Journal of Neuroscience Research 31:34&358 (1992)
Plasticity of the Serotonergic Innervation of the Dorsal Horn of the Rat Spinal Cord Following Neonatal Capsaicin Treatment L. Marlier, P. Poulat, N. Rajaofetra, F. Sandillon, and A. Privat Developement, Plasticit6 et Vieillissement du Systeme Nerveux, U.S.T.L. Case Coumer 106, Montpellier Cedex 05, France chemical (Buck et al., 1981; Holzer et al., 1988). Moreover, C afferent fibers, which mediate chemogenic pain, are particularly, and dose-dependently, lesioned by neonatal capsaicin treatment (Nagy et al., 1983). The disappearance of C unmyelinated afferent fibers is accompanied by a loss of several neuropeptides in both dorsal root ganglia (DRG) and the dorsal horn of the spinal cord (Nagy et al., 1981; Jancs6 et al., 1981; Buck et al., 1981; Priestley et al., 1982). Among these peptides, substance P and calcitonin gene-related peptide (CGRP) appear to be frequently colocalized in DRG (Skofitsch and Jacobowitz, 1985). Nevertheless, if the dose of capsaicin classically used (i.e., 50 mg/kg) destroys about 90% of C fibers, it also destroys some thin myelinated AI fibers whose neurotransmitter(s) remain unknown (Nagy et al., 1983). In addition, this commonly used treatment leads to an incomplete loss of substance P, i.e., 55% (Nagy et al., 1983) or 67% (Skofitsch and Jacobowitz, 1985); in the latter study, CGRP reduction was the same as substance P. As substance P-fibers in the dorsal horn originates from three regions, namely, DRG (Jesse1 et al., 1978), intrinsic cells of the dorsal horn (Senba et al., 1982) and bulbospinal pathways (Hokfelt et al., 1977), the loss of substance P in the dorsal horn cannot directly represent the loss of C fibers while this correlation appears to be acceptable for CGRP Key words: calcitonin gene-related peptide, sub- fibers whose origin, in the dorsal horn, is restricted to the stance P,nociception, electron microscopy, immuno- DRG (Gibson et al., 1984). cytochemistry If several research groups have investigated the effects of neonatal capsaicin treatment on the neuropeptide content of primary afferents, little is known about the INTRODUCTION consequences of capsaicin treatment on bulbospinal Capsaicin (8-methyl-N-vanillyl-6-nonenamide),a aminergic pathways. Among amines, serotonin is constituent of red peppers, is a widely used tool in studying primary nociceptive afferent pathways (see review in Buck and Burks, 1986). Whereas little is known about its exact mechanisms of action, capsaicin effects appear to Received June 4, 1990; revised July 30, 1991; accepted August 5 , be mediated by a severe destruction of primary afferent 1991. sensory neurons (Jancsb et al., 1977). Rats treated at Address reprint requests to L. Marlier, Developement, Plasticit6 et birth with capsaicin exhibit a reduced response to nox- Vieillissement du Systerne Nerveux, U.S.T.L.-Case Courrier 106, ious stimuli, mainly thermal, but also mechanical or Place Eugkne Bataillon, 34095 Montpellier Cedex 05, France.
Neonatal capsaicin treatments (25 or 50 mg/kg, 12, 24, or 48 hr after birth given subcutaneously) were applied in order to follow by immunocytochemical techniques the postnatal development and plasticity of the serotonergic system in the dorsal horn of the rat spinal cord. Two markers of the lesions of C primary afferents induced by capsaicin were tested by immunocytochemical detection: substance P and calcitonin gene-related peptide (CGRP). We show that the internal part of substantia gelatinosa (lamina Ili) which does not contain serotonergic fibers in intact or vehicle-treated rats is invaded within a few days after capsaicin treatment by serotonergic fibers apparently sprouting from the deepest laminae. Moreover, these fibers often establish axodendritic synapses while synapses are rare in intact animals in the whole dorsal horn. This reorganization is stable whatever the dose of capsaicin used or the moment chosen for its injection. On the other hand, while lesions of substance P-ergic fibers appeared quite stable, partial recovery of CGRP innervation was found after 3 to 6 months, especially with the low dose of capsaicin. We discuss the ability of the serotonergic system innervating the dorsal cord either to find new targets or to fill vacated sites when one of its putative targets is removed.
0 1992 Wiley-Liss, Inc.
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known, like noradrenaline, to modulate the nociception (see review in Besson and Chaouch, 1987). Serotonergic pathways originate from the caudal raphe nuclei, namely, nucleus raphe magnus, obscurus, and pallidus and terminate in the dorsal horn of the spinal cord (Dahlstrom and Fuxe, 1964; Basbaum and Fields, 1979). In raphe nuclei, serotonin has been shown to coexist with several peptides and, among them, with substance P (Chan-Palay, 1979a,b; Hokfelt et al., 1978; Johansson et al., 1981; MCnetrey and Basbaum, 1987); that is the third origine for substance P afferent fibers in the dorsal horn as described above. According to the cytoarchitectonic organization of the dorsal horn of the rat spinal cord (Molander et al., 1984, 1989), serotonergic innervation has been located to laminae I, 110, and 111 to V, while lamina IIi appears almost devoid of innervation (Maxwell et al., 1983; Marlier et al., 1990, 1991). We recently showed that, after subcutaneous neonatal capsaicin treatment (50 mg/kg, 48 hr after birth), this pattern was modified, lamina IIi being invaded by serotonergic immunoreactive profiles (Marlier et al., 1990). Moreover, previous work by Holzer et al. (1981) reported, using a biochemical assay, an increase in the serotonergic content of the rat dorsal horn after neonatal capsaicin treatment. Nevertheless, controversy exists, as other authors have not reported such an increase after capsaicin treatment (Singer et al., 1982). Such a reorganization of the serotonergic innervation of the rat spinal cord would illustrate, firstly, the plasticity of the system, but, secondly, it implies that new interactions with other components of the dorsal horn, such as other fibers (myelinated or not), or cells, might occur in the internal part of lamina 11. The aim of this study was, primarily, to investigate, with the use of immunocytochemistry, the modification of the innervation of the lamina IIi by serotonergic axons. Immunocytochemistry was also carried at the ultrastructural level in order to provide additional data upon the cytological reorganization. Moreover, we investigated the effect of a low dose of capsaicin which would produce a lesion of C fibers without inducing any lesions on Ai fibers (Nagy et al., 1983). Finally, in order to interact as early as possible with the postnatal ontogeny of both primary and serotonergic afferent fibers, we tested the effect of capsaicin injections immediately after birth. As substance P- and CGRP-like immunoreactivities appear to be severely reduced after capsaicin (Skofitsch and Jacobowitz, 1985), we used these two markers as an index of the severity of the lesions. The latter and their consequences on serotonergic innervation were followed over a period of 6 months in order to evaluate the stability of the peptidergic lesions on the one hand, and the reorganization of the serotonergic system, on the other hand.
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MATERIALS AND METHODS Capsaicin Treatment Capsaicin (Sigma) was injected subcutaneously into newborn Sprague Dawley rats (IFFA CREDO) in a single injection of 25 pl, 12 (POS), 24 (Pl), or 48 hr (P2) after birth. Injections consisted of 25 (C25) or 50 mg/kg (C50) capsaicin freshly prepared and diluted in 10% alcohol-10% Tween 80 in saline according to Jancs6 and coworkers (1977). All the pups in a given litter received the same dose of capsaicin at the same time, while control littermates received 25 pl of vehicle. Six combinations of capsaicin dose and injection times were studied, at 6 survival periods ranging from 1 week to 6 months: three groups of pups received 25 mg/kg (C25) at 12 (C25/J0.5), 24 h (C25/J1) or 48 hr after birth (C25/J2) and three groups received 50 mg/kg (C50) at 12 (C50/J0.5), 24 (C50/J1) or 48 hr after birth (C50/J2). Some combinations, especially for survival periods of l week and 6 months, were not analyzed in detail as the number of pups treated varied from one litter to another. We therefore prefemed to remove a few groups from this study rather than reduce the number of animals per stage. Assessment of Capsaicin Injury In order to test for impaired chemosensitivity of the cornea following capsaicin treatment, the animals were tested for protective wipings of the forepaws. A capsaicin solution (100 pg/ml in 10% alcohol in saline) was instillated into the eyes according to Szolcsanyi et al. (1975). Such a behavioral test is only possible on rats more than 2 weeks old whose eyelids are open. Capsaicin-treated rats respond to capsaicin instillation with rare protective movements, compared with vehicle-treated ones, which react actively. Immunocytochemistry Animals from each group and control rats, were sacrificed after 1 , 2 , and 3 weeks, and 1 , 3 , and 6 months after birth. For immunocytochemistry , rats were sacrificed after deep ether anesthesia (1- and 2-week-old rats) or after pentobarbital (50 mg/kg) anesthesia (older rats) by intracardiac perfusion of fixative. After thoracotomy , 2 ml/kg of sodium nitrite (5% in water)-heaprine (500 UI) were intracardially injected. Animals were then perfused as follows: 1/100 ml/kg of washing solution consisting of cacodylic acid (50 mM)-sodium metabisulfite (MBS = 50 mM) buffer at pH 7.6; 2/1 Wkg of glutaraldehyde 5% in the same buffer (Geffard et al., 1985). The animals were then placed for 2-4 hr at 4 ° C after which the spinal cords were removed and postfixed for 24 hr in fresh fixative. Transverse vibratome sections
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Marlier et al. TABLE I. Immunocytochemical Data* 1 week
2 weeks
HT
SP
CGRP
5-HT
C25/JO.5 C25/J1 C25iJ2 C5O/JO.5 C50/J 1 C50/J2
++ ++ ++ NT ++
NT
NT
NT
+ + ++ NT ++
+ + + NT +
SP
CGRP
5-
+
++ ++ NT +
+ ++
++ NT ++
+ ++
+
+ + + ++
++
+ ++ + ++ + ++
+
++ ++
1 month
3 weeks C2WJ0.5 C25/J1 C25/J2 C50/J0.5 C50/J1 C50/J2
+ ++
NT
++ ++ ++ + + ++
+ ++ ++ + ++
+
3 months
+ + ++ + ++ ++
+
+ ++ + + ++
6 months
~
C25iJ0.5
(+I
(+I
NT
C25iJ 1
++
NT
NT
(+) ++ + (+I (+I + + + + C25IJ2 NT NT NT + + ++ C50/J0.5 + + ++ ++ + ++ C50lJ 1 NT NT NT ++ + ++ C50iJ2 ++ + ++ *For SP and cGRP, + indicates a mild and + + a marked decrease of immunoreactivity in the superficial layers of the dorsal horn. For 5.HT, + and + + correspond, respectively, to a
discrete and an important reorganization of the innervation pattern, leading to invasion of lamina IIi. NT = not tested.
(50 pm thick) were performed in Tris (50 mM)-MBS (50 mM) buffer (pH 7.4), and then transferred to trypsinEDTA (Gibco) for 5 min (except sections from 1- and 2-weeks-old rats) and rinsed in the Tris-MBS buffer. For serotonin immunocytochemistry only, a step of sodium borohydride (10 mM for 8 min in the same buffer) was added. Sections were then incubated in primary sera at 4°C overnight. Immunocytochemical detection of substance P (Amersham antiserum diluted 1:4,000), calcitonin gene-related peptide (CGRP; Amersham antiserum diluted 1:4,000), and serotonin (antiserum prepared by M. Geffard, 1985, diluted 1:20,000) was assayed in the same buffer added in non-specific antiserum (goat; NSS = 1%) and Triton X-100 (0.1%). Triton was omitted for sections from 1- and 2-week-old pups and for those used for electron microscopy. Specificity of these antisera have previously been discussed (Marlier et al., 1990). Detection of the primary antiserum obtained in rabbits was realized according to Sternberger (1979). The processing consisted successively of goat anti-rabbit antiserum (Nordica; 1% in Tris 50 mM-saline added in NSS 1% for 30 min at room temperature), PAP antiserum in the same conditions (Biosys). The peroxidase product was revealed with diaminobenzidine (0.05%) and hydrogen
peroxide (0.01%) in the same buffer. Finally, sections were transferred in 50 mM phosphate buffer at pH 7.6. For light microscopic studies, sections were deposited on slides, dried overnight, cleared in xylene, and mounted with Gurr-DPX. For each stage, we graded ( + ), + , or + the extent of the lesions for peptides and the invasion of lamina IIi for serotonin. These data are reported in Table I.
+
Electron Microscopy Sections were postfixed in 4% osmium tetroxide in 0.1 M phosphate buffer, dehydrated in graded alcohol, then in acetone and placed for 1 hr to araldite-acetone (1 :1). They were transferred overnight in araldite-acetone (3:1) at room temperature. After two baths of 2 hr at 37°C in araldite, sections were flat-embedded in araldite. After reembedding on cylindrical blocks, ultrathin sections were cut with a diamond knife, collected on grids and counterstained with uranyl-acetate and lead-citrate. RESULTS General Considerations The use of the test described by Szolcsanyi et al. (1975) allowed us clearly to identify the lesioned animals
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Fig. 1 . Immunocytochemical detection of CGRP (A,B) and serotonin (C,D) in the spinal cord of 1-week-old rats. A,C: Control animals. B,D: Animals lesioned with capsaicin (50
mg/kg, 48 hr after birth; C50/J2). Arrows point to serotonergic immunoreactive fibers invading the superficial laminae of the dorsal horn. A,B: Bars equal 100 pm. C,D: Bars equal 50 pm.
only after 3 weeks, as the eyelids are closed before then. Animals that received 50 mk/kg (C50 groups) exhibited, most of the time, only a few wiping movements of the forepaws. On the other hand, control animals responded to the application of capsaicin in the eye by vigorous movements. An ‘‘intermediate” behaviour was obtained with animals of the C25 groups. We did not try to establish further correlations between the response to the test and the moment chosen for the injection of capsaicin. Nevertheless, all treated animals which behaved like the controls (i.e., by vigorous wiping) were excluded from the study. With regard to the different immunocytochemical detections studied, we never observed any significant difference between control (vehicle-treated) and intact animals, whatever the stage of the study (Table I). Before 1 month, both substance P-ergic and serotonergic innervation of the dorsal horn exhibited variable degrees of maturity, while CGRP-ergic innervation dis-
played a pattern comparable to that of an adult after 2 weeks. The detailed analysis, stage by stage, of the light microscopic study is reported below. For electron microscopy, we chose to illustrate the l month stage.
1-Week-Old Rats For substance P, control animals exhibited a discrete innervation of the dorsal horn. Immunoreactivity was severely reduced in all groups of capsaicin-treated rats. For CGRP, we noticed in control rats, as stated above, an immunoreactive pattern close to that of the adult: laminae I and I1 already appeared strongly immunoreactive, while isolated fibers could be traced in the deepest laminae (i.e., I11 and IV; Fig. 1A). In capsaicintreated rats, the staining was slightly reduced in C25 groups, while C50 groups exhibited a strong reduction in the number of immunostained fibers (Fig. 1B).
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For serotonin, we observed in the controls rare fibers in the superficial laminae of the dorsal horn (Fig. 1C). In all capsaicin-treated animals many fibers invaded the superficial laminae (Fig. 1D).
2-Week-Old Rats Control sections stained for substance P showed a band of immunoreactivity corresponding to laminae I and I1 while the deepest laminae were unstained, except for a few growing fibers (Fig. 2A). Sections from capsaicin-treated rats exhibited a reduction of this staining with minor variations among the different groups (Fig. 2B). For CGRP, control animals had an immunoreactive pattern qualitatively similar to that of adults (Fig. 2C). On lesioned animals, immunoreactivity was markedly decreased (Fig. 2D), with, here too, minor variations between groups. The immunoreactive pattern of serotonin was clearly trilaminar in controls as in adults. Immunoreactive fibers were present in the outer laminae (I and IIo), and in the deepest lamine (111, IV) of the dorsal horn, and were separated by a clear band tentatively corresponding to lamina Ili of the adult in which only rare fibers were seen (Fig. 2E). All the capsaicin-lesioned animals showed fibers sprouting in this clear band, which appeared to arise from the deepest laminae (Fig. 2F). Minor variations were seen between groups. 3-Week-Old Rats The immunoreactive pattern for substance P in control sections was now organized as in adults: numerous immunoreactive fibers were located in laminae I and I1 while they were rare in the deepest laminae (Fig. 3D). Capsaicin-treated groups showed a decrease in the staining intensity in a dose-dependent manner (Fig. 3E,F). It is interesting to note that in all capsaicin-treated rats, the dorsolateral funiculus (DLF) remained immunoreactive (arrows), testifying the preservation of bulbospinal substance P fibers. The same dose-dependency versus capsaicin holds true for CGRP (Fig. 3A-C). The pattern of serotonin immunoreactivity showed the same modifications as described in previous stages when compared with controls (Fig. 3G): immunoreactive fibers were seen within the clear band in treated rats (Fig. 3H). As this pattern had now reached an adult status, the clear band can now be equated with lamina IIi, the internal part of substantia gelatinosa. Once again, no major differences were found between groups of capsaicintreated rats regarding the reorganization of the serotonergic immunoreactive pattern. However, C25 groups, which showed relatively minor lesions for peptides compared with C50 groups, exhibited, except when com-
pared to the C50/J2 combination, a more marked modification of the serotonergic pattern.
1-Month-Old Rats The extent of the lesions for substance P and CGRP were approximately the same as in the previous stage (Fig. 4A-D). For substance P, a relation between the time of the injection and the extent of the lesions now appears, the latest injections giving better results than the earliest. For serotonin, we also noticed, in all groups of treated animals, immunoreactive fibers within laminae IIi (Fig. 4F), while the clear band was conspicuous in the controls (Fig. 4E). 2-Month-Old Rats The intensity of immunostaining for substance P reached a plateau in control animals with the highest intensity in lamina I (Fig. 5D). The dose-dependency is maintained for capsaicin treatment (Fig. 5E,F), as well as the relation between the extent of the lesions and the time of the injection. For CGRP, the picture was generally similar to that of previous stages (Fig. 5A-C), but the extent of the lesions was reduced when compared with substance P at the same survival period but also when compared with CGRP at 1 month. The lesions remained most extensive in the lateral part of the dorsal horn. Finally, the serotonergic immunoreactive pattern was in each case disrupted versus controls (Fig. 5G) with fibers sprouting in lamina 111 (Fig. 5H). 6-Month-Old Rats For the three series studied (C25/J 1, C5O/JO.5, and C50/J2) the results were the same as those found after 3 months for the three neurotransmitters. Electron Microscopy Electron microscopy studies of serotonin immunoreactivity were performed after a survival period of 1 month, in the C50/J2 group. In the controls, the relationships already described for serotonergic endings in the rat spinal cord (Maxwell et al., 1983; Marlier et al., submitted) were confirmed. Indeed, serotonergic immunoreactive profiles were found in laminae I, 110,111, and IV, while extremely rare profiles occurred in lamina IIi. Profiles were usually medium-sized (diameter of about 1 pm); half of them contained vesicles, but among the latter no more than 20% established conventional synapses. These were type TI synapses primarily located on medium-sized, non-vesicular dendrites (Fig. 6B), but rare axo-somatic synapses were also found (Fig. 6C). Most of the serotonergic boutons were dome-shaped (Fig. 6A), but scalloped boutons
A
B
C
D
Fig. 2. Immunocytochemical detection of substance P (A,B), CGRP (C,D), and serotonin (E,F) in 2-week-old rats. A,C,E: Control animals. B,D,F: Animals treated with capsaicin (50 mglkg, 48 hr after birth; C50/J2). In A, arrow points to a
growing fiber invading lamina I1 from the surface. In E, the indications refer to laminae according to Molander. A,B,E,F: Bars equal 50 p.m. C,D: Bars equal 100 p.m.
A
D
B
E
C
F
G
H
Fig. 3. Immunocytochemical detection of CGRP (A-C), substance P (D-F), and serotonin (G,H) in 3-week-old rats, illustrating the dose-dependency for CGRP and substance P. A,D,G: Control animals. B,C,E,F,H: Animals treated with
capsaicin; 25 mg/kg, 12 hr after birth (C23J0.5) in B and E or 50 mg/kg, 48 hr after birth (C50/J2) in C, F, and H. Arrows point to DLF on sections stained for substance P. A-F: Bars equal 100 pm. G,H: Bars equal 50 pm.
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A
B
C
D
E
F
Fig. 4. Immunocytochemical detection of substance P (A,B), CGRP (C,D), and serotonin (E,F) in 1-month-old rats. A,C,E: Control animals. B,D,F: Animals treated with capsaicin-25 mg/kg, 48 hr after birth (C25/J2) for substance P (B), 50 mg/
kg, 24 hr after birth (C50/J1) for CGRP (D), and 50 mg/kg, 48 hr after birth (C50/J2) for serotonin (F). A-D: Bars equal 100 km. E,F: Bars equal 50 km.
also occurred. The rare profiles establishing a synapse were principally of dome-shaped type. After neonatal capsaicin treatment, serotonergic profiles found in laminae I, 110, I11 and IV exhibited the same characteristics as in control animals. Contrary to
the controls, in the internal part of substantia gelatinosa (lamina IIi), serotonergic profiles were numerous, they were small (diameter < 1 pm) , mainly dome-shaped, and rich in vesicles. More than half of the vesicular profiles were in synaptic contact with avesicular small-
Fig. 5 . Irnrnunocytochernical detection of CGRP (A-C), substance P (D-F), and serotonin (G,H) in 3-month-old rats, illustrating the dose-dependency for CGRP and substance P. A,D,G: Control animals. B ,C,E,F,H: Animals treated with
capsaicin-25 rng/kg, 24 hr after birth (C25/J1) in B, E, and H, or 50 rng/kg, 48 hr after birth (C50/J2) in C and F. A-F; Bars equal 100 prn. G,H; Bars equal 50 prn.
Fig. 6 . Electron-microscopic detection of serotonin in 1-month-old animals. A X : From control animals. D-F: From rats treated with capsaicin (50 mg/kg, 48 hr after birth; C50/J2). Bars equal 250 nm.
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sized dendrites, and most often on dendritic spines (Fig. 6D-F) .
DISCUSSION Postnatal Development of Peptidergic Systems The data reported here give some indications on the postnatal ontogeny of substance P and CGRP in the rat spinal cord. First, we confirm here the previous report from Senba and coworkers (1982) on the protracted ontogeny of substance P in the rat spinal cord. Indeed, the plexus of substance P fibers in laminae I and I1 is discrete at birth. Its fine organization and final density reach an adult pattern only after about 3 weeks. Surprisingly, the DLF is lightly stained up to 3 weeks, indicating an incomplete maturation of the bulbospinal pathways that contain substance P (Hokfelt et al., 1977). Secondly, for CGRP, we have shown that projections to the dorsal horn reach an adult pattern soon after birth. Once again, the density of the plexus of CGRP fibers in laminae I and I1 in one hand, and of the fibers in laminae I11 and IV on the other hand, is moderate at birth but will reach an adult level within two weeks. As substance P and CGRP are often colocalized in the afferent fibers (Skofitsch and Jacobowitz, 1985) this small variation in timing is worth noting as it can correspond to a different expression or maturation of these two peptides within the same fiber. However, as substance P fibers have three putative origins, the delay reported by us for the complete maturation of substance P fibers might also reflect differences between the maturation of these three systems. Meanwhile, biochemical assays for substance P after neonatal capsaicin treatment reported a 67% loss, i.e., a value close to that found after dorsal rhizotomy (Skoftisch and Jacobowitz, 1985). The same authors found a similar decrease for CGRP after rhizotomy. As CGRP is only found in DRG afferents, it may be concluded that, as the decrease is the same as for substance P, most of substance P in fibers within the dorsal horn originates from DRG afferent innervation. Thus, substance P staining found in DLF would not correspond to these bulbospinal fibers whose targets are theoretically the superficial laminae of the dorsal horn; these substance P fibers within the DLF would, in turn, have other deeper targets. These positively stained fibers in the DLF might also correspond to the remaining substance P measured by biochemical assays after capsaicin or rhizotomy . Effects of Neonatal Capsaicin Treatments on Substance P- and CGRP-Containing Fibers Examination of sections from rats treated at birth with capsaicin show, first, that both substance P and
CGRP fibers are destroyed early by the toxin. Indeed, all the combinations “dose/injection time” tested showed, after one week, a reduction of the staining for both peptides. As stated above, the DLF remained stained for substance P, proving that capsaicin does not appear to have any effect on substance P contained in bulbospinal pathways. The extent of the lesions induced by capsaicin appeared the same for substance P or CGRP within the first month. Later, differences occurred; after 3 to 6 months, the lesions of CGRP fibers are reduced when compared with earlier stages. This phenomenon appeared most conspicuous for the low dose of capsaicin. On the other hand, for substance P, the extent of the lesions was the same all along the study. Such differences between the stability of the capsaicin lesions of substance P and CGRP systems, confirm a recent study by Hammond and Ruda (1989) that evidenced a partial recovery of CGRP innervation, while substance P fibers remained reduced. Another interesting point is the dose-dependency that appeared after 3 weeks. Indeed, for both substance P and CGRP, treatments with 50 mg/kg resulted in more extended lesions than with 25 mg/kg. It is also interesting to note that the age of the injection modified the extent of the lesions: the latest date (52) gave more complete lesions than the two earliest (J0.5 or Jl). Thus, the combination of these two parameters (namely, dose and age) leads to the conclusion that the classically used dose of 50 mg/kg injected 48 hr after birth gives the most severe lesions of both substance P and CGRP afferent fibers, and that these lesions are more stable for substance P than for CGRP.
Postnatal Development of Serotonergic System As previously described by Rajaofetra and coworkers (1989), the maturation of serotonergic projections within the dorsal horn of the rat spinal cord is incomplete at birth. Indeed, fibers are present 1 week after birth in the dorsal horn, but superficial laminae are free of immunoreactivity . This supports the theory of Rajaofetra concerning an initial innervation of the dorsal horn arising from a ventral pool, the participation of the dorsal bulbospinal pathway coursing through the DLF being delayed (personal communication). Innervation of the dorsal horn by serotonergic fibers is therefore found first only in laminae 111 to V. In a second phase, that is after two weeks, fibers are seen in lamina I and in the outer part of lamina 11, the internal part of substantia gelatinosa (IIi) remaining devoid of fibers. The development of this peripheral innervation is rapid. The adult pattern is reached both qualitatively and quantitatively at one month.
Neonatal Capsaicin Treatment
Effects of Neonatal Capsaicin Treatments on Serotonergic Innervation of the Dorsal Horn After 1 week, whatever the dose of capsaicin, some serotonergic fibers were present in the superficial laminae of the dorsal horn, contrary to the controls. After 2 weeks, the clear band, devoid of serotonergic immunoreactivity, present in controls, was absent in all capsaicin-treated animals. This pattern remained unchanged throughout the study, that is, at the age of 6 months. The occlusion of lamina IIi by serotonergic fibers appeared similar whatever the dose and the time chosen for the injection. This appears, in turn, like an “all or nothing phenomenon” whose induction is linked to the removal of C afferent fibers. This specific pattern had not been reported in the study of Jancsd et al. (1981), who did not find modifications of the serotonergic innervation after neonatal capsaicin treatment. These ectopic fibers found in treated rats appeared to sprout from lamina 111. When examining with the electron microscope sections taken from 1-month-old rats (C50/J2 group), the main difference between control and treated animals was found in lamina IIi. Briefly, in superficial laminae, few synapses were found (less than 20% of vesicle-containing profiles) as in cerebral cortex (SCguCla et al., 1989) or in cerebellum (Beaudet and Sotelo, 1981). They were located on medium-sized dendrites and rarely on soma (Maxwell et al., 1983; Marlier et al., 1991). After capsaicin treatment, fibers were seen in lamina IIi. They were thin, the varicosities contained vesicles, and, surprisingly, most of them contributed to the formation of synapses. Post-synaptic structures were most often dendritic spines. Such newly established relationships bear some similarities with those described in agranular cerebellum by Beaudet and Sotelo (1981), who found many more serotonergic fibers establishing synapses than in the normal cerebellum. They proposed that the reactive serotonergic system evolves from a “non-junctional” into a “junctional” system. Our data support this theory, as fibers seen in lamina IIi seem to sprout from the deepest laminae, and change their classical relationships when they find new targets in this region. Interestingly, a loss of type I and type I11 serotonergic receptors has been reported by Hamon’s group after neonatal capsaicin treatment (Daval et al., 1987; Hamon et al., 1989). The nature of the putative receptors engaged in the newly established synapses is unknown. Another point of interest is the nature of the postsynaptic element contacted by serotonergic fibers in lamina IIi. Several groups have investigated the altered distribution of primary afferent fibers after neonatal capsaicin treatment. For instance, Ribeiro-Da-Silva and Coimbra (1984) reported that most of type I synaptic glomeruli were absent in lamina I1 in adults after neona-
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tal capsaicin treatment, thus supporting the origin of type I glomeruli from unmyelinated primary afferents. Others found that lamina 111 was invaded by thick primary afferent fibers which are normally restricted to laminae I11 and IV (RCthelyi et al., 1986) or that aberrant myelinated primary afferents invaded the substantia gelatinosa 10 days after birth (Beal and Knight, 1987). Taken together these data lead to the conclusion that substantia gelatinosa underwent profound rearrangement. We propose two explanations: 1. The primary afferents that normally ended in laminae I11 or IV where they could be in relation with serotonergic fibers, attracted them to lamina IIi. 2. The removal by capsaicin of normal targets for serotonin (i.e., C fibers) triggered sprouting of serotonergic fibers, which established new relations with abnormal or unusual afferents in lamina IIi, including synaptic sites left vacant by the lesion of primary afferents. In any case, neonatal treatment with capsaicin appears to be a suitable model for studying the plasticity of serotonergic projections to the rat dorsal horn. The identification of new post-synaptic elements is the next step in the investigation of the ability of the serotonergic system to reorganize its projections following the removal of its target. Does that reorganization contribute to the compensation of the missing component in the array of sensory modes?
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