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Transplantation of embryonic raphe cells regulates the modifications of the GABAergic phenotype occurring in the injured spinal cord
Neuroscience Vol. 95, No. 1, pp. 173–182, 2000 173 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
Regulation of spinal GABAergic neurons in transplanted rats
Pergamon PII: S0306-4522(99)00412-1 www.elsevier.com/locate/neuroscience
TRANSPLANTATION OF EMBRYONIC RAPHE CELLS REGULATES THE MODIFICATIONS OF THE GABAERGIC PHENOTYPE OCCURRING IN THE INJURED SPINAL CORD A. DUMOULIN, A. PRIVAT* and M. GIME´NEZ Y RIBOTTA INSERM Unite´ 336, Universite´ Montpellier II, Place Euge`ne Bataillon, BP 106, 34095 Montpellier Cedex 05, France
Abstract—Transection of the spinal cord yields a permanent deficit due to the interruption of descending and ascending tracts which subserve the supraspinal control of spinal cord functions. We have shown previously that transplantation below the level of the section of embryonic monoaminergic neurons can promote the recovery of some segmental functions via a local serotonergic and noradrenergic reinnervation. Moreover, the up-regulation of the corresponding receptors resulting from the section was corrected by the transplants. The aim of the present work was to determine whether such a graft could also influence nonmonoaminergic local neurons, the GABAergic interneurons of the spinal cord. Following spinal cord transection, the number of cells which express glutamate decarboxylase (mol. wt 67,000) messenger RNA—a marker of GABA synthesis—increased significantly below the lesion compared with the intact animal. In contrast, in lesioned animals which had been grafted one week later with raphe neuroblasts, this number was close to control level. These post-grafting modifications were further associated with increased GABA immunoreactivity in the host tissue. These data suggest that the graft of embryonic raphe cells which compensates the deficit of serotonin in the distal segment also regulates the expression of the GABAergic phenotype in the host spinal cord. This regulation could be mediated by the reestablishment of a local functional innervation by both serotonin and GABAergic transplanted neurons and/or by trophic factors released from the embryonic cells. It appears then that grafted cells influence the host tissue in a complex manner, through the release and/or regulation of several neurotransmitter systems. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: GAD, serotonin, spinal cord transection, non-radioactive in situ hybridization, rat.
Glutamate decarboxylase (GAD) is the rate-limiting synthesizing enzyme of GABA. Two isoforms of the protein exist, encoded by different genes and with mol. wts of 65,000 and 67,000 (GAD65 and GAD67, respectively). 10 The presence of either or both isoform mRNAs and/or proteins is sufficient to characterize a neuron as GABAergic. GAD65 is found mainly in synaptosomal fractions and can be membrane associated. 47 This isoform would preferentially synthesize most of the synaptically released neurotransmitter and be responsible for short-term modifications of GABA release. In contrast, GAD67 is a cytosolic protein thought to be responsible for the metabolic pool of GABA, which provides GABA for the cellular energetic metabolism. It is also involved in long-term modifications of GABAergic inhibition 11,19 and/or non-vesicular release of GABA. 46 In the immature CNS, GABA has been reported to have an excitatory action 5,30,32 and to probably act as a neurotrophin. 2,22,49 In the adult rat CNS, in contrast, it elicits a hyperpolarizing response in neurons and is considered as the main inhibitory neurotransmitter. However, GABA may still have some neurotrophic properties in the adult rat CNS, as described in the peripheral nervous system for the cervical ganglion. 6,57 Following injury, an up-regulation of the GABAergic system has been observed in several models
(see Ref. 27 for a review). Such a post-lesional response of GABAergic neurons mainly involves the 67,000 mol. wt isoform, but in many cases, the mechanisms and function of this up-regulation remain unclear. In the spinal cord, we have recently shown an increased expression of GAD67 mRNA after neonatal deafferentation by capsaicin. 13 Moreover, in the adult spinal cord, the expression of GAD mRNAs, mainly but not exclusively GAD67, was induced following rhizotomy and transection in neurons which were not detected as GABAergic in intact animals. 7,14 Finally, an excitatory action of GABA has been reported in vitro following neuronal trauma. 53 Thus, GABA shows a rather complex pattern of action throughout development and following injury. In the spinal cord, serotonin (5-HT) exerts a modulatory influence on pain, 40 sensorimotor 55 and autonomic functions (see Ref. 56 for a review). Spinal serotonergic fibres originate mostly in the brainstem, from caudal raphe nuclei (B1–B3). 3 Following transection, these fibres degenerate in the distal segment of the spinal cord, which becomes devoid of 5-HT. A graft of embryonic raphe cells in the denervated segment leads to a specific 5-HT reinnervation 16,36 that contributes to the recovery of some of the deficits induced by the loss of supraspinal 5-HT control. 33–36 This specific serotonergic reinnervation allows the recovery of rhythmic locomotor activities 15,58 by initiating and maintaining activity bursts in the lumbar central pattern generator, which has been located at the L1–L2 level in neonatal rats using an in vitro model of the spinal cord. 4 The aim of the present work was thus to determine whether transplanted embryonic raphe cells regulate only and directly the effectors of autonomic and motor systems in the cord, or whether they also regulate the
*To whom correspondence should be addressed. Tel.: 1 33-4-67-14-33-86; fax: 1 33-4-67-14-33-18. E-mail address: [email protected] (A. Privat) Abbreviations: DIG, digoxigenin; E, embryonic day; EDTA, ethylenediaminetetra-acetate; GAD, glutamate decarboxylase; 5-HT, serotonin (5hydroxytryptamine); PBS, phosphate-buffered saline; p.g., post-grafting; PIPES, piperzine-N,N 0 bis(2-ethane-sulphonic acid),1,4-piperazinediethane sulphonic acid. 173
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Animals were deeply anaesthetized under equithesin (3 mg/kg, i.p.) and underwent a laminectomy at the spinal thoracic level T8–T9. The spinal cord and the dura were completely transected with microscissors. Transection was considered complete after retraction of the spinal stumps, which left the ventral surface of the vertebral bone exposed. No spinal tissue was removed. The different layers of the musculature were sutured and the skin was closed with wound clips. Animals received gentamycin (1.4 mg/kg, i.m.) until the fifth day following surgery. Urinary retention during the first week was counteracted by manual compression on the bladder.
buffer [50% formamide, 5 × hybridization salts (750 mM NaCl, 25 mM EDTA, 25 mM PIPES, pH 6.8), 1 × Denhardt’s solution, 0.2% sodium dodecyl sulphate, 0.25 mg/ml salmon sperm DNA, 0.25 mg/ml Poly A] at 378C for 2 h. They were hybridized overnight at 508C in a hybridization solution containing 0.2 ng/ml of DIGlabelled probe. After hybridization, the sections were incubated with RNAase A (50 mg/ml, Sigma) for 25 min and washed down to a final stringency of 0.1 × standard saline citrate/10 mM sodium thiosulphate at 558C for 30 min. For probe detection, hybridized sections were processed according to the protocol of Trembleau and Roche, 52 including (i) an overnight incubation of the sections at 48C in the alkaline phosphatase-conjugated DIG antibody, diluted 1:5000 in 100 mM Tris–HCl (pH 7.5), 1 M NaCl, 2 mM MgCl2, 2% goat serum and 0.1% Triton X-100, and (ii) a colorimetric reaction with nitroblue tetrazolium (0.35 mg/ml), 5-bromo-4-chloro-3-indolyl phosphate (0.18 mg/ml) and levamisole (0.24 mg/ml). Satisfactory staining was obtained within 2–4 h. Probe specificity was demonstrated by the absence of labelling either using a sense probe (GAD65) or using the antisense-labelled probe in the presence of a 50-fold excess of nonlabelled probe (GAD67). For statistical analysis, 10–12 sections per animal were analysed for the two markers. The total number of labelled cells in each section processed either for GAD65 or GAD67 mRNA hybridization was counted. Statistical analysis of the results was performed using the non-parametric Mann–Whitney comparison test (InStat, GraphPad, San Diego, CA, U.S.A.). For quantification of labelled cells within the graft, six to 12 fields (6.25 × 10 22 mm 2) per animal were taken into account and a Student’s t-test was performed (InStat).
Cell suspension
Immunohistochemistry
The embryonic donor tissue was from the same inbred strain as the host animals. Dissociated cell suspensions were prepared from embryonic day 14 (E14) rat brainstem, as described by Ko¨nig et al. 20 The caudal rhombencephalon, extending from the pontine flexure to the cervical end of the spinal cord and containing the B1–B3 raphe nuclei, was dissected in Hanks’ saline solution (Gibco). A lateral band of tissue was cut on each side and discarded. The cells were dissociated mechanically by gentle pipetting in calcium/magnesium-free Puck’s solution (Gibco). The suspension was then centrifuged at 80 × g for 10 min, resuspended in minimal essential culture medium (Gibco) and adjusted to a final concentration of 50,000 cells/ml.
Intact (n 2), transected and transected/grafted animals were perfused intracardially at three weeks (n 2 and 4, respectively) or six weeks after transection (n 2 and 4, respectively) with 5% glutaraldehyde (in 50 mM sodium metabisulphite/50 mM cacodylate buffer, pH 7.5) and spinal cords were removed. Coronal sections (40 mm thick) from spinal cord segments between transection and transplantation sites, as well as throughout the graft, were cut with a Vibratome and processed either for single 5-HT or GABA immunodetection or for double 5-HT/GABA immunostaining.
GABAergic phenotype of interneurons in the transected spinal cord of adult rats. EXPERIMENTAL PROCEDURES
Animals Thirty-seven adult female Sprague–Dawley rats weighing 200– 240 g (Iffa–Credo, France) were used in this study. Three groups of animals (intact, n 5; transected, n 14; transected/grafted, n 18) and two survival delays [three and six weeks post-transection, i.e. two and five weeks post-grafting (p.g.), respectively] were considered. Animals were cared for and surgically handled in accordance with the European Communities Council Directive (24 November 1986, 86/609/EEC). Spinal cord transection
Grafting Animals were grafted one week after transection following the procedure described by Rajaofetra et al., 35 this period being optimal for graft development. A second laminectomy was performed at the T11–T12 spinal level and 4 ml of the cell suspension was injected into the spinal cord (1 mm below the pial surface) with a metallic needle (0.4 mm in diameter) connected to a Hamilton microsyringe. The needle was withdrawn 2 min after the end of the injection to avoid suspension reflux. The musculature was then sutured and the animals were treated as described above to allow recovery. In situ hybridization Intact (n 3), transected and transected/grafted rats were perfused transcardially at three (n 5 for both) and six weeks following transection (n 5 for both) with phosphate-buffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde in PBS. The level and completeness of transection were confirmed post mortem. Coronal floating sections (30 mm thick) from spinal cord segments between transection and transplantation sites, as well as sections throughout the portion containing the graft, were cut on a cryostat, collected in a cryoprotective solution 25 and stored at 2208C. Sections from at least one intact, one transected and one transected/grafted animal (same survival delay) were processed in the same experiment for GAD mRNA detection. The transcription of digoxygenin (DIG)-labelled cRNA probes and the in situ hybridization procedures for GAD65 and GAD67 mRNAs have been described in detail elsewhere. 7 In brief, GAD65 and GAD67 recombinant plasmids, generously provided by A. Tobin (UCLA), were linearized and cRNA probes were transcribed and labelled by the incorporation of a DIG–UTP every 20–25 nucleotides (DIG RNA Labelling Kit, Boehringer Mannheim, Mannheim, Germany). Sections were incubated in proteinase K for 8 min (1 mg/ml; Sigma, St Quentin Fallavier, France), rinsed and incubated in hybridization
Single peroxidase immunostaining. Sections were pretreated with trypsin–EDTA (0.25%, 5 min, Gibco) and H2O2 (0.5%, 10 min). They were then successively incubated (i) with a mouse monoclonal antibody against GABA (1:500; Chemicon) or with a rabbit polyclonal antibody against 5-HT (1:20,000; Immunotech) for 48 h at 48C; (ii) either with a rabbit anti-mouse immunoglobulin G (1:100; DAKO) or with a goat anti-rabbit immunoglobulin G (1:200; Tilburg) for 1 h at room temperature; (iii) either with a mouse peroxidase–antiperoxidase complex (1:100; DAKO) or a rabbit peroxidase–antiperoxidase complex (1:200; DAKO) for 1 h at room temperature. All the antibodies were diluted in PBS containing 0.1% saponin and 1% nonspecific goat serum. Immunoreactivity was revealed with 0.1% 3,3 0 diaminobenzidine diluted in 0.05% Tris buffer (pH 7.3), in the presence of 0.2% H2O2. After rinsing, sections were mounted with DePeX. The method used for controls consisted of omitting the primary antibody and applying the secondary antibody alone. Double fluorescence immunostaining. Following pretreatment with 1% sodium borohydride (45 min) and incubation with the mouse monoclonal anti-GABA and the rabbit polyclonal anti-5-HT, sections of the graft level only were incubated for 2 h at 48C with the corresponding secondary antibodies, including an anti-mouse immunoglobulin G conjugated with Cy3 (1:800; Sigma) and an anti-rabbit immunoglobulin G conjugated with fluorescein isothiocyanate (1:200; Sigma). After rinsing, sections were mounted in Mowiol (Calbiochem) and observed under a Zeiss axioskop fluorescence microscope, equipped with the appropriate filter set. RESULTS
In grafted animals, the location of the transplant and its interface with the host spinal cord were clearly recognized, both in tissues processed for in situ hybridization and for
Regulation of spinal GABAergic neurons in transplanted rats
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Fig. 1. In situ hybridization of GAD transcripts in spinal cord sections from a transplanted animal. Two weeks after transplantation, numerous cells within the graft (delimited with a dotted line in A and B, and shown at a higher magnification in C and D) are labelled for GAD65 (A, C) and GAD67 mRNAs (B, D). In the host spinal cord, only a few lightly GAD65 mRNA-labelled cells are detected (arrows in C), while a number of neurons is intensely stained for GAD67 transcripts (arrows in D). Neurons labelled for GAD65 and GAD67 mRNAs (arrows in E and F, respectively) are also detected in some cell suspension that leaked from the injection site and remained apposed to the white matter (wm). Scale bars 250 mm (B), 100 mm (D), 50 mm (F).
immunocytochemistry. The transplant was generally well developed and located in or close to the dorsal horns. Transplant
In one grafted animal, the injection needle pierced the cord along a dorsal-to-ventral axis and the cellular suspension partly leaked to an extraparenchymal location apposed to the ventral white matter. Both GAD65 and GAD67 mRNAs were also detected in this tissue (Fig. 1E, F).
In situ hybridization. At both two and five weeks after transplantation (i.e. three and six weeks after transection), numerous cells within the graft showed GAD65 and GAD67 mRNA labelling (Fig. 1). No significant difference was found either between the number of cells expressing the two markers or between two and five weeks p.g. (Table 1). Differences in the staining intensities of GAD mRNAs were sometimes observed, but could not be correlated to a specific delay.
Immunohistochemistry. Immunodetection of GABA revealed a number of small, round positive somata dispersed among the transplanted cells, as well as a very dense network of GABA-immunostained fibres extending throughout the graft (Fig. 2A). However, whether these fibres arose from grafted GABAergic embryonic neurons or from host spinal GABAergic neurons is not known.
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Table 1. Number of glutamate decarboxylase messenger RNA-labelled cells detected within the graft Animal no.
GAD65
GAD67
Two weeks p.g. 1 2 3 4 5
n.d. n.d. 31.2 ^ 5.1 26.0 ^ 2.7 22.5 ^ 1.7
n.d. n.d. 39.5 ^ 6.0 28.5 ^ 0.8 29.4 ^ 2.2
Five weeks p.g. 1 2 3 4 5
n.d. 27.0 ^ 1.4 17.8 ^ 1.2 17.1 ^ 1.9 35.3 ^ 1.6
n.d. 26.1 ^ 1.5 19.1 ^ 1.1 17.1 ^ 1.0 28.0 ^ 0.7*
The number of labelled cells within the same graft does not differ significantly between the two markers at either two or five weeks p.g., except for one animal (no. 5, five weeks p.g.). Each value is the mean ^ S.E.M. for an area of 6.25 × 10 22 mm 2; five to 12 determinations per animal were considered. n.d., not determined. *P , 0.05 (Student’s t-test).
Serotonergic immunoreactive cell bodies were also detected within the graft at both survival delays, most of them being large multipolar neurons (Fig. 2B). 5-HT-immunostained fibres also formed a dense network within
the graft, and isolated fibres extended at some distance into the gray matter of the host spinal cord (not shown). Double fluorescent immunodetection of GABA and 5-HT conformed to the single peroxidase immunostaining description for each marker and further indicated that no co-localization between the two markers occurred either in cell bodies or in fibres within the graft (Fig. 3). Spinal cord Observations were restricted to the region located between the transection and transplantation sites. In situ hybridization. Glutamate decarboxylase-65 messenger RNA. In transected, non-grafted rats, the number of GAD65 mRNA-labelled cells showed no statistical difference with intact rats at three weeks after transection (Fig. 4A, B, Table 2). By contrast, in transected/grafted animals, a very significant decrease (P , 0.001) was detected in the number of labelled cells at this survival delay (two weeks p.g.) compared with intact animals. At five weeks p.g., no significant difference was found between transected and transected/ grafted rats. Glutamate decarboxylase-67 messenger RNA. In transected animals, a very significant increase (P , 0.001)
Fig. 2. Immunodetection of GABA (A) and 5-HT (B) in spinal cord sections from a transplanted animal (two weeks p.g.). (A) Numerous GABA-immunoreactive cell bodies are observed within the graft (arrows). Positive somata are also detected in the host spinal cord (arrowheads). A dense network of GABApositive fibres is detected in the graft, as opposed to layers II/III of the host spinal cord, which contain only scarce GABA-positive fibres (asterisk). (B) In contrast to GABA immunoreactivity, 5-HT innervation originates only from transplanted 5-HT cells (arrows). Scale bar 100 mm.
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Regulation of spinal GABAergic neurons in transplanted rats
Fig. 3. Confocal paired images of a spinal cord section from a transplanted animal double immunostained for 5-HT and GABA. Two weeks after transplantation, 5-HT and GABA immunostainings are associated with distinct somata and fibres located within the graft (arrows and arrowheads point to 5-HT- and GABA-immunostained somata, respectively). Notice that 5-HT-immunoreactive spots are present on the 5-HT-positive cell bodies, but not on GABA-positive ones. Scale bar 25 mm.
in the number of GAD67 mRNA-expressing cells was observed at three and six weeks compared with intact animals (Fig. 4C, D, Table 2). By contrast, in transected/grafted animals, the number of GAD67 mRNA-labelled cells observed at two weeks p.g. showed no significant difference with control values from intact animals. By five weeks p.g., the number of stained cells was still reduced compared with transected animals, although the decrease was not as marked as at the previous survival delay. Immunohistochemistry. In transected animals, GABA immunoreactivity was less marked than in control intact animals, particularly within the superficial dorsal layers, where very few positive cell bodies were detected (Fig. 5B). In contrast, in the transected/grafted animals, numerous GABA-positive cell bodies were detected in this region (Fig. 5C). Moreover, the GABAergic innervation in the deep dorsal and ventral layers of the gray matter appeared to be denser than in non-grafted animals, particularly in the ventral horn around the motoneuronal somata (Fig. 5C2). In transected animals, the spinal cord below the lesion was totally devoid of 5-HT immunoreactivity. In contrast, in grafted animals, numerous 5-HT-immunoreactive fibres arising from the graft were detected at both survival delays in the host spinal cord. Ten millimetres above the graft, isolated 5-HT-positive fibres were seldom detected at two weeks p.g. and showed a quite random distribution. At five weeks p.g., 5-HT-positive fibres detected at this distance appeared to concentrate in the region surrounding the central canal, in the motoneuronal pools and in the intermediolateral column (whenever present; Fig. 6). Very few fibres were detected in the superficial dorsal layers. A reinnervation by 5-HT-positive fibres was also observed caudally to the graft, at the lumbar level.
DISCUSSION
The present observations demonstrate that: (i) the embryonic raphe transplant contains different neuronal populations, expressing either 5-HT or GABA immunoreactivities; (ii) after complete section, there is an increase in the number of host neurons that express GAD67 and the graft is able to reverse these modifications in the GABAergic phenotype; (iii) an increase in GABA content is detected in cell bodies and fibres of the spinal cord following grafting. Furthermore, detection of 5-HT-immunostained cells within the transplant fully confirmed our previous studies, 33–35 including the reinnervation of original 5-HT target areas in the spinal cord, 3 although reinnervation of the superficial dorsal horn was less marked in the present study. Characterization of transplanted cells An interesting finding of this study is the detection of numerous GABAergic neurons among the transplanted cells. Since cellular suspensions apposed to the white matter (thus having actual minimal contact with the host spinal cord) showed labelling for both GAD mRNAs similar to the one observed in the integrated grafts, it can be assumed that the expression of this phenotype is related to the intrinsic properties of the embryonic tissue rather than to its interactions with the host. To our knowledge, an embryonic raphe showing such a high content of GABAergic cells has not been described before. However, this finding is not really surprising, since the GABAergic system develops early and, by E14, GABAergic fibres are already present in the raphe 21 at a time when serotonergic neurons begin to differentiate. It has also been reported that GABA acts as a neurotrophic factor on immature neurons from the brain and spinal cord, as a chemoattractant and by inducing differentiation. 2,22,49 Furthermore, it has
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Fig. 4. In situ hybridization of GAD transcripts in spinal cord sections from a transected (A, C) and a transected/grafted animal (B, D). Five weeks after transplantation, heavily stained GAD65 mRNA-expressing cells (arrows) are detected in the region surrounding the central canal in both transected (A) and transected/grafted animals (B), while some cells showing a fainter labelling (arrowheads) are located throughout layers I–VII. In contrast, very numerous GAD67 mRNA-expressing cells are detected in the transected animal (arrows in C) in most layers of the gray matter, whereas the number and staining intensity of the labelled cells is decreased markedly in the grafted animal (arrows in D). Scale bar 100 mm.
recently been demonstrated that GABA enhances cell survival and increases neurite development of 5-HT neurons in cultures of E14 brainstem cells. 24 Moreover, in the dorsal raphe nucleus of adult rats, it is admitted that
local GABAergic innervation modulates 5-HT neuronal activity. 51,54 Thus, the presence of a high number of GABAergic cells in the transplant is coherent with the influence of GABA upon 5-HT neurons at the developmental
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Table 2. Total number of glutamate decarboxylase messenger RNA-labelled cells in spinal cord sections from control, transected and transected/grafted animals Control (n 3) GAD65 GAD67
32.3 ^ 1.2 53.7 ^ 2.4
Transected
Transected/grafted
Week 3 (n 5)
Week 6 (n 5)
31.9 ^ 2.6 75.2 ^ 3.8***
27.2 ^ 1.7* 80.5 ^ 3.8***
Week 3 (n 5)
Week 6 (n 5)
18.9 ^ 0.9***††† 57.2 ^ 3.2†††
33.5 ^ 2.1 65.8 ^ 2.7**††
The number of GAD65 mRNA-expressing cells detected in transected animals is quite similar to those of control animals, while in grafted animals it significantly decreases at three, but not six, weeks (two and five weeks p.g., respectively). The number of cells labelled for GAD67 mRNA is highly increased in transected animals, but returns towards control levels in grafted animals. This effect is maximal by three weeks after transection (two weeks p.g.). Each value is the mean ^ S.E.M. of 10–12 determinations per animal. Statistical analysis was performed using the Mann–Whitney test (nonparametric). *P , 0.05, **P , 0.01, ***P , 0.001 between injured (transected or transected/grafted) and control animals. ††P , 0.01, †††P , 0.001 between transected and transected/grafted animals at the same survival delay.
A1
B1
C1
A2
B2
C2
Fig. 5. GABA immunodetection in the spinal cord. In an intact animal, GABA-positive somata are detected in the dorsal horn (A1, arrows) and GABA-positive fibres in the ventral horn (A2, arrowheads). In a transected animal, a decrease in the labelling of GABA is observed in both the dorsal horn, where only a few cell bodies are detected (B1, arrow), and the ventral horn (B2, arrowhead). In a grafted animal, by five weeks p.g. GABA staining is restored in the host spinal cord. Scale bar 50 mm.
and adult stages. No co-localization between 5-HT and GABA was detected in this study either in embryonic neurons (E14) cultured for 24 h (unpublished observations) or in the grafted tissue at short or long survival delays (two and five weeks p.g.), but such a co-localization has been reported to occur in only a small percentage of raphe neurons (3.6% in the B3 nucleus). 18,50 Raphe cells grafted in the lesioned spinal cord may lack factors and/or connections that would have been provided by their natural environment and which may induce differences in phenotypic expression, even though another co-localization which occurs naturally in the raphe (5-HT and substance P) has been detected in the same transplantation model. 35 Finally, 5-HT-containing terminals were detected apposed to 5-HT-positive cell bodies in the graft. Interestingly enough, 5-HT has been shown to act as a differentiation factor on a raphe-derived neuronal cell line (RN46A) and could possibly be involved in an autocrine regulation loop via the 5-HT1A receptor. 8
Putative mechanisms of action of transplanted cells on the GABAergic neurons of the host spinal cord We have demonstrated previously that an embryonic raphe graft leads specifically to the recovery of some of the deficits in transected animals, acting on motoneuronal output 15 and recovering reflexes. 33,34 Since intrinsic GABAergic neurons are involved in the control of motoneuronal output, 28,31 their activity may have been modified by the lesion and then by the newly established connections. Indeed, GABAergic innervation of the motoneuronal pools appears to be increased following grafting as compared to transected rats. Several mechanisms can be considered for the regulation of the GABAergic activity in the host spinal cord. A first possibility is that embryonic tissue may provide multiple diffusible factors, including GABA, which may influence the surrounding spinal neurons. The injured environment in which raphe cells are transplanted may thus benefit from the trophins and factors released by the graft that enhance host network
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Fig. 6. 5-HT immunodetection in the spinal cord. In an intact animal, a specific 5-HT innervation is detected in target areas: the intermediolateral column (A) and the region surrounding the central canal (C), which are devoid of 5-HT immunoreactivity in transected animals (not shown). In transected/grafted animals, a 5-HT innervation pattern is re-established at five weeks p.g. (B, D). Scale bar 50 mm.
reorganization. However, it appears unlikely that the regulatory effect of the transplant observed as early as two weeks p.g. at a distance of about 10 mm from the injection site could be mediated by the diffusion of those factors up to such a distance. An alternative possibility could thus be that those factors diffusing from the graft have direct regulatory effects on interneurons located within the host spinal cord close to the graft which, in turn, influence the activity of more distant neurons to which they are directly or indirectly connected. The regulation of GABAergic neurons seen here might also, and most probably, be a consequence of the innervation of host spinal cord cells by various axonal fibres arising from the graft, including serotonergic fibres. A strong reinnervation of the host spinal cord by 5-HT fibres has been described previously 15,33–36 and is consistent with a direct regulation of distant GABAergic neurons by means of synaptic or nonsynaptic connections, as happens in the intact spinal cord. 37 As in the case of the 5-HT innervation, a supraspinal GABAergic innervation exists. 1,17,29 Descending fibres from the rostral ventromedial medulla, which includes the nucleus raphe magnus (B3), project mainly to layers IV/V and I/IIo, while some of the GABAergic descending fibres (13.8%) directly contact GABA-positive dendrites and cell bodies. 1 Following transection and transplantation, an additional specific GABAergic innervation of the spinal cord could arise from the grafted GABAergic neurons, in a similar way to the 5-HT innervation seen here. Regulation of glutamate decarboxylase messenger RNA and GABA by the transplant We show here that the modifications of the GABAergic neurons observed in the distal segment following transection
mainly involved the GAD67 isoform. The increased expression of the GAD67 mRNA isoform is a feature shared by the neurons of different CNS structures following injury, including the hippocampus, 12 cerebellum 23 and striatum. 49 In the present study, we show that the influence of a spinal cord transection on the expression of GAD67 messenger was reversed by transplantion of embryonic raphe cells into the denervated spinal cord, re-establishing the number of labelled neurons close to controls. However, GAD67 mRNA levels and GABA cellular content appear to be inversely correlated. A similar inverse correlation was observed between GAD67 and GABA immunoreactivities of spinal cord neurons in a model of sciatic nerve chronic constriction, 9 suggesting a regulation by negative feedback. In fact, increased GABA levels induced by vigabatrin have been shown to specifically decrease GAD67 protein levels in vitro 38 and in vivo, 39,44 although GAD67 mRNA levels were not affected, indicating a posttranslational regulation or/and changes in protein stability. In our model, compared with pharmacological manipulation, injury may have modified the input to GABAergic cells, thus inducing the up-regulation of GAD67 gene transcription levels. As GAD67 is probably involved in non-synaptic release of GABA, the over-expression of GAD67 mRNA may reflect an increase in this activity as a response to decreased GABA in the injured tissue. In transplanted animals, GAD67 mRNA could thus be down-regulated by the additional GABAergic and serotonergic innervation. Finally, it may also represent an injury-induced modification of the phenotype of neurons towards immature features, since virtually all spinal neurons express the GABAergic phenotype during development, 26,41,48 or simply reflect an increased metabolism of the cell. Another finding of this study is that the levels of GAD65 mRNA are also affected by the transplantation procedure,
Regulation of spinal GABAergic neurons in transplanted rats
although in an opposite manner to that of GAD67 mRNA. As the two GAD isoforms derive from different genes and their 5 0 untranslated regions show little homology, their expression is probably regulated by different mechanisms and factors. In fact, GADs may even respond differently within the same neuron, according to the afferent/efferent activities of the neuron or to the pathways in which it is involved (see Ref. 46 for a review). This is probably also the case in the spinal cord, since GAD65 and GAD67 are preferentially expressed in the dorsal and ventral horns, respectively. 14 This would explain the different responses of the two GADs to grafting procedures. Among the models of CNS injury in which mRNA levels are up-regulated, the denervation of the striatum appears to be of particular interest. 45 Destruction of dopaminergic fibres originating in the substantia nigra and projecting to the striatum leads to an increase in GAD67 mRNA levels and protein activity at four weeks post-injury. 42 Interestingly enough, it has been shown that, as in the spinal cord, (i) GAD67 mRNA increase is only detected in a population of neurons expressing low levels of this messenger under normal conditions 46 and (ii) a graft of embryonic substantia nigra, containing GABAergic neurons, is able to reverse these levels towards
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control values. 43 In the striatum, the graft regulates the injuryinduced GAD67 over-expression by re-establishing a local control on GABAergic neurons. Although spinal cord transection elicits a much more complex response than striatum deafferentation, it is very likely that, in the present case, a regulation of this gene by embryonic raphe cells transplantation results from similar reorganizing mechanisms. However, it remains to be shown that, if that is the case, the regulation of the GABAergic phenotype demonstrated here contributes to the restoration of spinal functions by embryonic raphe grafts. In any case, the present results shed some light on the complex phenomena which are triggered by a spinal cord lesion, and which can be corrected to some extent by transplanted embryonic neurons. Further studies should decide whether the GABAergic component which is illustrated here plays a specific role in the central pattern generator 15 responsible for the locomotion pattern, or whether it plays a more general role in the homeostasis of spinal cord functions. Acknowledgements—We are indebted to Ge´rard Alonso for his pertinent comments. Thanks are also due to Isabelle Chaudieu and Norbert Chauvet for helpful discussions. This work was supported by INSERM, BIOMED, AFM, IRME and VERTICALE.
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Regulation of spinal GABAergic neurons in transplanted rats
Pergamon PII: S0306-4522(99)00412-1 www.elsevier.com/locate/neuroscience
TRANSPLANTATION OF EMBRYONIC RAPHE CELLS REGULATES THE MODIFICATIONS OF THE GABAERGIC PHENOTYPE OCCURRING IN THE INJURED SPINAL CORD A. DUMOULIN, A. PRIVAT* and M. GIME´NEZ Y RIBOTTA INSERM Unite´ 336, Universite´ Montpellier II, Place Euge`ne Bataillon, BP 106, 34095 Montpellier Cedex 05, France
Abstract—Transection of the spinal cord yields a permanent deficit due to the interruption of descending and ascending tracts which subserve the supraspinal control of spinal cord functions. We have shown previously that transplantation below the level of the section of embryonic monoaminergic neurons can promote the recovery of some segmental functions via a local serotonergic and noradrenergic reinnervation. Moreover, the up-regulation of the corresponding receptors resulting from the section was corrected by the transplants. The aim of the present work was to determine whether such a graft could also influence nonmonoaminergic local neurons, the GABAergic interneurons of the spinal cord. Following spinal cord transection, the number of cells which express glutamate decarboxylase (mol. wt 67,000) messenger RNA—a marker of GABA synthesis—increased significantly below the lesion compared with the intact animal. In contrast, in lesioned animals which had been grafted one week later with raphe neuroblasts, this number was close to control level. These post-grafting modifications were further associated with increased GABA immunoreactivity in the host tissue. These data suggest that the graft of embryonic raphe cells which compensates the deficit of serotonin in the distal segment also regulates the expression of the GABAergic phenotype in the host spinal cord. This regulation could be mediated by the reestablishment of a local functional innervation by both serotonin and GABAergic transplanted neurons and/or by trophic factors released from the embryonic cells. It appears then that grafted cells influence the host tissue in a complex manner, through the release and/or regulation of several neurotransmitter systems. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: GAD, serotonin, spinal cord transection, non-radioactive in situ hybridization, rat.
Glutamate decarboxylase (GAD) is the rate-limiting synthesizing enzyme of GABA. Two isoforms of the protein exist, encoded by different genes and with mol. wts of 65,000 and 67,000 (GAD65 and GAD67, respectively). 10 The presence of either or both isoform mRNAs and/or proteins is sufficient to characterize a neuron as GABAergic. GAD65 is found mainly in synaptosomal fractions and can be membrane associated. 47 This isoform would preferentially synthesize most of the synaptically released neurotransmitter and be responsible for short-term modifications of GABA release. In contrast, GAD67 is a cytosolic protein thought to be responsible for the metabolic pool of GABA, which provides GABA for the cellular energetic metabolism. It is also involved in long-term modifications of GABAergic inhibition 11,19 and/or non-vesicular release of GABA. 46 In the immature CNS, GABA has been reported to have an excitatory action 5,30,32 and to probably act as a neurotrophin. 2,22,49 In the adult rat CNS, in contrast, it elicits a hyperpolarizing response in neurons and is considered as the main inhibitory neurotransmitter. However, GABA may still have some neurotrophic properties in the adult rat CNS, as described in the peripheral nervous system for the cervical ganglion. 6,57 Following injury, an up-regulation of the GABAergic system has been observed in several models
(see Ref. 27 for a review). Such a post-lesional response of GABAergic neurons mainly involves the 67,000 mol. wt isoform, but in many cases, the mechanisms and function of this up-regulation remain unclear. In the spinal cord, we have recently shown an increased expression of GAD67 mRNA after neonatal deafferentation by capsaicin. 13 Moreover, in the adult spinal cord, the expression of GAD mRNAs, mainly but not exclusively GAD67, was induced following rhizotomy and transection in neurons which were not detected as GABAergic in intact animals. 7,14 Finally, an excitatory action of GABA has been reported in vitro following neuronal trauma. 53 Thus, GABA shows a rather complex pattern of action throughout development and following injury. In the spinal cord, serotonin (5-HT) exerts a modulatory influence on pain, 40 sensorimotor 55 and autonomic functions (see Ref. 56 for a review). Spinal serotonergic fibres originate mostly in the brainstem, from caudal raphe nuclei (B1–B3). 3 Following transection, these fibres degenerate in the distal segment of the spinal cord, which becomes devoid of 5-HT. A graft of embryonic raphe cells in the denervated segment leads to a specific 5-HT reinnervation 16,36 that contributes to the recovery of some of the deficits induced by the loss of supraspinal 5-HT control. 33–36 This specific serotonergic reinnervation allows the recovery of rhythmic locomotor activities 15,58 by initiating and maintaining activity bursts in the lumbar central pattern generator, which has been located at the L1–L2 level in neonatal rats using an in vitro model of the spinal cord. 4 The aim of the present work was thus to determine whether transplanted embryonic raphe cells regulate only and directly the effectors of autonomic and motor systems in the cord, or whether they also regulate the
*To whom correspondence should be addressed. Tel.: 1 33-4-67-14-33-86; fax: 1 33-4-67-14-33-18. E-mail address: [email protected] (A. Privat) Abbreviations: DIG, digoxigenin; E, embryonic day; EDTA, ethylenediaminetetra-acetate; GAD, glutamate decarboxylase; 5-HT, serotonin (5hydroxytryptamine); PBS, phosphate-buffered saline; p.g., post-grafting; PIPES, piperzine-N,N 0 bis(2-ethane-sulphonic acid),1,4-piperazinediethane sulphonic acid. 173
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Animals were deeply anaesthetized under equithesin (3 mg/kg, i.p.) and underwent a laminectomy at the spinal thoracic level T8–T9. The spinal cord and the dura were completely transected with microscissors. Transection was considered complete after retraction of the spinal stumps, which left the ventral surface of the vertebral bone exposed. No spinal tissue was removed. The different layers of the musculature were sutured and the skin was closed with wound clips. Animals received gentamycin (1.4 mg/kg, i.m.) until the fifth day following surgery. Urinary retention during the first week was counteracted by manual compression on the bladder.
buffer [50% formamide, 5 × hybridization salts (750 mM NaCl, 25 mM EDTA, 25 mM PIPES, pH 6.8), 1 × Denhardt’s solution, 0.2% sodium dodecyl sulphate, 0.25 mg/ml salmon sperm DNA, 0.25 mg/ml Poly A] at 378C for 2 h. They were hybridized overnight at 508C in a hybridization solution containing 0.2 ng/ml of DIGlabelled probe. After hybridization, the sections were incubated with RNAase A (50 mg/ml, Sigma) for 25 min and washed down to a final stringency of 0.1 × standard saline citrate/10 mM sodium thiosulphate at 558C for 30 min. For probe detection, hybridized sections were processed according to the protocol of Trembleau and Roche, 52 including (i) an overnight incubation of the sections at 48C in the alkaline phosphatase-conjugated DIG antibody, diluted 1:5000 in 100 mM Tris–HCl (pH 7.5), 1 M NaCl, 2 mM MgCl2, 2% goat serum and 0.1% Triton X-100, and (ii) a colorimetric reaction with nitroblue tetrazolium (0.35 mg/ml), 5-bromo-4-chloro-3-indolyl phosphate (0.18 mg/ml) and levamisole (0.24 mg/ml). Satisfactory staining was obtained within 2–4 h. Probe specificity was demonstrated by the absence of labelling either using a sense probe (GAD65) or using the antisense-labelled probe in the presence of a 50-fold excess of nonlabelled probe (GAD67). For statistical analysis, 10–12 sections per animal were analysed for the two markers. The total number of labelled cells in each section processed either for GAD65 or GAD67 mRNA hybridization was counted. Statistical analysis of the results was performed using the non-parametric Mann–Whitney comparison test (InStat, GraphPad, San Diego, CA, U.S.A.). For quantification of labelled cells within the graft, six to 12 fields (6.25 × 10 22 mm 2) per animal were taken into account and a Student’s t-test was performed (InStat).
Cell suspension
Immunohistochemistry
The embryonic donor tissue was from the same inbred strain as the host animals. Dissociated cell suspensions were prepared from embryonic day 14 (E14) rat brainstem, as described by Ko¨nig et al. 20 The caudal rhombencephalon, extending from the pontine flexure to the cervical end of the spinal cord and containing the B1–B3 raphe nuclei, was dissected in Hanks’ saline solution (Gibco). A lateral band of tissue was cut on each side and discarded. The cells were dissociated mechanically by gentle pipetting in calcium/magnesium-free Puck’s solution (Gibco). The suspension was then centrifuged at 80 × g for 10 min, resuspended in minimal essential culture medium (Gibco) and adjusted to a final concentration of 50,000 cells/ml.
Intact (n 2), transected and transected/grafted animals were perfused intracardially at three weeks (n 2 and 4, respectively) or six weeks after transection (n 2 and 4, respectively) with 5% glutaraldehyde (in 50 mM sodium metabisulphite/50 mM cacodylate buffer, pH 7.5) and spinal cords were removed. Coronal sections (40 mm thick) from spinal cord segments between transection and transplantation sites, as well as throughout the graft, were cut with a Vibratome and processed either for single 5-HT or GABA immunodetection or for double 5-HT/GABA immunostaining.
GABAergic phenotype of interneurons in the transected spinal cord of adult rats. EXPERIMENTAL PROCEDURES
Animals Thirty-seven adult female Sprague–Dawley rats weighing 200– 240 g (Iffa–Credo, France) were used in this study. Three groups of animals (intact, n 5; transected, n 14; transected/grafted, n 18) and two survival delays [three and six weeks post-transection, i.e. two and five weeks post-grafting (p.g.), respectively] were considered. Animals were cared for and surgically handled in accordance with the European Communities Council Directive (24 November 1986, 86/609/EEC). Spinal cord transection
Grafting Animals were grafted one week after transection following the procedure described by Rajaofetra et al., 35 this period being optimal for graft development. A second laminectomy was performed at the T11–T12 spinal level and 4 ml of the cell suspension was injected into the spinal cord (1 mm below the pial surface) with a metallic needle (0.4 mm in diameter) connected to a Hamilton microsyringe. The needle was withdrawn 2 min after the end of the injection to avoid suspension reflux. The musculature was then sutured and the animals were treated as described above to allow recovery. In situ hybridization Intact (n 3), transected and transected/grafted rats were perfused transcardially at three (n 5 for both) and six weeks following transection (n 5 for both) with phosphate-buffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde in PBS. The level and completeness of transection were confirmed post mortem. Coronal floating sections (30 mm thick) from spinal cord segments between transection and transplantation sites, as well as sections throughout the portion containing the graft, were cut on a cryostat, collected in a cryoprotective solution 25 and stored at 2208C. Sections from at least one intact, one transected and one transected/grafted animal (same survival delay) were processed in the same experiment for GAD mRNA detection. The transcription of digoxygenin (DIG)-labelled cRNA probes and the in situ hybridization procedures for GAD65 and GAD67 mRNAs have been described in detail elsewhere. 7 In brief, GAD65 and GAD67 recombinant plasmids, generously provided by A. Tobin (UCLA), were linearized and cRNA probes were transcribed and labelled by the incorporation of a DIG–UTP every 20–25 nucleotides (DIG RNA Labelling Kit, Boehringer Mannheim, Mannheim, Germany). Sections were incubated in proteinase K for 8 min (1 mg/ml; Sigma, St Quentin Fallavier, France), rinsed and incubated in hybridization
Single peroxidase immunostaining. Sections were pretreated with trypsin–EDTA (0.25%, 5 min, Gibco) and H2O2 (0.5%, 10 min). They were then successively incubated (i) with a mouse monoclonal antibody against GABA (1:500; Chemicon) or with a rabbit polyclonal antibody against 5-HT (1:20,000; Immunotech) for 48 h at 48C; (ii) either with a rabbit anti-mouse immunoglobulin G (1:100; DAKO) or with a goat anti-rabbit immunoglobulin G (1:200; Tilburg) for 1 h at room temperature; (iii) either with a mouse peroxidase–antiperoxidase complex (1:100; DAKO) or a rabbit peroxidase–antiperoxidase complex (1:200; DAKO) for 1 h at room temperature. All the antibodies were diluted in PBS containing 0.1% saponin and 1% nonspecific goat serum. Immunoreactivity was revealed with 0.1% 3,3 0 diaminobenzidine diluted in 0.05% Tris buffer (pH 7.3), in the presence of 0.2% H2O2. After rinsing, sections were mounted with DePeX. The method used for controls consisted of omitting the primary antibody and applying the secondary antibody alone. Double fluorescence immunostaining. Following pretreatment with 1% sodium borohydride (45 min) and incubation with the mouse monoclonal anti-GABA and the rabbit polyclonal anti-5-HT, sections of the graft level only were incubated for 2 h at 48C with the corresponding secondary antibodies, including an anti-mouse immunoglobulin G conjugated with Cy3 (1:800; Sigma) and an anti-rabbit immunoglobulin G conjugated with fluorescein isothiocyanate (1:200; Sigma). After rinsing, sections were mounted in Mowiol (Calbiochem) and observed under a Zeiss axioskop fluorescence microscope, equipped with the appropriate filter set. RESULTS
In grafted animals, the location of the transplant and its interface with the host spinal cord were clearly recognized, both in tissues processed for in situ hybridization and for
Regulation of spinal GABAergic neurons in transplanted rats
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Fig. 1. In situ hybridization of GAD transcripts in spinal cord sections from a transplanted animal. Two weeks after transplantation, numerous cells within the graft (delimited with a dotted line in A and B, and shown at a higher magnification in C and D) are labelled for GAD65 (A, C) and GAD67 mRNAs (B, D). In the host spinal cord, only a few lightly GAD65 mRNA-labelled cells are detected (arrows in C), while a number of neurons is intensely stained for GAD67 transcripts (arrows in D). Neurons labelled for GAD65 and GAD67 mRNAs (arrows in E and F, respectively) are also detected in some cell suspension that leaked from the injection site and remained apposed to the white matter (wm). Scale bars 250 mm (B), 100 mm (D), 50 mm (F).
immunocytochemistry. The transplant was generally well developed and located in or close to the dorsal horns. Transplant
In one grafted animal, the injection needle pierced the cord along a dorsal-to-ventral axis and the cellular suspension partly leaked to an extraparenchymal location apposed to the ventral white matter. Both GAD65 and GAD67 mRNAs were also detected in this tissue (Fig. 1E, F).
In situ hybridization. At both two and five weeks after transplantation (i.e. three and six weeks after transection), numerous cells within the graft showed GAD65 and GAD67 mRNA labelling (Fig. 1). No significant difference was found either between the number of cells expressing the two markers or between two and five weeks p.g. (Table 1). Differences in the staining intensities of GAD mRNAs were sometimes observed, but could not be correlated to a specific delay.
Immunohistochemistry. Immunodetection of GABA revealed a number of small, round positive somata dispersed among the transplanted cells, as well as a very dense network of GABA-immunostained fibres extending throughout the graft (Fig. 2A). However, whether these fibres arose from grafted GABAergic embryonic neurons or from host spinal GABAergic neurons is not known.
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Table 1. Number of glutamate decarboxylase messenger RNA-labelled cells detected within the graft Animal no.
GAD65
GAD67
Two weeks p.g. 1 2 3 4 5
n.d. n.d. 31.2 ^ 5.1 26.0 ^ 2.7 22.5 ^ 1.7
n.d. n.d. 39.5 ^ 6.0 28.5 ^ 0.8 29.4 ^ 2.2
Five weeks p.g. 1 2 3 4 5
n.d. 27.0 ^ 1.4 17.8 ^ 1.2 17.1 ^ 1.9 35.3 ^ 1.6
n.d. 26.1 ^ 1.5 19.1 ^ 1.1 17.1 ^ 1.0 28.0 ^ 0.7*
The number of labelled cells within the same graft does not differ significantly between the two markers at either two or five weeks p.g., except for one animal (no. 5, five weeks p.g.). Each value is the mean ^ S.E.M. for an area of 6.25 × 10 22 mm 2; five to 12 determinations per animal were considered. n.d., not determined. *P , 0.05 (Student’s t-test).
Serotonergic immunoreactive cell bodies were also detected within the graft at both survival delays, most of them being large multipolar neurons (Fig. 2B). 5-HT-immunostained fibres also formed a dense network within
the graft, and isolated fibres extended at some distance into the gray matter of the host spinal cord (not shown). Double fluorescent immunodetection of GABA and 5-HT conformed to the single peroxidase immunostaining description for each marker and further indicated that no co-localization between the two markers occurred either in cell bodies or in fibres within the graft (Fig. 3). Spinal cord Observations were restricted to the region located between the transection and transplantation sites. In situ hybridization. Glutamate decarboxylase-65 messenger RNA. In transected, non-grafted rats, the number of GAD65 mRNA-labelled cells showed no statistical difference with intact rats at three weeks after transection (Fig. 4A, B, Table 2). By contrast, in transected/grafted animals, a very significant decrease (P , 0.001) was detected in the number of labelled cells at this survival delay (two weeks p.g.) compared with intact animals. At five weeks p.g., no significant difference was found between transected and transected/ grafted rats. Glutamate decarboxylase-67 messenger RNA. In transected animals, a very significant increase (P , 0.001)
Fig. 2. Immunodetection of GABA (A) and 5-HT (B) in spinal cord sections from a transplanted animal (two weeks p.g.). (A) Numerous GABA-immunoreactive cell bodies are observed within the graft (arrows). Positive somata are also detected in the host spinal cord (arrowheads). A dense network of GABApositive fibres is detected in the graft, as opposed to layers II/III of the host spinal cord, which contain only scarce GABA-positive fibres (asterisk). (B) In contrast to GABA immunoreactivity, 5-HT innervation originates only from transplanted 5-HT cells (arrows). Scale bar 100 mm.
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Regulation of spinal GABAergic neurons in transplanted rats
Fig. 3. Confocal paired images of a spinal cord section from a transplanted animal double immunostained for 5-HT and GABA. Two weeks after transplantation, 5-HT and GABA immunostainings are associated with distinct somata and fibres located within the graft (arrows and arrowheads point to 5-HT- and GABA-immunostained somata, respectively). Notice that 5-HT-immunoreactive spots are present on the 5-HT-positive cell bodies, but not on GABA-positive ones. Scale bar 25 mm.
in the number of GAD67 mRNA-expressing cells was observed at three and six weeks compared with intact animals (Fig. 4C, D, Table 2). By contrast, in transected/grafted animals, the number of GAD67 mRNA-labelled cells observed at two weeks p.g. showed no significant difference with control values from intact animals. By five weeks p.g., the number of stained cells was still reduced compared with transected animals, although the decrease was not as marked as at the previous survival delay. Immunohistochemistry. In transected animals, GABA immunoreactivity was less marked than in control intact animals, particularly within the superficial dorsal layers, where very few positive cell bodies were detected (Fig. 5B). In contrast, in the transected/grafted animals, numerous GABA-positive cell bodies were detected in this region (Fig. 5C). Moreover, the GABAergic innervation in the deep dorsal and ventral layers of the gray matter appeared to be denser than in non-grafted animals, particularly in the ventral horn around the motoneuronal somata (Fig. 5C2). In transected animals, the spinal cord below the lesion was totally devoid of 5-HT immunoreactivity. In contrast, in grafted animals, numerous 5-HT-immunoreactive fibres arising from the graft were detected at both survival delays in the host spinal cord. Ten millimetres above the graft, isolated 5-HT-positive fibres were seldom detected at two weeks p.g. and showed a quite random distribution. At five weeks p.g., 5-HT-positive fibres detected at this distance appeared to concentrate in the region surrounding the central canal, in the motoneuronal pools and in the intermediolateral column (whenever present; Fig. 6). Very few fibres were detected in the superficial dorsal layers. A reinnervation by 5-HT-positive fibres was also observed caudally to the graft, at the lumbar level.
DISCUSSION
The present observations demonstrate that: (i) the embryonic raphe transplant contains different neuronal populations, expressing either 5-HT or GABA immunoreactivities; (ii) after complete section, there is an increase in the number of host neurons that express GAD67 and the graft is able to reverse these modifications in the GABAergic phenotype; (iii) an increase in GABA content is detected in cell bodies and fibres of the spinal cord following grafting. Furthermore, detection of 5-HT-immunostained cells within the transplant fully confirmed our previous studies, 33–35 including the reinnervation of original 5-HT target areas in the spinal cord, 3 although reinnervation of the superficial dorsal horn was less marked in the present study. Characterization of transplanted cells An interesting finding of this study is the detection of numerous GABAergic neurons among the transplanted cells. Since cellular suspensions apposed to the white matter (thus having actual minimal contact with the host spinal cord) showed labelling for both GAD mRNAs similar to the one observed in the integrated grafts, it can be assumed that the expression of this phenotype is related to the intrinsic properties of the embryonic tissue rather than to its interactions with the host. To our knowledge, an embryonic raphe showing such a high content of GABAergic cells has not been described before. However, this finding is not really surprising, since the GABAergic system develops early and, by E14, GABAergic fibres are already present in the raphe 21 at a time when serotonergic neurons begin to differentiate. It has also been reported that GABA acts as a neurotrophic factor on immature neurons from the brain and spinal cord, as a chemoattractant and by inducing differentiation. 2,22,49 Furthermore, it has
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Fig. 4. In situ hybridization of GAD transcripts in spinal cord sections from a transected (A, C) and a transected/grafted animal (B, D). Five weeks after transplantation, heavily stained GAD65 mRNA-expressing cells (arrows) are detected in the region surrounding the central canal in both transected (A) and transected/grafted animals (B), while some cells showing a fainter labelling (arrowheads) are located throughout layers I–VII. In contrast, very numerous GAD67 mRNA-expressing cells are detected in the transected animal (arrows in C) in most layers of the gray matter, whereas the number and staining intensity of the labelled cells is decreased markedly in the grafted animal (arrows in D). Scale bar 100 mm.
recently been demonstrated that GABA enhances cell survival and increases neurite development of 5-HT neurons in cultures of E14 brainstem cells. 24 Moreover, in the dorsal raphe nucleus of adult rats, it is admitted that
local GABAergic innervation modulates 5-HT neuronal activity. 51,54 Thus, the presence of a high number of GABAergic cells in the transplant is coherent with the influence of GABA upon 5-HT neurons at the developmental
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Regulation of spinal GABAergic neurons in transplanted rats
Table 2. Total number of glutamate decarboxylase messenger RNA-labelled cells in spinal cord sections from control, transected and transected/grafted animals Control (n 3) GAD65 GAD67
32.3 ^ 1.2 53.7 ^ 2.4
Transected
Transected/grafted
Week 3 (n 5)
Week 6 (n 5)
31.9 ^ 2.6 75.2 ^ 3.8***
27.2 ^ 1.7* 80.5 ^ 3.8***
Week 3 (n 5)
Week 6 (n 5)
18.9 ^ 0.9***††† 57.2 ^ 3.2†††
33.5 ^ 2.1 65.8 ^ 2.7**††
The number of GAD65 mRNA-expressing cells detected in transected animals is quite similar to those of control animals, while in grafted animals it significantly decreases at three, but not six, weeks (two and five weeks p.g., respectively). The number of cells labelled for GAD67 mRNA is highly increased in transected animals, but returns towards control levels in grafted animals. This effect is maximal by three weeks after transection (two weeks p.g.). Each value is the mean ^ S.E.M. of 10–12 determinations per animal. Statistical analysis was performed using the Mann–Whitney test (nonparametric). *P , 0.05, **P , 0.01, ***P , 0.001 between injured (transected or transected/grafted) and control animals. ††P , 0.01, †††P , 0.001 between transected and transected/grafted animals at the same survival delay.
A1
B1
C1
A2
B2
C2
Fig. 5. GABA immunodetection in the spinal cord. In an intact animal, GABA-positive somata are detected in the dorsal horn (A1, arrows) and GABA-positive fibres in the ventral horn (A2, arrowheads). In a transected animal, a decrease in the labelling of GABA is observed in both the dorsal horn, where only a few cell bodies are detected (B1, arrow), and the ventral horn (B2, arrowhead). In a grafted animal, by five weeks p.g. GABA staining is restored in the host spinal cord. Scale bar 50 mm.
and adult stages. No co-localization between 5-HT and GABA was detected in this study either in embryonic neurons (E14) cultured for 24 h (unpublished observations) or in the grafted tissue at short or long survival delays (two and five weeks p.g.), but such a co-localization has been reported to occur in only a small percentage of raphe neurons (3.6% in the B3 nucleus). 18,50 Raphe cells grafted in the lesioned spinal cord may lack factors and/or connections that would have been provided by their natural environment and which may induce differences in phenotypic expression, even though another co-localization which occurs naturally in the raphe (5-HT and substance P) has been detected in the same transplantation model. 35 Finally, 5-HT-containing terminals were detected apposed to 5-HT-positive cell bodies in the graft. Interestingly enough, 5-HT has been shown to act as a differentiation factor on a raphe-derived neuronal cell line (RN46A) and could possibly be involved in an autocrine regulation loop via the 5-HT1A receptor. 8
Putative mechanisms of action of transplanted cells on the GABAergic neurons of the host spinal cord We have demonstrated previously that an embryonic raphe graft leads specifically to the recovery of some of the deficits in transected animals, acting on motoneuronal output 15 and recovering reflexes. 33,34 Since intrinsic GABAergic neurons are involved in the control of motoneuronal output, 28,31 their activity may have been modified by the lesion and then by the newly established connections. Indeed, GABAergic innervation of the motoneuronal pools appears to be increased following grafting as compared to transected rats. Several mechanisms can be considered for the regulation of the GABAergic activity in the host spinal cord. A first possibility is that embryonic tissue may provide multiple diffusible factors, including GABA, which may influence the surrounding spinal neurons. The injured environment in which raphe cells are transplanted may thus benefit from the trophins and factors released by the graft that enhance host network
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Fig. 6. 5-HT immunodetection in the spinal cord. In an intact animal, a specific 5-HT innervation is detected in target areas: the intermediolateral column (A) and the region surrounding the central canal (C), which are devoid of 5-HT immunoreactivity in transected animals (not shown). In transected/grafted animals, a 5-HT innervation pattern is re-established at five weeks p.g. (B, D). Scale bar 50 mm.
reorganization. However, it appears unlikely that the regulatory effect of the transplant observed as early as two weeks p.g. at a distance of about 10 mm from the injection site could be mediated by the diffusion of those factors up to such a distance. An alternative possibility could thus be that those factors diffusing from the graft have direct regulatory effects on interneurons located within the host spinal cord close to the graft which, in turn, influence the activity of more distant neurons to which they are directly or indirectly connected. The regulation of GABAergic neurons seen here might also, and most probably, be a consequence of the innervation of host spinal cord cells by various axonal fibres arising from the graft, including serotonergic fibres. A strong reinnervation of the host spinal cord by 5-HT fibres has been described previously 15,33–36 and is consistent with a direct regulation of distant GABAergic neurons by means of synaptic or nonsynaptic connections, as happens in the intact spinal cord. 37 As in the case of the 5-HT innervation, a supraspinal GABAergic innervation exists. 1,17,29 Descending fibres from the rostral ventromedial medulla, which includes the nucleus raphe magnus (B3), project mainly to layers IV/V and I/IIo, while some of the GABAergic descending fibres (13.8%) directly contact GABA-positive dendrites and cell bodies. 1 Following transection and transplantation, an additional specific GABAergic innervation of the spinal cord could arise from the grafted GABAergic neurons, in a similar way to the 5-HT innervation seen here. Regulation of glutamate decarboxylase messenger RNA and GABA by the transplant We show here that the modifications of the GABAergic neurons observed in the distal segment following transection
mainly involved the GAD67 isoform. The increased expression of the GAD67 mRNA isoform is a feature shared by the neurons of different CNS structures following injury, including the hippocampus, 12 cerebellum 23 and striatum. 49 In the present study, we show that the influence of a spinal cord transection on the expression of GAD67 messenger was reversed by transplantion of embryonic raphe cells into the denervated spinal cord, re-establishing the number of labelled neurons close to controls. However, GAD67 mRNA levels and GABA cellular content appear to be inversely correlated. A similar inverse correlation was observed between GAD67 and GABA immunoreactivities of spinal cord neurons in a model of sciatic nerve chronic constriction, 9 suggesting a regulation by negative feedback. In fact, increased GABA levels induced by vigabatrin have been shown to specifically decrease GAD67 protein levels in vitro 38 and in vivo, 39,44 although GAD67 mRNA levels were not affected, indicating a posttranslational regulation or/and changes in protein stability. In our model, compared with pharmacological manipulation, injury may have modified the input to GABAergic cells, thus inducing the up-regulation of GAD67 gene transcription levels. As GAD67 is probably involved in non-synaptic release of GABA, the over-expression of GAD67 mRNA may reflect an increase in this activity as a response to decreased GABA in the injured tissue. In transplanted animals, GAD67 mRNA could thus be down-regulated by the additional GABAergic and serotonergic innervation. Finally, it may also represent an injury-induced modification of the phenotype of neurons towards immature features, since virtually all spinal neurons express the GABAergic phenotype during development, 26,41,48 or simply reflect an increased metabolism of the cell. Another finding of this study is that the levels of GAD65 mRNA are also affected by the transplantation procedure,
Regulation of spinal GABAergic neurons in transplanted rats
although in an opposite manner to that of GAD67 mRNA. As the two GAD isoforms derive from different genes and their 5 0 untranslated regions show little homology, their expression is probably regulated by different mechanisms and factors. In fact, GADs may even respond differently within the same neuron, according to the afferent/efferent activities of the neuron or to the pathways in which it is involved (see Ref. 46 for a review). This is probably also the case in the spinal cord, since GAD65 and GAD67 are preferentially expressed in the dorsal and ventral horns, respectively. 14 This would explain the different responses of the two GADs to grafting procedures. Among the models of CNS injury in which mRNA levels are up-regulated, the denervation of the striatum appears to be of particular interest. 45 Destruction of dopaminergic fibres originating in the substantia nigra and projecting to the striatum leads to an increase in GAD67 mRNA levels and protein activity at four weeks post-injury. 42 Interestingly enough, it has been shown that, as in the spinal cord, (i) GAD67 mRNA increase is only detected in a population of neurons expressing low levels of this messenger under normal conditions 46 and (ii) a graft of embryonic substantia nigra, containing GABAergic neurons, is able to reverse these levels towards
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control values. 43 In the striatum, the graft regulates the injuryinduced GAD67 over-expression by re-establishing a local control on GABAergic neurons. Although spinal cord transection elicits a much more complex response than striatum deafferentation, it is very likely that, in the present case, a regulation of this gene by embryonic raphe cells transplantation results from similar reorganizing mechanisms. However, it remains to be shown that, if that is the case, the regulation of the GABAergic phenotype demonstrated here contributes to the restoration of spinal functions by embryonic raphe grafts. In any case, the present results shed some light on the complex phenomena which are triggered by a spinal cord lesion, and which can be corrected to some extent by transplanted embryonic neurons. Further studies should decide whether the GABAergic component which is illustrated here plays a specific role in the central pattern generator 15 responsible for the locomotion pattern, or whether it plays a more general role in the homeostasis of spinal cord functions. Acknowledgements—We are indebted to Ge´rard Alonso for his pertinent comments. Thanks are also due to Isabelle Chaudieu and Norbert Chauvet for helpful discussions. This work was supported by INSERM, BIOMED, AFM, IRME and VERTICALE.
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