Neuroscience Letters, 128 (1991) 9-12 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100311D NSL 07829
Modification of serotonergic immunoreactive pattern in the dorsal horn of the rat spinal cord following dorsal root rhizotomy central nervous system. Lionel Marlier, Philippe Poulat, Narivelo Rajaofetra and Alain Privat INSERM U-336, Dbveloppement, Plasticitb et Vieillissement du Syst~rne Nerveux, U.S.T.L., Montpellier (France) (Received 7 August 1990; Revised version received 21 March 1991; Accepted 25 March 1991)
Key words: Rhizotomy; Primary sensory neuron; Serotonin; Calcitonin gene-related peptide; Spinal dorsal horn; Plasticity We investigated by immunocytochemistry the reorganisation of serotonergic endings in the dorsal horn of the rat spinal cord following thoracolumbar unilateral rhizotomy. The extent of the removal of primary afferent fibres was testified 2 weeks and 4 months after surgery by the disappearance of almost all calcitonin gene-related peptide (CGRP) immunoreactivity in the dorsal horn. Two weeks after surgery, serotonin immunoreactivity was severely reduced within the whole dorsal horn. Then, after 4 months, we observed a regrowth of serotonergic axons giving rise to an immunoreactive pattern close to that of intact animals, except in lamina IIi which was spanned by growing fibres. Thus, the removal of putative targets of serotonergic axons in the dorsal horn induces a transient loss of immunoreactivity, which illustrates the interaction between primary afferents and descending bulbo-spinal fibres.
Bulbo-spinal serotonergic pathways which project to the dorsal horn have been extensively implicated in the modulation of pain transmission [1, 2, 14]. The anatomical substratum of this interaction can be defined as a non synaptic system since, in the rat, serotonergic terminals exhibit a paucity of synaptic contacts within the dorsal horn [10, 11]. Meanwhile, certain serotonergic receptors (5-HT1A and 5-HT3) have been localised on primary sensory neurons [4, 6]. The destruction of C fibres by capsaicin leads to a reduction of the density of these receptors [4, 6]. The latter argues for an unequivocal interrelationship between serotonergic terminals and primary sensory neurons which, in turn, indicates the involvement of serotonin in nociception at spinal level. We showed recently that after the specific destruction of C nociceptive afferent fibres (by neonatal capsaicin treatment), the trilaminar pattern of serotonergic immunoreactivity in the superficial layers of the dorsal horn is disrupted. Indeed, lamina Iio, which normally contains only a few serotonergic profiles is invaded by serotonin (5-HT) immunoreactive (IR) fibres in capsaicintreated rats [9]. Thus, in this model, the plasticity of the serotonergic system was evidenced as a consequence of the removal of one of its putative targets in the dorsal horn: the C unmyelinated nociceptive afferents. In addition, in another experimental model, the arthritic rat, we Correspondence: L. Marlier, INSERM U-336, Drveloppement, Plasticit6 et Vieillissement du Systrme Nerveux, U.S.T.L., Place E. BatailIon, 34080 Montpellier Cedex 02, France.
also observed modifications of 5-HT immunoreactivity, which was increased within the whole dorsal horn without occlusion of lamina Iio. Concomitantly we found an increase in substance P and calcitonin gene-related peptide (CGRP) immunoreactivities [8]. Thus, in two 'opposite' experimental models, the 5-HT-IR pattern was shown to be modified as a consequence of the manipulation of the primary afferents. Since dorsal rhizotomy has also been proposed as a model of chronic pain [7], we found it interesting to study in this model the possible modifications of serotonergic immunoreactivity in the dorsal horn. A report by Colado et al. has shown, caudally to the level of the rhizotomy (T3-L5 versus C5-T2), a conspicuous reduction of serotonin whereas no significant variations occurred at the level of the rhizotomy [3]. In the present experiment, male Sprague-Dawley rats (IFFA CREDO) were anaesthetized with equithesin and a laminectomy was performed on three vertebrae in order to give access to T10-L5 dorsal roots. Under the operating microscope, the dura was opened and 5 to 6 roots were sectioned unilaterally with fine iridectomy scissors. The animals received penicillin (50,000 IU, s.c.) on days 1 and 2 following surgery. They were individually housed and rapidly recovered, and no significant changes were observed in their behaviour. The first signs of autotomy were noticed within 2 or 3 weeks after the operation. The extent of autotomy of the denervated hindpaw sometimes led us to sacrifice the animals (for immunocytochemical studies), before the wound became
10
ii~ ~ i~ii!ii~ ii~iii~il,ii~!ii!~i
Fig. 1. Immunocytochemical detection of C G R P (A,B) and serotonin (C,D) 2 weeks after rhizotomy at lumbar level. A, C: controls; arrowhead in C indicates the clear band (lamina IIi) almost devoid of 5-HT immunoreactivity. B,D: operated side. Bars = 100/tm.
infected. Interestingly enough, some animals exhibited only a few signs of autotomy and local application of antiseptics was sufficient to cure the superficial skin wound. Two groups of 3 animals were sacrificed respectively 15 days (group 1) and 4 months (group 2) after surgery. These rats had all exhibited transient signs of autotomy, proof of an excellent denervation of the hindlimb. The animals were sacrificed by intracardiac perfusion of glutaraldehyde (5%) in cacodylic acid (50 mM)-sodium metabisulfite (50 mM) buffer at pH 7.6 under deep pentobarbital anaesthesia. The spinal cord was removed, postfixed, and processed for the immunocytochemical detection of 5-HT and CGRP. Briefly, 50 /~m thick vibratome sections were incubated overnight at 4°C in primary antiserum directed against 5-HT (1:20,000) [5] or CGRP (Amersham; 1/4,000). Detection of the primary antibody was performed with the peroxidase-antiperoxidase (PAP) method, using diaminobenzidine (DAB) as a chromogen for peroxidase [8-10]. Fig. 1 shows the immunoreactivity for 5-HT and CGRP 2 weeks after rhizotomy in the contralateral and ipsilateral sides of the surgery. Both 5-HT- and CGRPIR patterns appeared on the contralateral side similar to those found in intact animals (not shown). Thus, 5-HTIR fibres were found on the one hand in laminae I-Iio
and, on the other hand, in laminae III-VI, separated by a clear band almost devoid of serotonergic profiles which approximately correspond to lamina IIi [9-12]. CGRP immunoreactivity was intense in laminae I-II while a few fibres were also seen in the deepest laminae. On the rhizotomized side, CGRP immunoreactivity had almost completely disappeared in laminae I-II, indicating the completion of the rhizotomy. For serotonin, a conspicuous loss of immunoreactivity occurred in lamina I and in a large band along the dorsal column, corresponding approximately to the medial half of the dorsal horn (Fig. 1). This same pattern was found in several sections in all animals, all along the deafferented lumbar segments (identified by the lack of CGRP-IR profile in matching sections). After 4 months, a marked reduction of CGRP was still evident on the rhizotomized side, while in the contralateral side no significant changes occurred compared to the previous stage (Fig. 2). For serotonin we observed, in the side ipsilateral to the lesion, a marked modification of the pattern seen at two weeks: numerous fibres arising from deep layers had invaded the superficial ones. Thus, in this area, the staining intensity was quite comparable to that of the contralateral side but differences remain in the disposition of fibres, with noticeably the occlusion of lamina IIi. On the contralateral side,
Fig. 2. Same as Fig. 1, 4 months after rhizotomy performed at low thoracic level.Arrow in D points to lamina IIi which is invaded by serotonergic immunoreactivefibres. Note on rhizotomisedsides the atrophy of the neck of the dorsal horn. Bars= 100 gm.
comparison with intact animals showed some modifications: sprouting fibres were frequently seen (not shown) with a pattern reminiscent of the development of the system [13]. Considering these data, it appears that serotonergicIR pattern underwent a biphasic modification after rhizotomy. First, a severe loss of immunoreactivity was seen 2 weeks after surgery, then this reduction was compensated, as seen after 4 months. Moreover, no apparent modifications were seen on the contralateral side for CGRP, while sprouting 5-HT-IR fibres were seen here after 4 months. Biochemical assays reported by Colado et al. failed to detect any modification for 5-HT at the level of the rhizotomy 2 weeks after surgery [3]. Since the modifications we reported here are very slight in proportion to the total amount of 5-HT in the whole gray matter, this discrepancy is not surprising. Thus, as suggested by these authors, immunocytochemistry appears to be a suitable tool to investigate fine reorganisations in a restricted region of the dorsal horn. When we compare these reorganisations with those found in other experimental models such as neonatal capsaicin treatment [9] and adjuvantinduced arthritis [8], several differences must be pointed out. At variance, in the capsaicin model, the chemical removal of C primary afferents results in a moderate reor-
ganisation in the serotonergic pattern within the dorsal horn but not, as after rhizotomy, in a large reduction in the number of immunoreactive profiles. Thus, complete removal of the dorsal root (rhizotomy) induces a different reaction of 5-HT axons which differs from that obtained after partial removal of dorsal root afferents. In arthritic rats, 5-HT immunoreactivity was not decreased during the onset of the disease but, on the contrary, increased. In addition, since after rhizotomy the loss of 5-HT affects both superficial and deep layers of the dorsal horn (and especially their medial-most extension), this model provides further evidence of the either direct or indirect interactions that exist between serotonergic bulbo-spinal neurons and the primary afferents in the dorsal horn. However, one should bear in mind the paucity of synaptic contacts involving serotonergic axons within the dorsal horn of the spinal cord, while conversely numerous serotonergic receptors belonging to several subtypes are present. Taken together, these data indicate firstly that two neuronal systems which are not synaptically linked can exhibit marked inter-relations. Secondly, it appears that, depending on the nature and the extent of the modification of the target, the reaction of 5-HT afferents may exhibit a different pattern and a different timecourse. This exemplifies again the important contribu-
12
tion of monoaminergic neurons to the plasticity of the central nervous system. The authors wish to thank M. Geffard for his generous gift of serotonin antiserum and J.R. Teilhac for artwork. This study was supported by grants from IRME and AFM, and a D. Heuman fellowship to N.R. and FRM fellowship to L.M.. 1 Basbaum, A.I. and Fields, H.L., The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. Comp. Neurol., 187 (1979) 513 532. 2 Besson, J.M. and Chaouch, A., Peripheral and spinal mechanisms of nociception, Physiol. Rev., 67 (I 987) 67-186. 3 Colado, M.I., Arnedo, A., Peralta, E. and Del Rio, J., Unilateral rhizotomy decreases monoamine levels in the rat spinal cord, Neurosci. Lett., 87 (1988) 302-306. 4 Daval, G., Verg6, D., Basbaum, A.I., Bourgoin, S. and Hamon, M., Autoradiographic evidence of serotoninl binding site on primary afferent fibres in the dorsal horn of the rat spinal cord, Neurosci. Lett., 83 (1987) 71-76. 5 Geffard, M., Henrick-Rock, A.M., Dulluc, J. and Seguela, P., Antisera against small neurotransmitter like molecules, Neurochem. Int., 7 (1985) 403~413. 6 Hamon, M., Gallissot, M.C., Menard, F., Gozlan, H., Bourgoin, S. and Verg6, D., 5-HT3 receptor binding sites are on capsaicin-sensitive fibres in the rat spinal cord, Eur. J. Pharmacol., 164 (1989) 315 322.
7 Lombard, M.C., Nashold, B.S. and Albe-Fessard, D., Deafferentation hypersensitivity in the rat after dorsal rhizotomy: a possible model of chronic pain, Pain, 6 (1979) 163-174. 8 Marlier, L., Poulat, P., Rajaofetra, N. and Privat, A., Modifications of serotonin-, substance P-, and calcitonin-gene related peptide-like immunoreactivities in the dorsal horn of the spinal cord of arthritic rats: a quantitative immunocytochemical study, Exp. Brain Res., in press. 9 Marlier, L., Rajaofetra, N., Poulat, P. and Privat, A., Modification of serotonergic innervation of the rat spinal cord dorsal horn after neonatal capsaicin treatment, J. Neurosci. Res., 25 (1990) 112 118. 10 Marlier, L., Sandillon, F., Poulat, P., Rajaofetra, N., Geffard, M. and Privat, A., Serotonergic innervation of the dorsal horn of the rat spinal cord: a light- and electron-microscope immunocytochemical study, J. Neurocytol., 20 (1991) 310-322. 11 Maxwell, D.J., Leranth, C. and Verhofstad, A.A.J., Fine structure of serotonin-containing axons in the marginal zone of the rat spinal cord, Brain Res., 266 (1983) 253-259. 12 Molander, C., Xu, Q. and Grant, G., The cytoarchitectonic organization of the spinal cord in the rat. I. The lower thoracic and lumbosacral cord, J. Comp. Neurol., 230 (1984) 133-141. 13 Rajaofetra, N., Sandillon, F., Geffard, M. and Privat, A., Pre- and post-natal ontogeny of serotonergic projections to the rat spinal cord, J. Neurosci. Res., 22 (1989) 305-321. 14 Rivot, J.P., Chaouch, A. and Besson, J.M., Nucleus raphe magnus modulation of response of rat dorsal horn neurons to unmyelinated fiber inputs: partial involvement of serotonergic pathways, J. Neurophysiol., 44 (1980) 1039- 1057.
Modification of serotonergic immunoreactive pattern in the dorsal horn of the rat spinal cord following dorsal root rhizotomy central nervous system. Lionel Marlier, Philippe Poulat, Narivelo Rajaofetra and Alain Privat INSERM U-336, Dbveloppement, Plasticitb et Vieillissement du Syst~rne Nerveux, U.S.T.L., Montpellier (France) (Received 7 August 1990; Revised version received 21 March 1991; Accepted 25 March 1991)
Key words: Rhizotomy; Primary sensory neuron; Serotonin; Calcitonin gene-related peptide; Spinal dorsal horn; Plasticity We investigated by immunocytochemistry the reorganisation of serotonergic endings in the dorsal horn of the rat spinal cord following thoracolumbar unilateral rhizotomy. The extent of the removal of primary afferent fibres was testified 2 weeks and 4 months after surgery by the disappearance of almost all calcitonin gene-related peptide (CGRP) immunoreactivity in the dorsal horn. Two weeks after surgery, serotonin immunoreactivity was severely reduced within the whole dorsal horn. Then, after 4 months, we observed a regrowth of serotonergic axons giving rise to an immunoreactive pattern close to that of intact animals, except in lamina IIi which was spanned by growing fibres. Thus, the removal of putative targets of serotonergic axons in the dorsal horn induces a transient loss of immunoreactivity, which illustrates the interaction between primary afferents and descending bulbo-spinal fibres.
Bulbo-spinal serotonergic pathways which project to the dorsal horn have been extensively implicated in the modulation of pain transmission [1, 2, 14]. The anatomical substratum of this interaction can be defined as a non synaptic system since, in the rat, serotonergic terminals exhibit a paucity of synaptic contacts within the dorsal horn [10, 11]. Meanwhile, certain serotonergic receptors (5-HT1A and 5-HT3) have been localised on primary sensory neurons [4, 6]. The destruction of C fibres by capsaicin leads to a reduction of the density of these receptors [4, 6]. The latter argues for an unequivocal interrelationship between serotonergic terminals and primary sensory neurons which, in turn, indicates the involvement of serotonin in nociception at spinal level. We showed recently that after the specific destruction of C nociceptive afferent fibres (by neonatal capsaicin treatment), the trilaminar pattern of serotonergic immunoreactivity in the superficial layers of the dorsal horn is disrupted. Indeed, lamina Iio, which normally contains only a few serotonergic profiles is invaded by serotonin (5-HT) immunoreactive (IR) fibres in capsaicintreated rats [9]. Thus, in this model, the plasticity of the serotonergic system was evidenced as a consequence of the removal of one of its putative targets in the dorsal horn: the C unmyelinated nociceptive afferents. In addition, in another experimental model, the arthritic rat, we Correspondence: L. Marlier, INSERM U-336, Drveloppement, Plasticit6 et Vieillissement du Systrme Nerveux, U.S.T.L., Place E. BatailIon, 34080 Montpellier Cedex 02, France.
also observed modifications of 5-HT immunoreactivity, which was increased within the whole dorsal horn without occlusion of lamina Iio. Concomitantly we found an increase in substance P and calcitonin gene-related peptide (CGRP) immunoreactivities [8]. Thus, in two 'opposite' experimental models, the 5-HT-IR pattern was shown to be modified as a consequence of the manipulation of the primary afferents. Since dorsal rhizotomy has also been proposed as a model of chronic pain [7], we found it interesting to study in this model the possible modifications of serotonergic immunoreactivity in the dorsal horn. A report by Colado et al. has shown, caudally to the level of the rhizotomy (T3-L5 versus C5-T2), a conspicuous reduction of serotonin whereas no significant variations occurred at the level of the rhizotomy [3]. In the present experiment, male Sprague-Dawley rats (IFFA CREDO) were anaesthetized with equithesin and a laminectomy was performed on three vertebrae in order to give access to T10-L5 dorsal roots. Under the operating microscope, the dura was opened and 5 to 6 roots were sectioned unilaterally with fine iridectomy scissors. The animals received penicillin (50,000 IU, s.c.) on days 1 and 2 following surgery. They were individually housed and rapidly recovered, and no significant changes were observed in their behaviour. The first signs of autotomy were noticed within 2 or 3 weeks after the operation. The extent of autotomy of the denervated hindpaw sometimes led us to sacrifice the animals (for immunocytochemical studies), before the wound became
10
ii~ ~ i~ii!ii~ ii~iii~il,ii~!ii!~i
Fig. 1. Immunocytochemical detection of C G R P (A,B) and serotonin (C,D) 2 weeks after rhizotomy at lumbar level. A, C: controls; arrowhead in C indicates the clear band (lamina IIi) almost devoid of 5-HT immunoreactivity. B,D: operated side. Bars = 100/tm.
infected. Interestingly enough, some animals exhibited only a few signs of autotomy and local application of antiseptics was sufficient to cure the superficial skin wound. Two groups of 3 animals were sacrificed respectively 15 days (group 1) and 4 months (group 2) after surgery. These rats had all exhibited transient signs of autotomy, proof of an excellent denervation of the hindlimb. The animals were sacrificed by intracardiac perfusion of glutaraldehyde (5%) in cacodylic acid (50 mM)-sodium metabisulfite (50 mM) buffer at pH 7.6 under deep pentobarbital anaesthesia. The spinal cord was removed, postfixed, and processed for the immunocytochemical detection of 5-HT and CGRP. Briefly, 50 /~m thick vibratome sections were incubated overnight at 4°C in primary antiserum directed against 5-HT (1:20,000) [5] or CGRP (Amersham; 1/4,000). Detection of the primary antibody was performed with the peroxidase-antiperoxidase (PAP) method, using diaminobenzidine (DAB) as a chromogen for peroxidase [8-10]. Fig. 1 shows the immunoreactivity for 5-HT and CGRP 2 weeks after rhizotomy in the contralateral and ipsilateral sides of the surgery. Both 5-HT- and CGRPIR patterns appeared on the contralateral side similar to those found in intact animals (not shown). Thus, 5-HTIR fibres were found on the one hand in laminae I-Iio
and, on the other hand, in laminae III-VI, separated by a clear band almost devoid of serotonergic profiles which approximately correspond to lamina IIi [9-12]. CGRP immunoreactivity was intense in laminae I-II while a few fibres were also seen in the deepest laminae. On the rhizotomized side, CGRP immunoreactivity had almost completely disappeared in laminae I-II, indicating the completion of the rhizotomy. For serotonin, a conspicuous loss of immunoreactivity occurred in lamina I and in a large band along the dorsal column, corresponding approximately to the medial half of the dorsal horn (Fig. 1). This same pattern was found in several sections in all animals, all along the deafferented lumbar segments (identified by the lack of CGRP-IR profile in matching sections). After 4 months, a marked reduction of CGRP was still evident on the rhizotomized side, while in the contralateral side no significant changes occurred compared to the previous stage (Fig. 2). For serotonin we observed, in the side ipsilateral to the lesion, a marked modification of the pattern seen at two weeks: numerous fibres arising from deep layers had invaded the superficial ones. Thus, in this area, the staining intensity was quite comparable to that of the contralateral side but differences remain in the disposition of fibres, with noticeably the occlusion of lamina IIi. On the contralateral side,
Fig. 2. Same as Fig. 1, 4 months after rhizotomy performed at low thoracic level.Arrow in D points to lamina IIi which is invaded by serotonergic immunoreactivefibres. Note on rhizotomisedsides the atrophy of the neck of the dorsal horn. Bars= 100 gm.
comparison with intact animals showed some modifications: sprouting fibres were frequently seen (not shown) with a pattern reminiscent of the development of the system [13]. Considering these data, it appears that serotonergicIR pattern underwent a biphasic modification after rhizotomy. First, a severe loss of immunoreactivity was seen 2 weeks after surgery, then this reduction was compensated, as seen after 4 months. Moreover, no apparent modifications were seen on the contralateral side for CGRP, while sprouting 5-HT-IR fibres were seen here after 4 months. Biochemical assays reported by Colado et al. failed to detect any modification for 5-HT at the level of the rhizotomy 2 weeks after surgery [3]. Since the modifications we reported here are very slight in proportion to the total amount of 5-HT in the whole gray matter, this discrepancy is not surprising. Thus, as suggested by these authors, immunocytochemistry appears to be a suitable tool to investigate fine reorganisations in a restricted region of the dorsal horn. When we compare these reorganisations with those found in other experimental models such as neonatal capsaicin treatment [9] and adjuvantinduced arthritis [8], several differences must be pointed out. At variance, in the capsaicin model, the chemical removal of C primary afferents results in a moderate reor-
ganisation in the serotonergic pattern within the dorsal horn but not, as after rhizotomy, in a large reduction in the number of immunoreactive profiles. Thus, complete removal of the dorsal root (rhizotomy) induces a different reaction of 5-HT axons which differs from that obtained after partial removal of dorsal root afferents. In arthritic rats, 5-HT immunoreactivity was not decreased during the onset of the disease but, on the contrary, increased. In addition, since after rhizotomy the loss of 5-HT affects both superficial and deep layers of the dorsal horn (and especially their medial-most extension), this model provides further evidence of the either direct or indirect interactions that exist between serotonergic bulbo-spinal neurons and the primary afferents in the dorsal horn. However, one should bear in mind the paucity of synaptic contacts involving serotonergic axons within the dorsal horn of the spinal cord, while conversely numerous serotonergic receptors belonging to several subtypes are present. Taken together, these data indicate firstly that two neuronal systems which are not synaptically linked can exhibit marked inter-relations. Secondly, it appears that, depending on the nature and the extent of the modification of the target, the reaction of 5-HT afferents may exhibit a different pattern and a different timecourse. This exemplifies again the important contribu-
12
tion of monoaminergic neurons to the plasticity of the central nervous system. The authors wish to thank M. Geffard for his generous gift of serotonin antiserum and J.R. Teilhac for artwork. This study was supported by grants from IRME and AFM, and a D. Heuman fellowship to N.R. and FRM fellowship to L.M.. 1 Basbaum, A.I. and Fields, H.L., The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. Comp. Neurol., 187 (1979) 513 532. 2 Besson, J.M. and Chaouch, A., Peripheral and spinal mechanisms of nociception, Physiol. Rev., 67 (I 987) 67-186. 3 Colado, M.I., Arnedo, A., Peralta, E. and Del Rio, J., Unilateral rhizotomy decreases monoamine levels in the rat spinal cord, Neurosci. Lett., 87 (1988) 302-306. 4 Daval, G., Verg6, D., Basbaum, A.I., Bourgoin, S. and Hamon, M., Autoradiographic evidence of serotoninl binding site on primary afferent fibres in the dorsal horn of the rat spinal cord, Neurosci. Lett., 83 (1987) 71-76. 5 Geffard, M., Henrick-Rock, A.M., Dulluc, J. and Seguela, P., Antisera against small neurotransmitter like molecules, Neurochem. Int., 7 (1985) 403~413. 6 Hamon, M., Gallissot, M.C., Menard, F., Gozlan, H., Bourgoin, S. and Verg6, D., 5-HT3 receptor binding sites are on capsaicin-sensitive fibres in the rat spinal cord, Eur. J. Pharmacol., 164 (1989) 315 322.
7 Lombard, M.C., Nashold, B.S. and Albe-Fessard, D., Deafferentation hypersensitivity in the rat after dorsal rhizotomy: a possible model of chronic pain, Pain, 6 (1979) 163-174. 8 Marlier, L., Poulat, P., Rajaofetra, N. and Privat, A., Modifications of serotonin-, substance P-, and calcitonin-gene related peptide-like immunoreactivities in the dorsal horn of the spinal cord of arthritic rats: a quantitative immunocytochemical study, Exp. Brain Res., in press. 9 Marlier, L., Rajaofetra, N., Poulat, P. and Privat, A., Modification of serotonergic innervation of the rat spinal cord dorsal horn after neonatal capsaicin treatment, J. Neurosci. Res., 25 (1990) 112 118. 10 Marlier, L., Sandillon, F., Poulat, P., Rajaofetra, N., Geffard, M. and Privat, A., Serotonergic innervation of the dorsal horn of the rat spinal cord: a light- and electron-microscope immunocytochemical study, J. Neurocytol., 20 (1991) 310-322. 11 Maxwell, D.J., Leranth, C. and Verhofstad, A.A.J., Fine structure of serotonin-containing axons in the marginal zone of the rat spinal cord, Brain Res., 266 (1983) 253-259. 12 Molander, C., Xu, Q. and Grant, G., The cytoarchitectonic organization of the spinal cord in the rat. I. The lower thoracic and lumbosacral cord, J. Comp. Neurol., 230 (1984) 133-141. 13 Rajaofetra, N., Sandillon, F., Geffard, M. and Privat, A., Pre- and post-natal ontogeny of serotonergic projections to the rat spinal cord, J. Neurosci. Res., 22 (1989) 305-321. 14 Rivot, J.P., Chaouch, A. and Besson, J.M., Nucleus raphe magnus modulation of response of rat dorsal horn neurons to unmyelinated fiber inputs: partial involvement of serotonergic pathways, J. Neurophysiol., 44 (1980) 1039- 1057.
