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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
Brain Research, 377 (1986) 397-402 Elsevier
397
BRE21652
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 DJAMEL CHITOUR, LUIS VILLANUEVA and DANIEL LE BARS Unit( de Recherches de Neurophysiologie Pharmacologique de I'INSERM (U. 161) 75014 Paris (France) (Accepted March 25th, 1986) Key words: pain - - morphine analgesia - - dorsal horn convergent neuron - - bulbo-spinal control - - dorsolateral funiculus (DLF)
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 electrophysiologicalstandpoint, 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 + I0.1% (n = 13) and reversal of these effects by naloxone was always observed. The A-fibre evoked responses, cuncurrently 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.
The analgesic effects of m o r p h i n e are m e d i a t e d by at least two distinct loci in the central nervous system, the spinal cord and supraspinal structures. The first site of action is well d o c u m e n t e d with biochemical, physiological, behavioural and clinical data: opioid binding sites 1 and immunoreactivity to leucine-enkephalin 15, m e t h i o n i n e - e n k e p h a l i n 2° and dynorphin 7 were found to be particularly a b u n d a n t within the superficial layers of the dorsal horn where thin peripheral afferent fibres terminate; direct measurements of the release of e n d o g e n o u s opioid-like substances from the spinal cord showed that noxious stimuli could trigger the activity of spinal opioidergic systemsS'4°; electrophysiological investigations revealed that all opioids tested so far depress neuronal dorsal horn activities related to pain (see refs in ref. 14). The behavioural and clinical observations of analgesia following the intrathecal administration of
morphine illustrates the potency of spinal opioidergic systems in the control of pain transmission (see refs. in ref. 39). The supraspinal action of opiates was demonstrated in behavioural studies where analgesic effects were observed following intraventricular or microinjection of m o r p h i n e in various sites. In current views 16'28'29, m o r p h i n e is s u p p o s e d to act at brainstem sites such as the periaqueductal grey of the midbrain and, m o r e caudally, the nucleus raph6 magnus and surrounding areas, to reinforce descending inhibitory control systems; this is supposed to be an additional means of producing a block of the spinal transmission of nociceptive messages. This hypothesis is largely based on indirect evidence such as the fact that both an analgesia in behavioural studies and inhibitions of dorsal horn nociceptive neurones can be elicited, in a naloxone-reversible m a n n e r , by elec-
Correspondence: D. Le Bars, Unit6 de Recherches de Neurophysiologie Pharmacologique de i'INSERM (U. 161), 2 rue d'Al6sia, 75014 Paris, France. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
398 trical stimulation of these areas (see refs. in refs. 6, 16, 28, 29). More direct evidence was provided by behavioural experiments 2,4,32 where analgesia induced by systemic morphine could be impaired by lesions of the DLF, suggesting that systemic morphine increased descending inhibitory controls travelling via the DLF. Electrophysiological studies by our group in intact anaesthetized rats have shown that intravenous morphine chlorhydrate depresses dose-dependently the C-fibre evoked responses of convergent neurones recorded at the lumbar level in the 1-10 mg/kg range 24, while responses to A-fibre were not affected. Since the mean effective dose for depressing by 50% (ED50) the C-fibre evoked responses was 6 mg/kg, we investigated in the present work the effects of the same dose of morphine under identical experimental conditions, except that bilateral lesions of the DLF were performed previously. If indeed, systemic morphine increases descending inhibitory controls travelling via the DLF, then depressant effects would be less than 50%. Experiments were performed on 13 Sprague-Dawley male rats weighing 230-250 g. Anaesthesia, surgical preparation, monitoring and recording have been previously described 22. Briefly, lumbar dorsal horn cell activities were recorded under gaseous anaesthesia (0.5% halothane in a nitrous oxide/oxygen mixture 2:1) and characterized using electrophysiological criteria proposed by Men6trey et al. 3°. Only convergent neurones were selected: they were driven by both noxious (pinch, radiant heat) and innocuous (hair movement, stroking) natural cutaneous stimuli applied on their peripheral excitatory receptive field located at the extremity of the ipsilateral hindpaw. Transcutaneous electrical pulses (2 ms width) via a pair of stainless steel needles inserted at the centre of the receptive field always evoked an initial response and a late discharge which, according to their latency and using the classification of Gasser and Erlanger 17 correspond to A-and C-fibre transmitted volleys respectively. The mean threshold for C-fibre activation was determined (T = 3.0 + 0.7 mA). The current intensity was then raised to a supraliminar value with a mean of 2.6 T yielding easily reproducible responses: on average, 8.6 + 2.6 A-fibre latency and 25.3 + 4.0 C-fibre latency spikes were evoked per stimulus.
The experimental procedure consisted of a sequence of 50 such suprathreshold stimuli (0.66 Hz) applied every 5 rain and for each sequence a poststimulus histogram was built. Two consecutive stable sequences were considered as the control prelesion responses. The dura was slit over the cervical spinal cord at the end of the second control sequence and two lesions including the DLF ipsilateral and controlateral to the recording site were made under a dissecting microscope by cutting the cord with a lancet diamond knife (A. Meyer Co.). Great caution was employed in sparing the local vascularization and the hiatus created by the section was filled by a thin slice of hemostatic gauze. Two postlesion sequences were carried out 30 rain after the lesions to minimize early possible changes induced by the surgery. A previous work 38 has indicated that this half-hour period was a sufficient time in this respect. This second set of two sequences was considered as the control postlesion premorphine value. Results were expressed with reference to the mean of these two sequences (see Fig. 2). Morphine chlorhydrate (6 mg/kg; i.v.) was then slowly injected and its effect observed 5 and 10 min later; previous experiments have shown that the effects of morphine on dorsal horn neurones exhibit a rapid onset 24. Then naloxone (0.8 mg/kg; i.v.) was administered to test the specificity of morphine action. At the conclusion of the experiments, the recording sites were marked by electrophoretic deposition of Pontamine sky blue, the cervical and lumbar spinal cord were removed and fixed by immersion in a 10% formalin solution for 72 h and these were then soaked in a 30% buffered sucrose solution for 48 h. Samples were frozen, cut in serial 100-ktm thick sections and Nissl-stained with cresyl violet or carmin (see Fig. 1 for an example). Cord lesions were reconstructed from camera lucida drawings of serial sections (see Figs. 1 and 2). Recording sites were found in laminae I V - V . This strict protocol was fully followed for the 13 units presented here. Fig. i provides an example of a typical experiment. The total extent of DLF lesion reconstructed from camera iucida drawings and an example of Nisslstained section of the cervical cord are shown. Thirty rain following this lesion, the C-fibre-evoked responses were not clearly modified while the subsequent administration of morphine (6 mg/kg; i.v.) re-
399
Control
0
250
~
~
500
after DLF lesion
0
250
500
after Morphine
0
250
500
after Naloxone
0
250
5 0 0 ms
Fig. 1. Example of the depressive effect of morphine (6 mg/kg; i.v.) upon the C-fibre evoked responses of a convergent neurone after bilateral lesions of the DLF. Poststimulus histograms (50 trials, bin width 5 ms) were built before (control) and following DLF lesions. Morphine was injected 30 min following lesions. Inset photograph: a Nissl-stained section of the cervical cord where lesions were made; inset drawing: the camera lucida reconstruction of the total extent of the lesions.
suited in strong depressive effects which were reversed by naloxone. Fig. 2A shows the extent of D L F lesions reconstructed from camera lucida drawings for the 12 remaining units. In all cases were the two D L F completely cut, with, in some cases, the more ventral part of the white matter and/or parts of the grey matter also being involved. Following these 13 lesions, the C-fibre response either increased (3 cases), decreased (4 cases) or remained constant within a 20% variation range (6 cases); on average, variations were less than 10%. Fig. 2B illustrates the mean quantitative data; the results are expressed in terms of percent value with reference, in each individual case, to the mean of the 2 pre-morphine responses. Five min following morphine administration, the Cfibre responses were lowered to 52.9 + 10.1% of control values. The depression was almost identical 10 min later and naloxone (0.8 mg/kg; i.v.) fully reversed these effects. There was no direct relationship between the effect of the lesion on C-fibre responses and the subsequent effect of morphine; the drug depressed to similar extents the previously increased, decreased or unaffected responses. In contrast, as previously described in non-transected animals 24, the concurrently recorded A-fibre responses were not affected (90.4 + 11.2% of control values, 5 min following morphine). In Fig. 3 the present results (black column) are compared to a previous work 24 performed in identical conditions, except that convergent neurones were recorded in rats with intact spinal cord. Results are expressed in percentage of depression observed 5
Bilateral DLF Lesions
B
Morphine
Naloxone
A> 100
==
E
~
50
N=13 0
// i
1'0
20 min.
Fig. 2. A: extent of individual lesions (C3-C4 segment); the thirtieth lesion is shown in Fig. 1. B: mean effects of morphine. (6 mg/kg; i.v.) on the responses of convergent neurones elicited by C-fibre activation in rats after bilateral DLF lesions. Results are expressed in terms of percentage with reference to the postlesion, premorphine C-fibre responses.
400 %-
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Fig. 3. Depression of C-fibre evoked responses induced by a single (6 mg/kg) dose of morphine in DLF lesioned rats (black bar) as compared with those previously obtained24 with increasing doses of morphine in intact animals (hatched area).
min following morphine-administration. Note the linear relationship found in a semilogarythmic scale between morphine dosage (x, range 1-9 mg/kg) and depressive effect (y): y = 46.6 log x +14.26 (r3a = 0.48; P < 0.01). The administration of 6 mg/kg (the ED50 in intact rats) in lesioned rats resulted in a 47.1 _+ 10.1% depression, which fits very well with the dose-response curve built from data in intact animals. It appears therefore that the depressive effect of systemic morphine on C-fibre responses of convergent neurones was not modified by a bilateral lesion of the DLF. In this respect, it is important to note that the bilateral lesions did not affect the responses themselves, thereby excluding a possible bias in our results. We have, therefore, no evidence in the present study for a lifting of tonic descending inhibition traveling through both DLF. However no definitive conclusion can be drawn since in another series of experiments performed under the same conditions 37, we observed a strong facilitation of the C-fibreevoked responses following a single lesion of the ipsilateral DLF; again a bilateral DLF lesion did not change such responses. Since the dose-response curve shown in Fig. 3 is
superimposable on the dose-response curve obtained in 'spinal' rats 27, one can conclude that the observed depressive effect of morphine is mainly due to a direct spinal effect. In fact the question of an eventual increase by morphine of bulbospinal inhibitory controls is a highly controversial subject 14"23. In electrophysiological experiments, two types of experimental protocols were used for the investigation of this problem: (1) comparison of the effects of systemic morphine upon responses of dorsal horn convergent neurones in 'spinal' and 'intact' preparations or 'decerebrate' preparations 13"19,21,24-26'33'35'36, (2) effects of microinjected morphine in PAG or NRM upon responses of dorsal horn convergent neurones 5'9-12"18"22. It is not our purpose here to discuss in detail these papers; suffice to say that methodological problems are not absent from some of them (see Discussion in refs. 9, 10, 35) and that results are highly confusing. In our hands, none of our results using both methodologies gave evidence for an increase by morphine of descending inhibitory controls 11'22-26. For example, the dose-response curves (1-10 mg/kg range) were found superimposable in the 'spinal' and 'intact' preparation 24. From the behavioural point of view it appears noteworthy to point out that while DLF lesions did decrease the effects of morphine upon reflex responses 2'32, they have no effect on morphine analgesia tested with a more integrated behaviour3~. Moreover, Rydenhag and Andersson 32 showed that the descending system, with strong influence on the tailflick response after morphine administration, shifts from the DLF to the ventral parts of cord below the ninth thoracic segment. These data, taken together with the present study and anatomical evidence that the DLF projects mainly, if not entirely, to the dorsal horn 3'34, strongly suggest that DLF lesions impaired the effects of morphine upon spinal motor functions, without modifying its effects upon the spinal transmission of nociceptive messages. We gratefully acknowledge preparation of the graphics by Eric Dehausse, preparation of the manuscript by Martine Hoch and English correction by John Leah. Support by INSERM.
401 REFERENCES 1 Atweh, S.E. and Kuhar, M.J., Autoradiographic localization of opiate receptors in rat brain. I. Spinal cord and medulla, Brain Research, 124 (1977) 53-67. 2 Barton, C., Basbaum, A.I. and Fields, H.L., Dissociation of supraspinal and spinal actions of morphine: a quantitative evaluation, Brain Research, 188 (1980) 487-498. 3 Basbaum, A.I., Clanton, C.H. and Fields, H.L., Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain-modulating systems, J. Comp. Neurol., 178 (1978) 209-224. 4 Basbaum, A.I., Marley, N.J.E., O'Keefe, J. and Clanton, C.H., Reversal of morphine and stimulus-produced analgesia by subtotal spinal cord lesions, Pain, 3 (1977) 43-56. 5 Bennett, G.J. and Mayer, D.J., Inhibition of spinal cord interneurons by narcotic microinjection and focal electrical stimulation in the periaqueductal central gray matter, Brain Research, 172 (1979) 243-257. 6 Besson, J.M., Oliveras, J.L., Chaouch, A. and Rivot, J.P., Role of the raph6 nuclei in stimulation producing analgesia. In B. Haber, S. Gabay, M.R. Issidorides and S.G.A. Alivisatos (Eds.), Serotonin. Current Aspects of Neurochemistry and Function, Plenum Press, New York, 1981, pp. 153-176. 7 Botticelli, L.J., Cox, B.M. and Goldstein, A., Immuno•reactive dynorphin in mammalian spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 7783-7786. 8 Cesselin, F., Le Bars, D., Bourgoin, S., Artaud, F., Gozlan, H., Clot, A.M., Besson, J.M. and Hamon, M., Spontaneous and evoked release of methionine-enkephalin-like material from the rat spinal cord in vivo, Brain Research, 339 (1985) 305-313. 9 Clark, S.L. and Ryall, R.W., The antinociceptive action of etorphine in the dorsal horn is due to a direct spinal action and not to activation of descending inhibition, Br. J. Pharmacol., 78 (1983) 307-319. 10 Clark, S.L., Edeson, R.O. and Ryall, R.W., The relative significance of spinal and supraspinal actions in the antinociceptive effect of morphine in the dorsal horn: an evaluation of the microinjection technique, Br. J. Pharmacol., 79 (1983) 807-818. 11 Dickenson, A.H. and Le Bars, D., Morphine microinjections into periaqueductal grey matter of the rat: effects on dorsal horn neuronal responses to C-fibre activity and diffuse noxious inhibitory controls, Life Sci., 33, suppl. 1 (1983) 549-552. 12 Du, H.J., Kitahata, L.M., Thalhammer, J.G. and Zimmermann, M., Inhibition of nociceptive neuronal responses in the cat's spinal dorsal horn by electrical stimulation and morphine microinjection in nucleus raph6 magnus, Pain, 19 (1984) 249-257. 13 Duggan, A.W., Griersmith, B.T. and North, R.A., Morphine and supraspinal inhibition of spinal neurones: evidence that morphine decreases tonic descending inhibition in the anaesthetized cat, Br. J. Pharmacol., 69 (1980) 461-466. 14 Duggan, A.W. and North, R.A., Electrophysiology of opioids, Pharmacol. Rev., 35 (1984) 219-281. 15 Elde, R., Hokfelt, T., Johansson, O. and Terenius, L., Immunohistochemical studies using antibodies to leucine-enkephalin: initial observations on the nervous system of the rat, Neurosciences, 1 (1976) 349-351.
16 Fields, H.L. and Basbaum, A T , Endogenous pain control mechanisms. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingstone, 1984, pp. 142-152. 17 Gasser, H.S. and Erlanger, J., The role played by the sizes of the constituent fibres of a nerve trunk in determining the form of its action potential wave, Am. J. Physiol., 80 (1927) 522-547. 18 Gebhart, G.F., Sandkiihler, J., Thalhammer, J.G. and Zimmermann, M., Inhibition in spinal cord of nociceptive information by electrical stimulation and morphine microinjection at identical sites in midbrain of the cat, J. Neurophysiol., 51 (1984)75-89. 19 Hanaoka, K., Ohtani, M., Toyooka, H., Dohi, S., Ghazisaidi, K., Taub, A. and Kitahata, L.M., The relative contribution of direct and supraspinal descending effects upon spinal mechanisms of morphine analgesia, J. Pharmacol. Exp. Ther., 207 (1978) 476-484. 20 H6kfelt, T., Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, C., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3081-3085. 21 Jurna, I. and Grossman, W., The effect of morphine on the activity evoked in ventrolateral tract axons of the cat spinal cord, Exp. Brain Res., 24 (1976) 473-484. 22 Le Bars, D., Dickenson, A.H. and Besson, J.M., Microinjection of morphine within nucleus raph6 magnus and dorsal horn neurone activities related to nociception in the rat, Brain Research, 189 (1980) 467-481. 23 Le Bars, D., Dickenson, A.H. and Besson, J.M., Opiate analgesia and descending control systems. In J.J. Bonica, U. Lindblom and A. Iggo (Eds.), Advances in Pain Research and Therapy, Vol. 5, Raven Press, New York, 1983, pp. 341-372. 24 Le Bars, D., Guilbaud, G., Chitour, D. and Besson J.M., Does systemic morphine increase descending inhibitory controls of dorsal horn neurones involved in nociception?, Brain Research, 202 (1980) 223-228. 25 Le Bars, D., Men6trey, D. and Besson, J.M., Effects of morphine upon the lamina V type cells activities in the dorsal horn of the decerebrate cat, Brain Research, 113 (1976) 293-310. 26 Le Bars, D., Men6trey, D., ConseiUer, C. and Besson, J.M., Comparaison chez le chat spinal et le chat d6c6r~br~, des effets de la morphine sur les activit6s des interneurones de type V de la corne dorsale de la moelle, C.R. Acad. Sci. (Paris), 273 (1974) 1369-1371. 27 Le Bars, D., Rivot, J.P., Guilbaud, G., Menetrey, D. and Besson, J.M., The depressive effect of morphine on the Cfibre response of dorsal horn neurones in the spinal rat pretreated or not by pCPA, Brain Research, 176 (1979) 337-353. 28 Liebeskind, J.C., Giesler, G., Jr. and Urca, G., Evidence pertaining to an endogenous mechanism of pain inhibition in the central nervous system. In Y. Zotterman (Ed.), Sensory Functions of the Skin, Pergamon Press, Oxford, 1976, pp. 561-573. 29 Mayer, D.J. and Price, D.D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379-404. 30 Menetrey, D., Giesler, G., Jr. and Besson, J.M., An analysis of responses properties of spinal cord dorsal horn neurones to non-noxious and noxious stimuli in the spinal rat, Exp. Brain Res., 27 (1977) 15-33. 31 Ryan, S.M., Watkins, L.R., Mayer, D.J. and Maier, S.F.,
402
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36
Spinal pain suppression mechanisms may differ for phasic and tonic pain, Brain Research, 334 (1985) 172-175. Rydenhag, B. and Andersson, S., Effect of DLF lesions at different spinal levels on morphine-induced analgesia, Brain Research, 212 (1981) 239-242. Satoh, M., Nakamura, N. and Takagi, H., Effect of morphine on bradykinin-induced unitary discharges in the spinal cord of the rabbit, Eur. J. Pharmacol., 16 (1971) 245-247. Skagerberg, G. and Bj6rklund, A., Topographic principles in the spinal projections of serotonergic and non-serotonergic brainstem neurons in the rat, Neuroscience, 15 (1985) 445-480. Soja, P.J. and Sinclair, J.G., Spinal vs supraspinal actions of morphine on cat spinal cord multireceptive neurones, Brain Research, 273 (1983) 1-7. Takagi, H., Satoh, M., Doi, T., Kawasaki, K. and Akaike, A., Indirect and direct depressive effects of morphine on
37
38
39 40
activation of lamina V cell of the spinal dorsal horn induced by intra-arterial injection of bradykinin, Arch. Int. Pharmacodyn. Ther., 221 (1976) 96-104. Villanueva, L., Chitour, D. and Le Bars, D., Involvement of the dorsolateral funiculus (DLF) in the descending spinal projections responsible for Diffuse Noxious Inhibitory Controls (DNIC) in the rat, J. Neurophysiol., in press. Villanueva, L., Peschanski, M., Calvino, B. and Le Bars, D., Ascending pathways in the spinal cord involved in triggering of diffuse noxious inhibitory controls (DNIC) in the rat, J. Neurophysiol., 55 (1986) 34-55. Yaksh, T.L., Spinal opiate analgesia. Characteristics and principles of action, Pain, 11 (1981) 293-346. Yaksh, T.L., Terenius, L., Nyberg, F., Jhamandas, K. and Wang, J.Y., Studies on the release by somatic stimulation from rat and cat spinal cord of active materials which displace dihydromorphine in an opiate-binding assay, Brain Research, 268 (1983) 119-128.
397
BRE21652
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 DJAMEL CHITOUR, LUIS VILLANUEVA and DANIEL LE BARS Unit( de Recherches de Neurophysiologie Pharmacologique de I'INSERM (U. 161) 75014 Paris (France) (Accepted March 25th, 1986) Key words: pain - - morphine analgesia - - dorsal horn convergent neuron - - bulbo-spinal control - - dorsolateral funiculus (DLF)
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 electrophysiologicalstandpoint, 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 + I0.1% (n = 13) and reversal of these effects by naloxone was always observed. The A-fibre evoked responses, cuncurrently 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.
The analgesic effects of m o r p h i n e are m e d i a t e d by at least two distinct loci in the central nervous system, the spinal cord and supraspinal structures. The first site of action is well d o c u m e n t e d with biochemical, physiological, behavioural and clinical data: opioid binding sites 1 and immunoreactivity to leucine-enkephalin 15, m e t h i o n i n e - e n k e p h a l i n 2° and dynorphin 7 were found to be particularly a b u n d a n t within the superficial layers of the dorsal horn where thin peripheral afferent fibres terminate; direct measurements of the release of e n d o g e n o u s opioid-like substances from the spinal cord showed that noxious stimuli could trigger the activity of spinal opioidergic systemsS'4°; electrophysiological investigations revealed that all opioids tested so far depress neuronal dorsal horn activities related to pain (see refs in ref. 14). The behavioural and clinical observations of analgesia following the intrathecal administration of
morphine illustrates the potency of spinal opioidergic systems in the control of pain transmission (see refs. in ref. 39). The supraspinal action of opiates was demonstrated in behavioural studies where analgesic effects were observed following intraventricular or microinjection of m o r p h i n e in various sites. In current views 16'28'29, m o r p h i n e is s u p p o s e d to act at brainstem sites such as the periaqueductal grey of the midbrain and, m o r e caudally, the nucleus raph6 magnus and surrounding areas, to reinforce descending inhibitory control systems; this is supposed to be an additional means of producing a block of the spinal transmission of nociceptive messages. This hypothesis is largely based on indirect evidence such as the fact that both an analgesia in behavioural studies and inhibitions of dorsal horn nociceptive neurones can be elicited, in a naloxone-reversible m a n n e r , by elec-
Correspondence: D. Le Bars, Unit6 de Recherches de Neurophysiologie Pharmacologique de i'INSERM (U. 161), 2 rue d'Al6sia, 75014 Paris, France. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
398 trical stimulation of these areas (see refs. in refs. 6, 16, 28, 29). More direct evidence was provided by behavioural experiments 2,4,32 where analgesia induced by systemic morphine could be impaired by lesions of the DLF, suggesting that systemic morphine increased descending inhibitory controls travelling via the DLF. Electrophysiological studies by our group in intact anaesthetized rats have shown that intravenous morphine chlorhydrate depresses dose-dependently the C-fibre evoked responses of convergent neurones recorded at the lumbar level in the 1-10 mg/kg range 24, while responses to A-fibre were not affected. Since the mean effective dose for depressing by 50% (ED50) the C-fibre evoked responses was 6 mg/kg, we investigated in the present work the effects of the same dose of morphine under identical experimental conditions, except that bilateral lesions of the DLF were performed previously. If indeed, systemic morphine increases descending inhibitory controls travelling via the DLF, then depressant effects would be less than 50%. Experiments were performed on 13 Sprague-Dawley male rats weighing 230-250 g. Anaesthesia, surgical preparation, monitoring and recording have been previously described 22. Briefly, lumbar dorsal horn cell activities were recorded under gaseous anaesthesia (0.5% halothane in a nitrous oxide/oxygen mixture 2:1) and characterized using electrophysiological criteria proposed by Men6trey et al. 3°. Only convergent neurones were selected: they were driven by both noxious (pinch, radiant heat) and innocuous (hair movement, stroking) natural cutaneous stimuli applied on their peripheral excitatory receptive field located at the extremity of the ipsilateral hindpaw. Transcutaneous electrical pulses (2 ms width) via a pair of stainless steel needles inserted at the centre of the receptive field always evoked an initial response and a late discharge which, according to their latency and using the classification of Gasser and Erlanger 17 correspond to A-and C-fibre transmitted volleys respectively. The mean threshold for C-fibre activation was determined (T = 3.0 + 0.7 mA). The current intensity was then raised to a supraliminar value with a mean of 2.6 T yielding easily reproducible responses: on average, 8.6 + 2.6 A-fibre latency and 25.3 + 4.0 C-fibre latency spikes were evoked per stimulus.
The experimental procedure consisted of a sequence of 50 such suprathreshold stimuli (0.66 Hz) applied every 5 rain and for each sequence a poststimulus histogram was built. Two consecutive stable sequences were considered as the control prelesion responses. The dura was slit over the cervical spinal cord at the end of the second control sequence and two lesions including the DLF ipsilateral and controlateral to the recording site were made under a dissecting microscope by cutting the cord with a lancet diamond knife (A. Meyer Co.). Great caution was employed in sparing the local vascularization and the hiatus created by the section was filled by a thin slice of hemostatic gauze. Two postlesion sequences were carried out 30 rain after the lesions to minimize early possible changes induced by the surgery. A previous work 38 has indicated that this half-hour period was a sufficient time in this respect. This second set of two sequences was considered as the control postlesion premorphine value. Results were expressed with reference to the mean of these two sequences (see Fig. 2). Morphine chlorhydrate (6 mg/kg; i.v.) was then slowly injected and its effect observed 5 and 10 min later; previous experiments have shown that the effects of morphine on dorsal horn neurones exhibit a rapid onset 24. Then naloxone (0.8 mg/kg; i.v.) was administered to test the specificity of morphine action. At the conclusion of the experiments, the recording sites were marked by electrophoretic deposition of Pontamine sky blue, the cervical and lumbar spinal cord were removed and fixed by immersion in a 10% formalin solution for 72 h and these were then soaked in a 30% buffered sucrose solution for 48 h. Samples were frozen, cut in serial 100-ktm thick sections and Nissl-stained with cresyl violet or carmin (see Fig. 1 for an example). Cord lesions were reconstructed from camera lucida drawings of serial sections (see Figs. 1 and 2). Recording sites were found in laminae I V - V . This strict protocol was fully followed for the 13 units presented here. Fig. i provides an example of a typical experiment. The total extent of DLF lesion reconstructed from camera iucida drawings and an example of Nisslstained section of the cervical cord are shown. Thirty rain following this lesion, the C-fibre-evoked responses were not clearly modified while the subsequent administration of morphine (6 mg/kg; i.v.) re-
399
Control
0
250
~
~
500
after DLF lesion
0
250
500
after Morphine
0
250
500
after Naloxone
0
250
5 0 0 ms
Fig. 1. Example of the depressive effect of morphine (6 mg/kg; i.v.) upon the C-fibre evoked responses of a convergent neurone after bilateral lesions of the DLF. Poststimulus histograms (50 trials, bin width 5 ms) were built before (control) and following DLF lesions. Morphine was injected 30 min following lesions. Inset photograph: a Nissl-stained section of the cervical cord where lesions were made; inset drawing: the camera lucida reconstruction of the total extent of the lesions.
suited in strong depressive effects which were reversed by naloxone. Fig. 2A shows the extent of D L F lesions reconstructed from camera lucida drawings for the 12 remaining units. In all cases were the two D L F completely cut, with, in some cases, the more ventral part of the white matter and/or parts of the grey matter also being involved. Following these 13 lesions, the C-fibre response either increased (3 cases), decreased (4 cases) or remained constant within a 20% variation range (6 cases); on average, variations were less than 10%. Fig. 2B illustrates the mean quantitative data; the results are expressed in terms of percent value with reference, in each individual case, to the mean of the 2 pre-morphine responses. Five min following morphine administration, the Cfibre responses were lowered to 52.9 + 10.1% of control values. The depression was almost identical 10 min later and naloxone (0.8 mg/kg; i.v.) fully reversed these effects. There was no direct relationship between the effect of the lesion on C-fibre responses and the subsequent effect of morphine; the drug depressed to similar extents the previously increased, decreased or unaffected responses. In contrast, as previously described in non-transected animals 24, the concurrently recorded A-fibre responses were not affected (90.4 + 11.2% of control values, 5 min following morphine). In Fig. 3 the present results (black column) are compared to a previous work 24 performed in identical conditions, except that convergent neurones were recorded in rats with intact spinal cord. Results are expressed in percentage of depression observed 5
Bilateral DLF Lesions
B
Morphine
Naloxone
A> 100
==
E
~
50
N=13 0
// i
1'0
20 min.
Fig. 2. A: extent of individual lesions (C3-C4 segment); the thirtieth lesion is shown in Fig. 1. B: mean effects of morphine. (6 mg/kg; i.v.) on the responses of convergent neurones elicited by C-fibre activation in rats after bilateral DLF lesions. Results are expressed in terms of percentage with reference to the postlesion, premorphine C-fibre responses.
400 %-
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Fig. 3. Depression of C-fibre evoked responses induced by a single (6 mg/kg) dose of morphine in DLF lesioned rats (black bar) as compared with those previously obtained24 with increasing doses of morphine in intact animals (hatched area).
min following morphine-administration. Note the linear relationship found in a semilogarythmic scale between morphine dosage (x, range 1-9 mg/kg) and depressive effect (y): y = 46.6 log x +14.26 (r3a = 0.48; P < 0.01). The administration of 6 mg/kg (the ED50 in intact rats) in lesioned rats resulted in a 47.1 _+ 10.1% depression, which fits very well with the dose-response curve built from data in intact animals. It appears therefore that the depressive effect of systemic morphine on C-fibre responses of convergent neurones was not modified by a bilateral lesion of the DLF. In this respect, it is important to note that the bilateral lesions did not affect the responses themselves, thereby excluding a possible bias in our results. We have, therefore, no evidence in the present study for a lifting of tonic descending inhibition traveling through both DLF. However no definitive conclusion can be drawn since in another series of experiments performed under the same conditions 37, we observed a strong facilitation of the C-fibreevoked responses following a single lesion of the ipsilateral DLF; again a bilateral DLF lesion did not change such responses. Since the dose-response curve shown in Fig. 3 is
superimposable on the dose-response curve obtained in 'spinal' rats 27, one can conclude that the observed depressive effect of morphine is mainly due to a direct spinal effect. In fact the question of an eventual increase by morphine of bulbospinal inhibitory controls is a highly controversial subject 14"23. In electrophysiological experiments, two types of experimental protocols were used for the investigation of this problem: (1) comparison of the effects of systemic morphine upon responses of dorsal horn convergent neurones in 'spinal' and 'intact' preparations or 'decerebrate' preparations 13"19,21,24-26'33'35'36, (2) effects of microinjected morphine in PAG or NRM upon responses of dorsal horn convergent neurones 5'9-12"18"22. It is not our purpose here to discuss in detail these papers; suffice to say that methodological problems are not absent from some of them (see Discussion in refs. 9, 10, 35) and that results are highly confusing. In our hands, none of our results using both methodologies gave evidence for an increase by morphine of descending inhibitory controls 11'22-26. For example, the dose-response curves (1-10 mg/kg range) were found superimposable in the 'spinal' and 'intact' preparation 24. From the behavioural point of view it appears noteworthy to point out that while DLF lesions did decrease the effects of morphine upon reflex responses 2'32, they have no effect on morphine analgesia tested with a more integrated behaviour3~. Moreover, Rydenhag and Andersson 32 showed that the descending system, with strong influence on the tailflick response after morphine administration, shifts from the DLF to the ventral parts of cord below the ninth thoracic segment. These data, taken together with the present study and anatomical evidence that the DLF projects mainly, if not entirely, to the dorsal horn 3'34, strongly suggest that DLF lesions impaired the effects of morphine upon spinal motor functions, without modifying its effects upon the spinal transmission of nociceptive messages. We gratefully acknowledge preparation of the graphics by Eric Dehausse, preparation of the manuscript by Martine Hoch and English correction by John Leah. Support by INSERM.
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