Journal of Neuroscience Research 25:112-118 (1990)
Modification of Serotonergic Innervation of the Rat Spinal Cord Dorsal Horn After Neonatal Capsaicin Treatment L. Marlier, N. Ra.iaofetra, P. Poulat, and A. Privat Laboratoire Neurobiologie du Developpement, UPR 41 CNRS U. 249 INSERM, EPHE, Institut de Biologic, Montpellier, France
Newborn rats were treated with Capsaicin. Seven weeks later, calcitonin gene-related peptide, substance P, and serotonin were detected immunocytochemically in the dorsal horn of the spinal cord. Peptide immunostaining, presumably located in primary afferents, was reduced in intensity, whereas the pattern of serotonin innervation was modified, suggesting a secondary reorganization due to the lesion of afferents. The possible mechanisms of this reorganization are discussed. Key words: CGRP, substance P, immunocytochemistry, plasticity INTRODUCTION Neonatal administration of capsaicin (8-methyl-Nvanillyl-6-nonenamide) results in neurochemical alterations in adult rats. The major injury due to the toxin is a selective and dose-dependent degeneration of distinct populations of primary sensory neurons involved in the mediation of chemogenic pain (Nagy et al., 1983). Capsaicin-treated rats show a decrease in noxious thermal, mechanical, and chemical stimuli transmitted by small, unmyelinated C fibers (Bucket al., 1986; Holzer, 1988). The most extensive and consistent lesions are produced by systemic treatment of newborn rats in which a single dose of 50 mg/kg results in an apparently permanent loss of 70 to 90% of C unmyelinated fibers. However, this treatment also affects the number of fine AS myelinated fibers (diameters < 2.5 pm) which is reduced by 40 to 60% (Nagy et al., 1983). Many AS fibers have diameters larger than 2.5 Fm and are unaffected by such a neonatal capsaicin treatment. This chemical deafferentation is accompanied by a loss of several peptides in the dorsal root ganglia such as substance P (SP) (Jessel et al., 1978) and calcitonin gene-related peptide (CGRP) (Skofitsch and Jacobowitz, 1985), both of which are contained in type B cells of the sensory ganglia, where they may be colocalized (Skofitsch and Jacobowitz, 1985). The major sources of CGRP in the spinal cord are 0 1990 Wiley-Liss, Inc.
the primary afferents (Gibson et al., 1984). There are two main origins for SP containing fibers in the dorsal horn:
1. The primary afferent fibers (probably C fibers) as described by Jessel et al. (1978); 2. The descending bulbospinal axons, some of which are contained in the dorsolateral funiculus and end in layers I and I1 (Hokfelt et al., 1977). Some of these may be colocalized in serotonergic raphe-spinal neurons (Johansson et al., 198 1). An additional source of SP could be spinal neurons, as it has been shown that SP-containing neurons exist in the dorsal horn (Hunt et a]., 1981). Several groups have investigated the effects of neonatal injections of capsaicin on the dorsal horn cytochemistry. Jancsd and coworkers (198 I ) , using immunocytochemical detection, showed that 50 mgikg of capsaicin on day 2 of life resulted in a decrease of SP staining in the dorsal cord, whereas the serotonin staining did not change. Using biochemical assays, Singer and coworkers (1982) reported that the same treatment produced a 75% decrease in the SP level without affecting the level of serotonin. At variance, Holzer et al. (1981) reported an increase in serotonin levels in the dorsal cord after capsaicin. The reduction of both SP and CGRP contents in the dorsal horn following neonatal capsaicin injection seems to be due to the destruction of C fibers, as higher brain structures and the ventral spinal horn are not affected (Skofitsch and Jacobowitz, 1985). In addition, work by Ruda and coworkers (1 982) have shown that monoaminergic axons are in contact with the neurons of layers I and I1 in the cat. Some of these neurons are enkephalinergic (Glazer and Basbaum, 1983). In the rat, Maxwell and coworkers (1983) reReceived May 10, 1989; revised July 24, 1989; accepted July 26, 1989. Address reprint requests to Lionel Marlier, Lab. Neurobiologie du Developpement. UPR 41 CNRS, U . 249 INSERM-EPHE, Institute de Biologie. Bd. Henri IV, 34060 Montpellier Cedex, France.
Serotonergic Innervation With Capsaicin
ported a paucity of synaptic junctions between serotonergic axons and other structures in the dorsal horn and suggested that the serotonergic system acts through nonsynaptic varicose structures that release serotonin into the extracellular space and in turn diffuse to a distant element. The question arises now as to the consequences of such chemical deafferentation of the dorsal horn: How does it reorganize its circuitry? To investigate the controversial findings on the reactions of serotonergic descending fibers to neonatal capsaicin treatment, we studied the laminar distribution of serotonergic fibers in the dorsal horn of treated and control rats using a highly specific antiserum.
MATERIALS AND METHODS Sprague-Dawley rats (IFFA-CREDO) were injected subcutaneously with 50 mg1kg capsaicin (Sigma), according to the protocol of Jancso et al. (1981), 48 hours after birth. Control litter mate rats received the vehicle (25 PI) consisting of 10% ethanol-l0% tween 80 in sterile saline (9 g/liter). Seven weeks after birth, treated animals were tested for impaired chemosensitivity of the eye in order to assess the behavioral specificity of sensory deprivation induced by capsaicin solution (Szolcsanyi et al., 1975). Briefly, the test consisted of placing 50 PI of a capsaicin solution (100 p.g/ml saline) on the cornea and counting the protective wiping movement of the forepaws. Control rats responded to the toxin with vigorous wipes compared with the treated rats, who rarely responded to the test. Control and lesioned rats were sacrificed under ether anesthesia by intracardiac perfusion with 5% glutaraldehyde in cacodylate (50 mM)-sodium metabisulfite (MBS: 1%) buffer. The lumbar spinal cord was dissected and postfixed overnight in the same fixative. Vibratomecut sections (50 km) were prepared in Tris (50 mM)MBS ( 1 %) buffer and then were transferred into trypsin (0.25%, Gibco) for 5 min and rinsed in Tris-MBS. For immunohistochemical serotonin detection, sections were incubated for 8 min in sodium borohydride (10 mM) in Tris-MBS buffer, rinsed, and then incubated with the primary antiserum. For CGRP and SP detection, the sections were immediatly incubated after trypsin. Both incubations were performed overnight at 4” C in tris-MBS added to a 1 % nonspecific serum (goat) with the following highly specific polyclonal antisera: serotonin antiserum diluted 1120,000 prepared and characterized by Geffard et al. (1985), CGRP, or SP antiserum diluted I / 4,000 (Amersham). The presence of the antigens was detected in Tris (50 mM)-saline buffer according to the peroxidase-antiperoxidase (PAP) method of Sternberger ( I 979) using DAB as a chromogen for peroxidase. Sections from control and treated rats were processed to-
113
gether to avoid differences induced by variations at each step. Sections were finally placed on slides, dehydrated and mounted with Gurr-DPX. SP antiserum has been shown to recognize horse, mouse, cat, monkey, human, and rat SP, but not bombesin, whereas CGRP antiserum recognizes rat, monkey, and human peptides without any other interaction with other peptides assayed (Amersham, personal communication, and present findings). Furthermore, in our hands, preadsorbtion of the antisera with commercial synthetic peptides resulted in abolition of immunostaining.
RESULTS Figure 1A shows CGRP immunostaining of a control animal at low magnification; a high intensity o f staining is present in layer I. Layer 110 is also heavily stained, particularly in the internal half, where layer I I i also appears labeled. Irnmunoreactive neurites were also observed to be coursing through layers IV and V. No CGRP cell body was seen in the dorsal horn, even after colchicine treatment (data not shown), although motoneurons appeared to be immunoreactive in the ventral horn. In a treated rat (Fig. IB), a large reduction of CGRP staining can be noticed: Fine fibers from deepest layers have disappeared, and in layers I and I1 the immunoreactivity is considerably reduced, particularly in the external part. No change occurred in other regions of the spinal cord. In control rats (Fig. IC), a high level of SP immunoreactivity was present in layers I and 11. Some immunostained fibers were observed in layer 111. Occasionally, SP-containing cell bodies were found in layers 11 and I11 after colchicine treatment only (not shown). Capsaicin-treated rats showed a homogeneous decrease of immunoreactivity (Fig. 1 D) in peripheral layers, although the dorsolateral funiculus (DLF) remained strongly immunostained (arrows). Sections from control rats stained for serotonin (Figs. lE, 2A, 3A) presented a trilaminar pattern in the dorsal horn: Layer I and the external part of layer I1 (110) were intensely immunoreactive. At variance, the internal part of layer I1 (Hi) showed sparse immunoreactive fibers (arrows). Finally, the deepest layers of the dorsal horn (I11 to V) contained many immunoreactive fibers, but their density was lower than that found in layers I and 110. At higher magnification (Figs. 2A, 3A,) the “clear band” corresponding to layer IIi appeared conspicuous, particularly with Nomarski interference contrast (Fig. 3A). In treated rats, this trilaminar pattern was disrupted (Figs. 1 F, 2B, 3B). Indeed, thin immunoreactive fibers were numerous in layer IIi, thus obliterating the “clearband” showed in controls. The density of immunoreactive fibers appeared similar in this layer to that of layers 111 and IV located underneath.
114
Marlier et al.
Fig. 1. Immunocytochemical detection of CGRP (A, B), substance P (C, D) and serotonin (E, F) in the dorsal horn of the spinal cord: A , C, and E represent control rats; B , D, and F are the neonatal capsaicin-treated rats. In C and D, arrows point to DLF, in E and F to layer Ili. Bar = 150 k m .
DISCUSSION As described above, there are two main sources of SP in the dorsal horn: 1) the primary afferents; and 2) the descending bulbospinal pathway, where SP may be colocalized with serotonin and also TRH and whose axons course in the DLF.
Previous reports from different laboratories have shown that, after neonatal capsaicin treatment, SP content of the dorsal horn is reduced by 50%, although 85% of the peptide disappear in the dorsal root ganglia (Nagy et al., 1981). We confirm here that the supraspinal source of SP in the dorsal horn remains unaffected by
Serotonergic Innervation With Capsaicin
115
Fig. 2. Immunocytochemical detection of serotonin in the dorsal horn of control (A) or neonatal capsaicin treated rat spinal cord (B). Bar = 150 p m .
capsaicin, as the staining of the DLF appears unchanged in treated rats when compared with controls. Thus, the present results show that SP immunoreactivity is only a partial indicator of the effect of neonatal capsaicin treatment and that CGRP appears to be a better marker of capsaicin-induced lesions than SP. Indeed, CGRP staining, corresponding exclusively to the afferent fibers, is considerably reduced in the dorsal horn of treated rats, particularly in the deepest layers and in the external part of the peripheral layers. Without quantitative data, we
cannot compare the extent of the chemical lesion on CGRP vs. SP in the dorsal horn, but it appears that most of the CGRP fibers were destroyed by capsaicin, as reported by Skofitsch and Jacobowitz (1985). With regard to serotonergic fibers, neonatal capsaicin treatment disrupted the trilaminar pattern of the dorsal horn, inducing fiber growth into layer IIi. A previous publication from Jancso and coworkers (1981) did not report such modifications in the lamination of serotonergic fibers. Indeed, the clear band of layer IIi was not
116
Marlier et al.
CONTROL
CAPSAlClN
Fig. 3. Immunocytochemical detection of serotonin in the dorsal horn of control or neonatal capsaicin-treated rat spinal cord (in Nomarski interference contrast). Bar = 10 k m .
reported in control animals in this study. However, other studies have shown this clear band in the rat dorsal horn (Hunt et al., 1981; Maxwell et al., 1983). In addition, the biochemical data of Holzer (198 l ) , indicated an in-
crease in the level of serotonin in rats treated neonatally with capsaicin. This increase could at least partly correspond to the local sprouting of fibers suggested by our immunocytochemical data. However, this local sprout-
Serotonergic Innervation With Capsaicin
ing cannot be totally responsible for the increase of 50% (2.99 2 0.4 vs. 4.48 ? 0.31) reported by Holzer and coworkers (1981). Actually, the lack of increase reported by Singer and coworkers (1982) in similar conditions would correspond more closely to our results, as only a fine microdissection of the dorsal horn could detect the subtle difference evidenced in our immunocytochemical study. Previous anatomical study from Jancso and colleagues (1981) did not show this change in serotonergic innervation of the rat spinal cord following neonatal capsaicin treatment. These authors used an immunofluorescence approach and, because the PAP method is known to be more sensitive, it is possible that the modifications we report here were undetectable with immunofluorescence. The question then arises as to what happens to serotonergic terminals in capsaicin-treated rats, whose C afferent fibers are largely destroyed. It has been shown by Nagy and Hunt (1983) that the largest AS fibers of the dorsal horn, which remained unaffected by neonatal capsaicin treatment, expanded their arborization pattern from layer 111 to layer 11. We may suppose that this reorganization could influence the serotonergic terminals, which possibly might follow the movement of AS fibers. This would mimic the mechanism by which serotonergic fibers invade the dorsal horn during embryogenesis (Rajaofetra et al., 1989). Such an hypothesis could be tested by studying the relationship between serotonergic terminals and AS fibers in control and capsaicin-treated rats. Because of the absence of a specific marker for A6 fibers, such a study will have to rest on a systematic ultrastructural examination of the dorsal horn of lesioned vs. control animals. In any case, this experimental paradigm appears to be a suitable model to study the reorganization and the plasticity of serotonergic afferents secondary to an experimental modification of primary afferents.
ACKNOWLEDGMENTS The authors acknowledge the help of J.R. Teilhac for art work, D. Dellmann for revision of the manuscript, and C. Bernii for secretarial assistance. Antiserotonin antibody was a generous gift of Dr. M. Geffard. This work has been supported by grants from IRME, AFM, DRET, and D. Heumann fund.
REFERENCES Buck SH, Burks TF (1986): The neuropharmacology of capsaicin: Review of some recent observations. Pharmacol Rev 38: 179226.
117
Geffdrd M, Henrich-Rock AM, Dulluc J (1985): Antisera against small neurotransmetter-like molecules. Neurochem Int 7:403410. Gibson SJ, Polak JM, Bloom SR, Sabate IM, Mulderry PM, Ghatel MA, McGregor GP, Morrison JFB, Kclly JS, Evans RM, Rosenfeld MG ( I 984): Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and eight other species. J Neurosci 4:3101-3111. Glazer EJ, Basbaum A1 (1984): Axons which take u p (3H]herotonin are presynaptic to enkephalin immunoreactive neurons in cat dorsal horn. Brain Res 298:386-391. Hokfelt T, Ljungdahl A , Terenus L, Elde R, Nilsson G (1977): Immunohistochemical analysis of peptide pathways posibly related to pain and analgesia: Enkephalin and substance P. Proc Natl Acad Sci USA 74:3081-3085. Holzer P ( 1988): Local effector functions of capsaicin-sensitive sensory nerve endings: Involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 24: 739 -768. Holzer P, Saria A, Skofitsch G , Lembeck F (1981): Increase in tissue concentrations of histamine and 5-hydroxytryptamine following capsaicin treatment of newborn rats. Life Sci 29:1099-
IIOS. Hunt SP, Kelly JS, Emson PC, Kimmel JR, Miller RJ, Wu JY (1981): An inimunohistochemical study of neuronal populations containing neuropeptides or gamma-aminobutyrate within the superficial layers of the rat dorsal horn. Neuroscience 6: 18831898. Jancsci G, Hokfelt T , Lundberg JM, Kiraly E, Halasz N, Nilsson G , Terenius L, Rehfeld J , Steinbusch H, Verhofstad A , Elde K, Said S, Brown M (1981): Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide and nomoamine neurons using antisera to substance P, gastrin/CCK, somatostatin, VIP, enkephalin, neurotensin and S-hydroxytiyptamine. J Neurocytol 10:963-980. Jessel T M , lversen LL, Cuello AC (1978): Capsaicin-induced depletion of substance P from primary sensory neurons. Brain Res 152:183-1 88. Johansson 0, Hdkfelt T , Pernow B, Jeffcoate SL, White N , Stcinbusch HW, Verhofstad AA, Emson PC, Spindel E (1981): Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance Pand thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord. Neuroscience 6:1857-1881. Maxwell DJ, Leranth C , Verhofstad AAJ (1983): Pine structure of serotonin-containing axons in the marginal zone of the rat spinal cord. Brain Res 266:253-259. Nagy J1, Hunt SP, Iversen LL, Emson PC (1981): Biochemical and anatomical observations on the degeneration of peptide-containing primary afferent neurons after neonatal capsaicin. Neuroscience 10:1923-1934. Nagy J1, Hunt S P ( 1983): The termination of primary afferents within the rat dorsal horn: Evidence for rearrangement following capsaicin treatment. J Comp Neurol 218:145-158. Nagy JI, Iversen LL, Goefert M, Chapman D, Hunt S P (1983): Dosedependent effects of capsaicin on primary sensory neurons in the neonatal rat. J Neurosci 3:399-406. Rajaofetra N, Sandillon F, Geffard M, Privat A (1989): Pre and postnatal ontogeny of serotonergic projections to the spinal cord. J Neurosci Res (in press). Ruda MA, Coffield J, Steinbusch HWM (1982): Immunocytochernical analysis of serotonergic axons in Laminae I and 11 of the lumbar spinal cord of the cat. J Neurosci 2:1660-1671.
118
Marlier et al.
Singer EA, Sperk G, Schmid R (1982): Capsaicin does not change tissue levels of glutamic acid, its uptake, or release in the rat spinal cord. J Neurochcm 38: 13x3-I 386. Skofitsch G, Jacobowitz M (1985): Calcitonin gene-related peptide coexists with substance P in capsaicin scnsitive neurons and sensory ganglia of the mt. Peptides 6:747-754.
Sternherger A (1979): “lmmunocytochemistry.” New York: Wiley and Sons. Szolcsanyi J , Jansco-Gabor A, Joo F (1975): Functional and fine structural characteristics of the sensory neuron blocking effect of capsaicin. Naunyn Schmiedebergs Arch Exp Pharrnacol287: IS7716 3 .
Modification of Serotonergic Innervation of the Rat Spinal Cord Dorsal Horn After Neonatal Capsaicin Treatment L. Marlier, N. Ra.iaofetra, P. Poulat, and A. Privat Laboratoire Neurobiologie du Developpement, UPR 41 CNRS U. 249 INSERM, EPHE, Institut de Biologic, Montpellier, France
Newborn rats were treated with Capsaicin. Seven weeks later, calcitonin gene-related peptide, substance P, and serotonin were detected immunocytochemically in the dorsal horn of the spinal cord. Peptide immunostaining, presumably located in primary afferents, was reduced in intensity, whereas the pattern of serotonin innervation was modified, suggesting a secondary reorganization due to the lesion of afferents. The possible mechanisms of this reorganization are discussed. Key words: CGRP, substance P, immunocytochemistry, plasticity INTRODUCTION Neonatal administration of capsaicin (8-methyl-Nvanillyl-6-nonenamide) results in neurochemical alterations in adult rats. The major injury due to the toxin is a selective and dose-dependent degeneration of distinct populations of primary sensory neurons involved in the mediation of chemogenic pain (Nagy et al., 1983). Capsaicin-treated rats show a decrease in noxious thermal, mechanical, and chemical stimuli transmitted by small, unmyelinated C fibers (Bucket al., 1986; Holzer, 1988). The most extensive and consistent lesions are produced by systemic treatment of newborn rats in which a single dose of 50 mg/kg results in an apparently permanent loss of 70 to 90% of C unmyelinated fibers. However, this treatment also affects the number of fine AS myelinated fibers (diameters < 2.5 pm) which is reduced by 40 to 60% (Nagy et al., 1983). Many AS fibers have diameters larger than 2.5 Fm and are unaffected by such a neonatal capsaicin treatment. This chemical deafferentation is accompanied by a loss of several peptides in the dorsal root ganglia such as substance P (SP) (Jessel et al., 1978) and calcitonin gene-related peptide (CGRP) (Skofitsch and Jacobowitz, 1985), both of which are contained in type B cells of the sensory ganglia, where they may be colocalized (Skofitsch and Jacobowitz, 1985). The major sources of CGRP in the spinal cord are 0 1990 Wiley-Liss, Inc.
the primary afferents (Gibson et al., 1984). There are two main origins for SP containing fibers in the dorsal horn:
1. The primary afferent fibers (probably C fibers) as described by Jessel et al. (1978); 2. The descending bulbospinal axons, some of which are contained in the dorsolateral funiculus and end in layers I and I1 (Hokfelt et al., 1977). Some of these may be colocalized in serotonergic raphe-spinal neurons (Johansson et al., 198 1). An additional source of SP could be spinal neurons, as it has been shown that SP-containing neurons exist in the dorsal horn (Hunt et a]., 1981). Several groups have investigated the effects of neonatal injections of capsaicin on the dorsal horn cytochemistry. Jancsd and coworkers (198 I ) , using immunocytochemical detection, showed that 50 mgikg of capsaicin on day 2 of life resulted in a decrease of SP staining in the dorsal cord, whereas the serotonin staining did not change. Using biochemical assays, Singer and coworkers (1982) reported that the same treatment produced a 75% decrease in the SP level without affecting the level of serotonin. At variance, Holzer et al. (1981) reported an increase in serotonin levels in the dorsal cord after capsaicin. The reduction of both SP and CGRP contents in the dorsal horn following neonatal capsaicin injection seems to be due to the destruction of C fibers, as higher brain structures and the ventral spinal horn are not affected (Skofitsch and Jacobowitz, 1985). In addition, work by Ruda and coworkers (1 982) have shown that monoaminergic axons are in contact with the neurons of layers I and I1 in the cat. Some of these neurons are enkephalinergic (Glazer and Basbaum, 1983). In the rat, Maxwell and coworkers (1983) reReceived May 10, 1989; revised July 24, 1989; accepted July 26, 1989. Address reprint requests to Lionel Marlier, Lab. Neurobiologie du Developpement. UPR 41 CNRS, U . 249 INSERM-EPHE, Institute de Biologie. Bd. Henri IV, 34060 Montpellier Cedex, France.
Serotonergic Innervation With Capsaicin
ported a paucity of synaptic junctions between serotonergic axons and other structures in the dorsal horn and suggested that the serotonergic system acts through nonsynaptic varicose structures that release serotonin into the extracellular space and in turn diffuse to a distant element. The question arises now as to the consequences of such chemical deafferentation of the dorsal horn: How does it reorganize its circuitry? To investigate the controversial findings on the reactions of serotonergic descending fibers to neonatal capsaicin treatment, we studied the laminar distribution of serotonergic fibers in the dorsal horn of treated and control rats using a highly specific antiserum.
MATERIALS AND METHODS Sprague-Dawley rats (IFFA-CREDO) were injected subcutaneously with 50 mg1kg capsaicin (Sigma), according to the protocol of Jancso et al. (1981), 48 hours after birth. Control litter mate rats received the vehicle (25 PI) consisting of 10% ethanol-l0% tween 80 in sterile saline (9 g/liter). Seven weeks after birth, treated animals were tested for impaired chemosensitivity of the eye in order to assess the behavioral specificity of sensory deprivation induced by capsaicin solution (Szolcsanyi et al., 1975). Briefly, the test consisted of placing 50 PI of a capsaicin solution (100 p.g/ml saline) on the cornea and counting the protective wiping movement of the forepaws. Control rats responded to the toxin with vigorous wipes compared with the treated rats, who rarely responded to the test. Control and lesioned rats were sacrificed under ether anesthesia by intracardiac perfusion with 5% glutaraldehyde in cacodylate (50 mM)-sodium metabisulfite (MBS: 1%) buffer. The lumbar spinal cord was dissected and postfixed overnight in the same fixative. Vibratomecut sections (50 km) were prepared in Tris (50 mM)MBS ( 1 %) buffer and then were transferred into trypsin (0.25%, Gibco) for 5 min and rinsed in Tris-MBS. For immunohistochemical serotonin detection, sections were incubated for 8 min in sodium borohydride (10 mM) in Tris-MBS buffer, rinsed, and then incubated with the primary antiserum. For CGRP and SP detection, the sections were immediatly incubated after trypsin. Both incubations were performed overnight at 4” C in tris-MBS added to a 1 % nonspecific serum (goat) with the following highly specific polyclonal antisera: serotonin antiserum diluted 1120,000 prepared and characterized by Geffard et al. (1985), CGRP, or SP antiserum diluted I / 4,000 (Amersham). The presence of the antigens was detected in Tris (50 mM)-saline buffer according to the peroxidase-antiperoxidase (PAP) method of Sternberger ( I 979) using DAB as a chromogen for peroxidase. Sections from control and treated rats were processed to-
113
gether to avoid differences induced by variations at each step. Sections were finally placed on slides, dehydrated and mounted with Gurr-DPX. SP antiserum has been shown to recognize horse, mouse, cat, monkey, human, and rat SP, but not bombesin, whereas CGRP antiserum recognizes rat, monkey, and human peptides without any other interaction with other peptides assayed (Amersham, personal communication, and present findings). Furthermore, in our hands, preadsorbtion of the antisera with commercial synthetic peptides resulted in abolition of immunostaining.
RESULTS Figure 1A shows CGRP immunostaining of a control animal at low magnification; a high intensity o f staining is present in layer I. Layer 110 is also heavily stained, particularly in the internal half, where layer I I i also appears labeled. Irnmunoreactive neurites were also observed to be coursing through layers IV and V. No CGRP cell body was seen in the dorsal horn, even after colchicine treatment (data not shown), although motoneurons appeared to be immunoreactive in the ventral horn. In a treated rat (Fig. IB), a large reduction of CGRP staining can be noticed: Fine fibers from deepest layers have disappeared, and in layers I and I1 the immunoreactivity is considerably reduced, particularly in the external part. No change occurred in other regions of the spinal cord. In control rats (Fig. IC), a high level of SP immunoreactivity was present in layers I and 11. Some immunostained fibers were observed in layer 111. Occasionally, SP-containing cell bodies were found in layers 11 and I11 after colchicine treatment only (not shown). Capsaicin-treated rats showed a homogeneous decrease of immunoreactivity (Fig. 1 D) in peripheral layers, although the dorsolateral funiculus (DLF) remained strongly immunostained (arrows). Sections from control rats stained for serotonin (Figs. lE, 2A, 3A) presented a trilaminar pattern in the dorsal horn: Layer I and the external part of layer I1 (110) were intensely immunoreactive. At variance, the internal part of layer I1 (Hi) showed sparse immunoreactive fibers (arrows). Finally, the deepest layers of the dorsal horn (I11 to V) contained many immunoreactive fibers, but their density was lower than that found in layers I and 110. At higher magnification (Figs. 2A, 3A,) the “clear band” corresponding to layer IIi appeared conspicuous, particularly with Nomarski interference contrast (Fig. 3A). In treated rats, this trilaminar pattern was disrupted (Figs. 1 F, 2B, 3B). Indeed, thin immunoreactive fibers were numerous in layer IIi, thus obliterating the “clearband” showed in controls. The density of immunoreactive fibers appeared similar in this layer to that of layers 111 and IV located underneath.
114
Marlier et al.
Fig. 1. Immunocytochemical detection of CGRP (A, B), substance P (C, D) and serotonin (E, F) in the dorsal horn of the spinal cord: A , C, and E represent control rats; B , D, and F are the neonatal capsaicin-treated rats. In C and D, arrows point to DLF, in E and F to layer Ili. Bar = 150 k m .
DISCUSSION As described above, there are two main sources of SP in the dorsal horn: 1) the primary afferents; and 2) the descending bulbospinal pathway, where SP may be colocalized with serotonin and also TRH and whose axons course in the DLF.
Previous reports from different laboratories have shown that, after neonatal capsaicin treatment, SP content of the dorsal horn is reduced by 50%, although 85% of the peptide disappear in the dorsal root ganglia (Nagy et al., 1981). We confirm here that the supraspinal source of SP in the dorsal horn remains unaffected by
Serotonergic Innervation With Capsaicin
115
Fig. 2. Immunocytochemical detection of serotonin in the dorsal horn of control (A) or neonatal capsaicin treated rat spinal cord (B). Bar = 150 p m .
capsaicin, as the staining of the DLF appears unchanged in treated rats when compared with controls. Thus, the present results show that SP immunoreactivity is only a partial indicator of the effect of neonatal capsaicin treatment and that CGRP appears to be a better marker of capsaicin-induced lesions than SP. Indeed, CGRP staining, corresponding exclusively to the afferent fibers, is considerably reduced in the dorsal horn of treated rats, particularly in the deepest layers and in the external part of the peripheral layers. Without quantitative data, we
cannot compare the extent of the chemical lesion on CGRP vs. SP in the dorsal horn, but it appears that most of the CGRP fibers were destroyed by capsaicin, as reported by Skofitsch and Jacobowitz (1985). With regard to serotonergic fibers, neonatal capsaicin treatment disrupted the trilaminar pattern of the dorsal horn, inducing fiber growth into layer IIi. A previous publication from Jancso and coworkers (1981) did not report such modifications in the lamination of serotonergic fibers. Indeed, the clear band of layer IIi was not
116
Marlier et al.
CONTROL
CAPSAlClN
Fig. 3. Immunocytochemical detection of serotonin in the dorsal horn of control or neonatal capsaicin-treated rat spinal cord (in Nomarski interference contrast). Bar = 10 k m .
reported in control animals in this study. However, other studies have shown this clear band in the rat dorsal horn (Hunt et al., 1981; Maxwell et al., 1983). In addition, the biochemical data of Holzer (198 l ) , indicated an in-
crease in the level of serotonin in rats treated neonatally with capsaicin. This increase could at least partly correspond to the local sprouting of fibers suggested by our immunocytochemical data. However, this local sprout-
Serotonergic Innervation With Capsaicin
ing cannot be totally responsible for the increase of 50% (2.99 2 0.4 vs. 4.48 ? 0.31) reported by Holzer and coworkers (1981). Actually, the lack of increase reported by Singer and coworkers (1982) in similar conditions would correspond more closely to our results, as only a fine microdissection of the dorsal horn could detect the subtle difference evidenced in our immunocytochemical study. Previous anatomical study from Jancso and colleagues (1981) did not show this change in serotonergic innervation of the rat spinal cord following neonatal capsaicin treatment. These authors used an immunofluorescence approach and, because the PAP method is known to be more sensitive, it is possible that the modifications we report here were undetectable with immunofluorescence. The question then arises as to what happens to serotonergic terminals in capsaicin-treated rats, whose C afferent fibers are largely destroyed. It has been shown by Nagy and Hunt (1983) that the largest AS fibers of the dorsal horn, which remained unaffected by neonatal capsaicin treatment, expanded their arborization pattern from layer 111 to layer 11. We may suppose that this reorganization could influence the serotonergic terminals, which possibly might follow the movement of AS fibers. This would mimic the mechanism by which serotonergic fibers invade the dorsal horn during embryogenesis (Rajaofetra et al., 1989). Such an hypothesis could be tested by studying the relationship between serotonergic terminals and AS fibers in control and capsaicin-treated rats. Because of the absence of a specific marker for A6 fibers, such a study will have to rest on a systematic ultrastructural examination of the dorsal horn of lesioned vs. control animals. In any case, this experimental paradigm appears to be a suitable model to study the reorganization and the plasticity of serotonergic afferents secondary to an experimental modification of primary afferents.
ACKNOWLEDGMENTS The authors acknowledge the help of J.R. Teilhac for art work, D. Dellmann for revision of the manuscript, and C. Bernii for secretarial assistance. Antiserotonin antibody was a generous gift of Dr. M. Geffard. This work has been supported by grants from IRME, AFM, DRET, and D. Heumann fund.
REFERENCES Buck SH, Burks TF (1986): The neuropharmacology of capsaicin: Review of some recent observations. Pharmacol Rev 38: 179226.
117
Geffdrd M, Henrich-Rock AM, Dulluc J (1985): Antisera against small neurotransmetter-like molecules. Neurochem Int 7:403410. Gibson SJ, Polak JM, Bloom SR, Sabate IM, Mulderry PM, Ghatel MA, McGregor GP, Morrison JFB, Kclly JS, Evans RM, Rosenfeld MG ( I 984): Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and eight other species. J Neurosci 4:3101-3111. Glazer EJ, Basbaum A1 (1984): Axons which take u p (3H]herotonin are presynaptic to enkephalin immunoreactive neurons in cat dorsal horn. Brain Res 298:386-391. Hokfelt T, Ljungdahl A , Terenus L, Elde R, Nilsson G (1977): Immunohistochemical analysis of peptide pathways posibly related to pain and analgesia: Enkephalin and substance P. Proc Natl Acad Sci USA 74:3081-3085. Holzer P ( 1988): Local effector functions of capsaicin-sensitive sensory nerve endings: Involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 24: 739 -768. Holzer P, Saria A, Skofitsch G , Lembeck F (1981): Increase in tissue concentrations of histamine and 5-hydroxytryptamine following capsaicin treatment of newborn rats. Life Sci 29:1099-
IIOS. Hunt SP, Kelly JS, Emson PC, Kimmel JR, Miller RJ, Wu JY (1981): An inimunohistochemical study of neuronal populations containing neuropeptides or gamma-aminobutyrate within the superficial layers of the rat dorsal horn. Neuroscience 6: 18831898. Jancsci G, Hokfelt T , Lundberg JM, Kiraly E, Halasz N, Nilsson G , Terenius L, Rehfeld J , Steinbusch H, Verhofstad A , Elde K, Said S, Brown M (1981): Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide and nomoamine neurons using antisera to substance P, gastrin/CCK, somatostatin, VIP, enkephalin, neurotensin and S-hydroxytiyptamine. J Neurocytol 10:963-980. Jessel T M , lversen LL, Cuello AC (1978): Capsaicin-induced depletion of substance P from primary sensory neurons. Brain Res 152:183-1 88. Johansson 0, Hdkfelt T , Pernow B, Jeffcoate SL, White N , Stcinbusch HW, Verhofstad AA, Emson PC, Spindel E (1981): Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance Pand thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord. Neuroscience 6:1857-1881. Maxwell DJ, Leranth C , Verhofstad AAJ (1983): Pine structure of serotonin-containing axons in the marginal zone of the rat spinal cord. Brain Res 266:253-259. Nagy J1, Hunt SP, Iversen LL, Emson PC (1981): Biochemical and anatomical observations on the degeneration of peptide-containing primary afferent neurons after neonatal capsaicin. Neuroscience 10:1923-1934. Nagy J1, Hunt S P ( 1983): The termination of primary afferents within the rat dorsal horn: Evidence for rearrangement following capsaicin treatment. J Comp Neurol 218:145-158. Nagy JI, Iversen LL, Goefert M, Chapman D, Hunt S P (1983): Dosedependent effects of capsaicin on primary sensory neurons in the neonatal rat. J Neurosci 3:399-406. Rajaofetra N, Sandillon F, Geffard M, Privat A (1989): Pre and postnatal ontogeny of serotonergic projections to the spinal cord. J Neurosci Res (in press). Ruda MA, Coffield J, Steinbusch HWM (1982): Immunocytochernical analysis of serotonergic axons in Laminae I and 11 of the lumbar spinal cord of the cat. J Neurosci 2:1660-1671.
118
Marlier et al.
Singer EA, Sperk G, Schmid R (1982): Capsaicin does not change tissue levels of glutamic acid, its uptake, or release in the rat spinal cord. J Neurochcm 38: 13x3-I 386. Skofitsch G, Jacobowitz M (1985): Calcitonin gene-related peptide coexists with substance P in capsaicin scnsitive neurons and sensory ganglia of the mt. Peptides 6:747-754.
Sternherger A (1979): “lmmunocytochemistry.” New York: Wiley and Sons. Szolcsanyi J , Jansco-Gabor A, Joo F (1975): Functional and fine structural characteristics of the sensory neuron blocking effect of capsaicin. Naunyn Schmiedebergs Arch Exp Pharrnacol287: IS7716 3 .
