ipsilateral hindpaw. The application of a sustained pinch to the excitatory receptive field resulted in an initial phasic activation of the neurone, which adapted to a stable tonic level of activity (mean 31.8 +/- 2.2 spikes/s). The levels of activity produced in this fashion were not appreciably different between the two types of preparation. In the intact anesthetized rat, the tonic activity produced by the sustained pinch could be strongly depressed by noxious conditioning stimuli applied to various parts of the body for all 10 neurones studied: heating the tail or pinching the contralateral hindpaw, the tail or a forepaw during 30 s each resulted in comparable inhibitions which had mean values in the order of 80% and which were always marked by post-effects lasting for upwards of 30 s. These inhibitory effects have been called Diffuse Noxious Inhibitory Controls (DNIC). In the spinal unanesthetized rat, the tonic activity was depressed to some extent by the same conditioning stimuli, for only 16/34 neurones studied. By comparison with the intact animals these inhibitions were weak, adapted to base-line levels within 30 s and were more marked for conditioning stimuli applied to structures proximal (tail, contralateral hindpaw) to the excitatory receptive field than for stimuli applied more distally (forepaws). The differences between the inhibitions found in the intact and spinal preparations were subsequently confirmed in a series of experiments in which single convergent neurones were studied before and after the pharmacological blocking of the cervical spinal cord in anaesthetized rats. The results in the spinal preparations provide evidence for the existence of some propriospinal modulatory processes, triggered by the onset of noxious stimulation and acting on convergent neurones. These processes appear to be different from those mediating DNIC, which have been shown to involve supraspinal structures, to concern all convergent neurones, to be very potent and associated with long-lasting post-effects whether the conditioning noxious stimuli are applied to parts of the body proximal or distal to the excitatory receptive field.

Pharmacological evidence for the involvement of serotonergic mechanisms in diffuse noxious inhibitory controls (DNIC).

The involvement of serotonergic mechanisms in diffuse noxious inhibitory controls (DNIC) acting on dorsal horn convergent neurones has been studied in the anaesthetized rat. 35 neurones activated by transcutaneous electrical stimulation of their hindpaw receptive fields giving clear large A-fibre and C-fibre responses were recorded. These activities were conditioned by DNIC, evoked by either noxious heat applied to the tail or noxious pinch of the nose. Cinanserin (4 mg/kg i.v.) and metergoline (5 mg/kg i.v.), serotonin (5-HT) receptor blockers, strongly reduced the inhibitory effects of DNIC whilst having no significant effect on the non-conditioned responses. 5-Hydroxytryptophan, a precursor of 5-HT synthesis, significantly potentiated the effect of DNIC. These results indicate an important role for descending serotonergic pathways in DNIC. The functional role of this system is discussed.

The encoding of thermal stimuli by diffuse noxious inhibitory controls (DNIC).

The relationship between stimulus intensity and its efficacy in inducing diffuse noxious inhibitory controls (DNIC) was investigated in anaesthetized rats by using thermal stimulation of the tail for conditioning dorsal horn convergent neuronal responses to C fibres emanating from the hindpaw extremity. The threshold for obtaining inhibition of the neuronal responses ranged between 40 and 44 degrees C and there was a highly significant correlation between noxious temperatures (44-52 degrees C) and the degree of inhibition. These data provide support for the notion of an involvement of inhibitory processes (DNIC) in the signalling of pain by convergent neurones.

The effect of systemic morphine upon diffuse noxious inhibitory controls (DNIC) in the rat: evidence for a lifting of certain descending inhibitory controls of dorsal horn convergent neurones.

The effects of exogenous opiates upon diffuse noxious inhibitory controls (DNIC) was investigated in intact anaesthetized rats. 58 convergent neurones, responding to both noxious and innocuous stimuli applied to their cutaneous receptive fields, were recorded at the lumbar level. These cells received A- and C-peripheral fibre inputs as shown by electrical stimulation of their receptive fields and were mainly located in the medial part of the dorsal horn. The immersion of the distal two-thirds of the tail in hot water (52 degrees C) induced strong inhibition of the responses to both A-(23%) and C-(69%) fibres. Post-effects of long duration were commonly observed after cessation of the conditioning stimulus. While systemic injection of morphine at a low dose-range (0.1-1 mg/kg) did not significantly affect the unconditioned responses, the DNIC-mediated inhibitions were profoundly altered. (a) DNIC of responses to C fibres were dose-dependently (P less than 0.01) lifted by morphine: (b) the post-effects observed after cessation of conditioning stimuli were dose-dependently (P less than 0.01) diminished; (c) DNIC of responses to A-fibre were similarly altered but this effect was less significant (P less than 0.05); (d) DNIC of responses to sustained moderate pressure were greatly diminished by morphine (P less than 0.01); and (e) these effects were specific since they were antagonized by the opiate antagonist, naloxone. In addition, they were shown to be stereospecific since while the dextrogyre stereoisomer, dextrorphan, was ineffective the levogyre derivative, levorphanol, induced a significant lifting of DNIC. It is concluded that morphine decreases the supraspinal inhibitory controls of dorsal horn convergent neurones, at least when these controls are triggered by noxious stimuli. Assuming that a basic somatosensory background activity (noise) is transmitted to higher centres by dorsal horn convergent neurones, and that the pain-signalling message is the contrast between the activity of the segmental pool of neurones induced by the noxious stimulus and the DNIC-mediated silence of the remaining neuronal population, it is proposed that, by a reduction in DNIC, low-dose morphine could restore the initial level of background activity, the final result being analgesia.

[Are bulbo-spinal serotonergic systems involved in the detection of nociceptive messages? (author's transl)]. / Les systèmes sérotoninergiques bulbo-spinaux jouent-ils un rôle dans la détection des messages nociceptifs?

Intensely noxious peripheral stimuli of the anaesthetized rat produce two changes in the activity of convergent dorsal horn units: the segmental neuronal pool is activated, while all other convergent neurones are inhibited. These Diffuse Noxious Inhibitory Controls (DNIC) are highly potent (60-80% inhibition) and suppress all convergent neuronal activity, whether spontaneous or evoked by noxious or nonnoxious stimuli. On the other hand, they have no effect on other dorsal horn cell types, including noxious-only and proprioceptive units. The "DNIC" circuits include at least one supraspinal relay since DNIC is not seen in spinal animals. Furthermore, they are greatly reduced by lesions of the Nucleus Raphé Magnus (NRM). It has been shown that this nucleus massively projects onto the spinal cord, in particular onto the dorsal horn, and that stimulation of the NRM results in convergent unit inhibition of the same degree of magnitude as with DNIC. The role of serotonergic mechanisms in DNIC can be demonstrated pharmacologically: pCPA pre-treatment (3 daily I.P. injections, 300 mg/kg) or cinanserin (4 mg/kg I.V.) both result in a potent decrease (50-80%). We have proposed that the nociceptive message from the convergent units could result in a contrast between activity of the activated segmental pool and silence of the remaining convergent units. If this hypothesis can be verified, then some raphé nuclei and brain stem serotonergic pathways may function as filters in the detection of nociceptive messages, allowing extraction of information from somatic background activity including the firing from peripheral mechanoreceptors. While superficially paradoxical in fact our hypothesis fits well with the observation of profound analgesia following NRM stimulation: indeed, this hypothetical contrast would be completely eliminated by NRM stimulation since both neuronal pools would then be inhibited.

Effect of naloxone upon diffuse noxious inhibitory controls (DNIC) in the rat.

Twenty-eight convergent neurones, responding to both noxious and innocuous stimuli applied to their cutaneous receptive fields were recorded at the lumbar level in anaesthetized intact rats. These cells received Aa and C fibre inputs as shown by electrical stimulation of their receptive fields, and were located in the medial part of the dorsal horn. (a) For 15 units, Diffuse Noxious Inhibitory Controls (DNCI)67,68 were investigated by applying noxious thermal stimuli (52 degrees C) to the distal two-thirds of the tail. This conditioning stimulus induced strong inhibition of the responses to both Aa (28%) and C (71%) fibres. Post-effects of long duration were commonly observed after cessation of the conditioning stimulus. The systemic injection of naloxone (0.3 mg/kg, i.v.) resulted in a partial reduction in these inhibitory effects with a decrease of about 50% for both Aa and C fibre response 10 min after naloxone administration. This was followed by a progressive recovery lasting 30 min. (b) 28 convergent units, including the 15 reported above, were recorded to investigate the effect of naloxone upon the unconditioned response. Responses to Aa fibre were unaffected, whereas the responses to C fibre were slightly (17%) but significantly increased by naloxone.

[Depression by morphine of various descending inhibitory controls modulating the transmission of nociceptive information at the spinal level in the rat]. / Dépression par la morphine de certains contrôles inhibiteurs descendants qui modulent, chez le rat, la transmission des messages nociceptifs au niveau spinal.

Recording from convergent neurones--i. e. those responding to both non-noxious and noxious cutaneous stimuli--in the dorsal horn of the intact anaesthetized Rat, two distinct effects are seen after application of noxious stimuli: there is an activation of units of the segmental pool, along with a very powerful inhibition of the remaining neuronal population (diffuse noxious inhibitory controls, DNIC). Morphine at doses inadequate to directly depress the activity of these units specifically blocks the inhibition. Since DNIC is dependent upon supraspinal mechanisms, these observations show that morphine is capable of depressing certain descending inhibitory controls, at least when these are induced by noxious peripheral stimuli.