Developmental Brain Research, 67 (1992) 237-246 © 1992 Elsevier Science Publishers B.V. All fights reserved. 0165-3806/92/$05.00
237
BRESD 51457
Pre- and postnatal development of noradrenergic projections to the rat spinal cord: an immunocytochemical study N. Rajaofetra a, P. Poulat a, L. Marlier a, M.
Geffard b and A. Privat a
aI.N.S.E.R.M. U336 (DPVSN)-EPHE (NBD), Montpellier (France) and bI.N.S.E.R.M. CJF 88-13, BP 66, Universit~ de Bordeaux, Bordeaux (France)
(Accepted 4 February 1992) Key words: Noradrenaline; Ontogeay; Spinal cord; Rat
Using immunocytochemistry with a specific antiserum against noradrenaline, the pre- and postnatal development of noradrenergic (NA) projections to the rat spinal cord was studied from embryonic day 16 (E16) to adulthood (the day following nocturnal mating being considered as E0). In this study, pregnant animals were pre-treated with the MAO inhibitor pargyline (200 mg/kg i.p.), whereas postnatal animals received 100 mg/kg. In vibratome sections, noradrenaline-immunoreactive (NA-IR) axons were seen to invade the spinal cord at El6, at cervical and upper thoracic levels, from the ventral funiculus. At El8, small caliber NA-IR fibers were present in the ventral horn at all cord levels, and some fibers were seen in the intermediolateral cell column (IML) at thoracic level. The growth of axons towards the dorsal horn became noticeable by postnatal day 0 (P0). At P3, fine beaded and radially orientated NA-IR fibers were observed at all levels. The pattern of NA innervation of the dorsal horn was similar to that of the adult by P7. The segregation of noradrenaline immunoreactivity in the ventral and dorsal horns, the IML and the periependymal area was more obvious at all levels by P14 and P20. From P30 the NA innervation was similar to that found in the adult spinal cord. Thus, noradrenaline, like serotonin, was present early in the spinal cord before the onset of specific functions. In addition to and prior to its transmitter function, it might play atrophic role in the neurogenesis of the spinal cord. INTRODUCTION The early development of the monoaminergic systems in the central nervous system of the rat was studied by Olson and Seiger as using a histofluorescence technique: noradrenaline was detected in the 11 m m embryo after serotonin and dopamine. However, very few studies had dealt in detail with the development of noradrenergic (NA) projections to the rat spinal cord. Moreover, they were analyzed with histofluorescence methods 9.t°'3s'43. The details of these N A projections had not yet been described with modern and specific immunocytochemical techniques, except for the postnatal study of A r a m a n t et al. 2 who used an antiserum against dopamine-fl-hydroxylase ( D B H ) to detect noradrenaline-like immunoreactire fibers. Moreover, the distributioa of spinal cord N A fibers had been thoroughly studied recently in the adult by immunocytochemistry, thanks to the availability of antibodies against the transmitter molecule 4°. The present report is the first to describe the day-today development of noradrenaline-immunoreactive (NAIR) fibers in the spinal cord from embryonic day 16 (E16) to adulthood, with immunocytochemistry, using an antiserum against noradrenaline ts'35. The findings of
this study are directly related to our strategy of intraspinal transplantation of embryonic N A - I R neurons 54. MATERIALS AND METHODS For this study, Sprague-Dawley OFA rats (lffa-C. ~do, France) at embryonic day (E) 16 to adulthood were used (the day following mating being considered as day 0). Pregnant females received the MAO inhibitor pargyline (200 mg/kg i.p.) 2 h before sacrifice. After laparotomy, fetuses were sacrificed daily from 16 to 20 days by intracardiac perfusion with a fixative composed of 5% glutaraldehyde in 50 mM sodium metabisulfite (SMB)-50 mM cacodylate buffer (pH 7.5). Postnatal animals (after 90 min of pargyline treatment (100 mg/kg i.p.) and under deep anesthesia with sodium pentobarbital (45 mg/kg, i.p.)), were perfused with the same fixative at 0, 3, 4, 7, 14, 20, 30 days, and at 2-3 months, as adults. For each age, at least three animals were used. After perfusion, the spinal cords were removed and post-fixed overnight in fresh fixative. Transverse sections (50 gm thick) were cut with a vibratome in Tris (50 mM)-SMB (50 mM) buffer at pH 7.6. at cervical, thoracic, lumbar and sacral levels. In addition, some longitudinal sections were also made at key stages. Noradrenaline immunocytochemistry was performed according to the general methods of our laboratory 41. In short, after treatment with trypsin-EDTA (0.25%, Gibco) for 30 s (E16-P7) and 5 min (P14-aduits), and sodium borohydride (10 raM) for 10 rain, sections were incubated with primary antiserum against noradrenaline Is at a dilution of 1:15,000 with the addition of 1% non-specific goat serum (NSS) and 0.1% Triton X-100 (P4-adults) for 48 h at 4°C in Tris-SMB buffer. Following rinses in "Iris saline buffer (50 raM) at pH 7.6, sections were processed for immunocytochemistry
Correspondence: N. Rajaofetra, I.N.S.E.R.M. U336 (DPVSN)-EPHE (NBD), Case courrier 106, U.S.T.L. Place Eug6ne Bataillon, 34095 Montpellier Cedex 05, France. Fax: (33) (67) 14 3318.
238 Fig. 3. Longitudinal section of the spinal cord at El6 showing NA-IR fibers (arrows) in the ventral funiculus at cervical level. (R) and (C) = rostral and caudal parts of the cord. Bar = 40 ~m. Fig. 4. At El7, immunoreactive NA-IR varicosities were seen in the medial part of the ventral horn (arrowheads) at cervical level. Arrow points to an NA-IR axon directed towards the central canal (CC). Bar = 40 lzm. Fig. 5. Transverse section of the cord at El8, showing at cervical level the density of NA innervation in the ventral horn (compare with Fig. 4). Bar = 40 l~m. Fig. 6. Transverse section of spinal cord at El9, showing at thoracic level numerous immunoreactive fibers invading the dorsal part of the central canal (CC). Bar = 30 #m. Figs. 7 and 8. Transverse sections of cervical spinal cord at sho~mg thin NA-IR fibers (arrows) in the deep part of the dorsal horn (Fig. 7), whereas in the ventral horn they were concentrated around motoneuron areas (Fig. 8). Bars = 50 l~m.
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El6 In longitudinal sections, NA-IR fibers were seen running in the basal plate (Fig. 3). In addition, an orthogonal angulation of some fibers was seen occasionally, directed towards the ventral horn at cervical and upper thoracic levels. On transverse sections, at the cervical level, fibers were seen in the entire thickness of the ventral funiculus. At upper thoracic level very few immunoreactive fibers were present and were mostly located in the ventral part of the cord. No immunoreactivity was seen in the lumbar spinal cord.
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In the present study transverse and longitudinal sections were used. Longitudinal sections were made in the horizontal plane. For the sake of clarity, schematic drawings in Figs. I and 2 summarize the results of this immunocytochemical study, showing the distribution of NA projections from E16 to P30.
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2 Figs. 1 and 2. Schematic drawings of the distribution of NA projections at different ages at cervical (C), thoracic (T), lumbar (L) and sacral (S) levels of the spinal cord. Bars -- 0.5 ram.
using the peroxidase-antiperoxidase (PAP) staining method46: they were successively incubated with goat anti-rabbit lgG (Nordica) and PAP (Dako) diluted 1:I00 in Tris saline-buffer added in 1% NSS for 30 min at room temperature. Under visual control, peroxidase deposit was revealed with 0.05% 3,3'-diaminobenzidine hy-
E17 At cervical level, numerous fibers were seen in the ventral funiculi. The ventral horns were invaded by thin immunoreactive fibers (Fig. 4). At the thoracic and lumbar levels, individual pioneering fibers were seen invading the grey matter. ,\t all levels, the lateral and dorsal horns were totally devoid of immunoreactivity. E18
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240 icose NA-IR fibers at all levels. The overall density of the ventral horn innervation increased progressively throughout the cord when compared with El7. Immunoreactive fine fibers were more numerous, particularly at cervical (Fig. 5) and upper thoracic levels where they were seen invading the medial part of the ventral horn. At the lower thoracic level, immunoreactive fibers were less numerous than in 'he upper thoracic cord. They also innervated the medial part of the ventral horn. However, at the lumbar level, immunoreactive fibers appeared much more numerous around the motoaeuron area, contrasting with the individual pioneering fibers detected at the sacral level at this age and in the lumbar horn at El7. In the dorsal and lateral funiculi, very few immunoreactive fibers were seen at the cervical and upper thoracic levels. At the mid-thoracic level, isolated NA-IR fibers were detected in the intermediolateral cell column (IML) and in the deep part of the dorsal horn.
El9 Immunoreactivity was increased around the motoneuron regions of the ventral horn at all levels, and this was particularly striking at the thoracic level. Again, at thoracic level, the dorsal part of the central canal (CC) corresponding to the dorsal autonomic nucleus showed a high concentration of fibers that began to form a commissure which linked the two IMLs horizontally (Fig. 6). E20 Immunoreactive fibers still increased in number in the ventral horn at all levels: they appeared thinner than at El9 except at lumbar level, where thick NA-IR fibers were concentrated in the medial part of the ventral horn (Fig. 8). A rostro-caudal density gradient was still present. At the thoracic level, immunoreactive fibers began to concentrate in the IML. Moreover, fine beaded fibers appeared around the CC, particularly in its dorsal part, to form a dorsal commissure. Finally, thin beaded NA-IR fibers were seen in layers III and IV of the dorsal horn at cervical and upper tho-
racic levels (Fig. 7).
Newborn The only difference from E20 was the further reinforcement of the lateral horn innervation. Indeed, longitudinal sections at thoracic level showed a typical organization of the IML, with parallel streams of fibers (Fig. 9) extending from the medial side of the cord around the CC. Occasionally, in the white matter, immunoreactive fibers with growth cones were still seen running longitudinally and at the cervical level, fine immunoreactive fibers were seen invading the whole ventral horn. At the lumbar and sacral levels, immunoreactive fibers were concentrated in the internal group of the ventral horn motoneurons. Growth cones were still evident at sacral level. In the dorsal horn, very few thin fibers were found in the superficial layers, and only at cervical level. P3 The increased innervation of the dorsal horn was a characteristic feature of this age. At all levels fine beaded and radially oriented NA-IR fibers were more conspicuous than at birth. At the cervical level, numerous immunoreactive fibers were seen innervating the dorsal horn following two patterns: layers III and IV were diffusely invaded from the central part of the cord, whereas fine fibers concentrated in layer I and the outer part of layer II were derived from the dorsal funiculus. At the lumbar level, individual fibers were seen in the external part of the dorsal horn. In the ventral horn, the density of the innervation of the motoneuron areas still increased over the background of the grey matter. A dense fine network of NA-IR fibers was observed all along the cord. /'4 The only difference from P3 was the progression of the dorsal horn innervation at lumbar level. Indeed, more fibers were seen invading layer I, as shown in Fig. 1.
Fig. 9. Longitudinal section of the thoracic spinal cord at PO showing the organi=ation of NA-IR fibers. Bar = 80 l*m. Figs. I0 and 11. Intrinsic NA-IR cell bodies located in the dorsal or dorsolate-al parts of the central canal (CC) of the high cervical spinal cord, at PI4 (Fig. 10) and P30 (Fig, IlL Bars - 80 ~um, Fig. 12. Longitudinal section of the thoracic spinal cord at P14 showing the increase of NA innervation intensity when comparing with that at PO (Fig. 9). Bar - 200 l~m. Figs. 13 and 15. Longitudinal sections of thoracic spinal cord at P30. Fig. 13. In the dorsal horn, NA-IR fibers oriented longitudinally were located in layer II. Bar = 200 ~m. Fig. 14. Longitudinal section of the thoracic spinal cord at P20, The periodicity of transversal orientation and the intensity of NA-IR fibers were more conspicuous than at PI4 (Fig. 12), Bar -- ~m. Fig. 15. Dense noradrenaline immunoreactivity was seen in the ventral horn, as in the adult cord. Bar -- 80/~m.
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242 pattern of radially oriented fibers in layers II and III at lower thoracic level. The organization of the IML was most prominent at thoracic levels.
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Figs. 16 and 17. Transverse sections of adult spinal cord showing the distribution and radial orientation (arrows) of NA-IR fibers in the dorsal horn (Fig. 16). Fig. 17 shows at low magnification NA distribution in the dorsal horn (DH), surrounding the central canal (CC), the intermediolateral cell column (IML), and around the motoneurons in the ventral horn (VH) at mid-thoracic level• Bars = 40/~m in Fig. 16, and 200 #m in Fig. 17.
243
P14 The pattern of innervation was now almost similar to that of the adult, especially for the dorsal horn, with an invasion of layers I and II by dense, thin, immunoreactive fibers at all levels following a rostro-caudal density gradient. For example, at the sacral level, immunoreactive fibers were less conspicuous than at other levels. The increased segregation of immunoreactive fibers in the ventral horn and the IML was conspicuous at all levels. At cervical and thoracic levels, fine immunoreactive fibers and varicosities were seen densely distributed around all groups of motoneurons in the ventral horn. They were particularly concentrated in the external part of the ventral horn at the lumbar level, while they were more prominent in the medial and internal parts at thoracic and sacral levels. Immunoreactive fibers were conspicuous in the IML at both thoracic and sacral levels. The periodic organization of the transversal fibers was more obvious than at P7. NA-IR perikarya were occasionally observed in the dorsal part of the CC (Fig. 10) at the cervical level only, and in the dorsolateral funiculus (in the reticular nucleus of the dorsal horn). However, the innervation did not appear to be stabilized at this age. Indeed, we observed several immunoreactive fibers showing unusual dilatations in the ventral horn at lumbar and sacral levels, suggestive of involutive processes. P20 The difference from P14 was the further overall increase in the density of innervation (compare Figs. 12 and 14). Specifically, the number of fine-beaded NA-IR fibers increased in the ventral horn at all levels when compared with that seen at P14. The IML presented a characteristic concentration of NA-IR profiles which were stained much more intensely than at P14 at thoracic (Fig. 14) and sacral levels. In the dorsal horn, at sacral level, immunoreactive fibers were more numerous and more intensely stained than at P14. P30 At this age, the innervation pattern was now identical to that of the adult. In the ventral horn, immunoreactive profiles appeared concentrated around the motoneurons, as seen in horizontal sections (Fig. 15). In the IML, at the thoracic level, the highest concentration of immunoreactive fibers was identical to that of the adult spinal cord as shown in Fig. 17. Specifically, periodic transverse connections between the dorsal au-
tonomic nucleus and IML were now more conspicuous than at previous stages. In the dorsal horn, at all levels, thin immunoreactive fibers ran longitudinally in layers I and II, as seen in horizontal sections (Fig. 13), whereas radially oriented fibers were seen coursing in the inner part of layer II and layer III, as seen in transverse sections of the adult cord (Fig. 16). Occasionally, a few NA-IR perikarya were found close to the central canal at upper cervical level (Fig. 11). DISCUSSION Direct immunocytochemical detection of noradrenaline was used in the present study to determine the timing of spinal cord innervation and to describe the dayto-day development and distribution of NA fibers. To date, studies of NA innervation of the cord, during development, or in the adult, have been conducted either with histochemical detection using formaldehydeinduced histofluorescence procedures 13 or with the immunocytochemical detection of noradrenaline synthesizing enzymes. In the adult, projections of locus coeruleus (LC) neurons to the spinal cord were first demonstrated by Dahlstr6m and Fuxe ~'~2. They were then confirmed by several studies t6'36'5°-53. Paralleily, neuroanatomical studies using a retrograde cell marker confirmed that numerous LC neurons projected to the spinal cord 4'21'29. The topography of NA spinal innervation was also detailed with histofluorescence by Mizukawa 34 and McLachlan and Oldfield 32. However, this latter technique did not permit unequivocal discrimination between fluorophores derived from NA and dopaminergic fibers6'45, and was not compatible with electron microscopy. Immunocytochemical detection of DBH, the final enzyme in the biosynthetic pathways of noradrenaline also permitted the detection of noradrenaline-containing neurons and of their spinal projections s'37 but DBH immunocytochemistry 2'16't7'5°'5 did not distinguish between dopaminergic, NA and adrenergic systems 24. This is of particular importance since adrenaline is present in the spinal cord. It has been shown unequivocally by immunocytochemical detection of phenylethanolamine-N-methyltransferase (PNMT) and its origin was traced to groups A1 and A2 of monoaminergic neurons, both in rats 5'25'33 and monkeys 7. Worth noting was the massive innervation of the IML, where preceding studies with histofluorescence or DBH immunocytochemistry could have mistaken it for noradrenaline 24. Similarly, during development, the histochemical studies of Olson and Seiger 3s, Seiger and Olson 43, Nygren
244 and Olson 36 and Commissiong 9't° could not discriminate between catecholamines. Moreover, these studies used less sensitive techniques than that ased here, since, for instance, Commissiong t° did not describe IML innervation before birth whereas we detected it as early as El9. In the present study, the use of an anti-noradrenaline antiserum was the method of choice, since cross-reactivity of this antiserum with other monoamines was excluded ts. The source of spinal cord NA fibers appeared essentially supraspinal. During development, NA innervation clearly followed a rostro-caudal gradient, and in addition, we did not detect intrinsic NA-IR perikarya, except at the upper cervical level, where they were continuous with medullary groups. The timing of spinal cord NA innervation is characteristic of monoaminergic systems. As for serotonin 4t, it is an early event in spinal cord ontogeny, as in other CNS areas 26. NA innervation was detected in the ventral horn before the corticospinal tract was established 4s. Similarly, in the dorsal horn NA innervation was present before the sensory system started functioning which was not present until the second postnatal week t4'~5, a period when NA innervation became prominent. Moreover, the sequence of NA spinal cord innervation which we detected, the ventral horn and IML preceding the dorsal horn, is in accordance with the sequence of maturation of motor, visceral and sensory systems 1~'39. When compared with serotonergic innervation, it appears slightly delayed, as 5-HT fibers were present in the cord as early as the 14th embryonic day 4~, This is true for all the specific targets, which were the same for the two amines, with a delay of 2-3 days. The initial pattern of innervation was the same for tile two amines, with an invasion of the grey matter by sharp angulation of fibers coursing in the ventral funiculus 4t' .,9. Similarly, the progressive segregation of fibers around motoneurons in the ventral horn, and the dual innervation of the dorsal horn were reminiscent of 5-HT innerration 4t.
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What is the functional implication of prenatal NA innervation? Is it, as suggested for 5-HT 3°'49, a neurotrophic factor or signal for target areas or, as has been hypothesized for monoamines, a 'differentiation regulator'22'23'27? In the ventral horn, for instance, it can be hypothesizdd that NA innereation could contribute to the stabilization of the motoneuron pool, which decreased after E15, and then remained stable from birth onwards 39. After birth, the basic innervation pattern was progressively completed and this corresponded to the period when the corticospinal aI:d the rubrospinal systems became functional 31'44'48. NA systems had potentially significant functions in somato-motor control and the regulation of autonomic and sensory functions: they may be involved in the modulation of complex motor activities 54, central generation of locomotion 19'2° and the modulation of spinal reflexes 1'36'47. Several studies have implicated the descending NA fibers in central autonomic control 3'2s'42. They were present and organized in the IML from E19 to adulthood at thoracic and sacral levels. These findings suggest that descending NA inputs play an important role in the regulation of sympathetic autonomic function, as well as of parasympathetic function. To summarize, the present study, the accuracy of which was made possible by the use of a specific antiserum, showed the early onset of NA innervation during spinal cord development. Such an early projection may correspond either to a neurotrophic influence on target regions, or to an early influence on spinal cord function. These two possibilities are not mutually exclusive. Further studies will be aimed at elucidating these functions, through early specific lesions and/or agonistic pharmacological manipulations.
Acknowledgemo~ts. The authors acknowledge J.-R. Teilhac for the art work. This work has been supported by D. Heumann Fund, IRME and AFM.
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6 7 8
9
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271-280. 38 Olson, L. and Seiger, A., Early prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations,Z. Anat. EntwickL Gesch., 137 (1972) 301-316. 39 OpP enheim' H.W., The absence of significantpost-natal motoneuron death in the brachial and lumbar spinal cord of the rat,J. Comp. Neurol., 246 (1986) 281-286. 40 Rajaofetra,N., Ridet, J.L., Poulat, P., Marlier, L., Sandillon, F., Geffard, M. and Privat, A., Immunocytochemical mapping of noradrenergic projections to the rat spinal cord with an antiserum against noradrenaliae,J. Neurocytol., submitted. 41 Rajaofetra,N., Sandillon, F., Geffard, M. and Privat,A., Preand post.natalontogeny of serotonergic projections to the rat spinal cord, J. Neurosci. Res., 22 (1989) 305-321. 42 Ryall, R.W. and deGroat, W.C., The microelectrophoreticadministrationof noradrenaline, 5.hydroxytryptamine, acetylcholine, and glycine to sacral parasympathetic preganglionic neuronS, Brain Res., 37 (1972). 43 Seiger, .~'.and Olson, L., Late prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations,Z. Anat. Entwickl. Gesch., 140 (1973) 281-318. 44 shieh,J.Y',Leong, S.K. and Wong, W.C., Origin of the rubrospinal tractin neonatal developing and mature rats,J. Comp. Neurol., 214 (1983) 79-86. 45 skagerberg,G., Bj6rklund, A., Lindvall,O. and Schmidt, R.H., origin and termination of the diencephalo-spinaldopamine systena in the rat, Brain Res. Bull., 9 (1982) 237-244. 46 sternberger,L.A., Hardy, P.H., Cuculis,J.J.and Meyer, H.G., The unlabeled antibody enzyme method of immunohistochemistrY. Preparation and properties of soluble antigen-antibody comple x horseradish-antihorseradishperoxidase and its use in identificationspirochetes,,/. Histochem. Cytochem., 18 (1970) 315-333. 47 strahlendorf,J.C., Strahlendorf,H.K., Kingsley, R.E., Gintautas, J" and Barnes, C.D., Facilitationof the lumbar monosynaptic reflexesby locus coeruleus stimulation,Neuropharmacology, 19 (1980) 225-230. 48 valentino, K.L., Jones, E.G. and Kane, S.A., Expression o~ glial fibrillary acidic protein immunoreactivity during develop-
246 ment of long fiber tracts in the rat central nervous system, Dev. Brain Res., 3 (1983) 317-336.
49 Wallace, J.A. and Lauder, J.M., Development of serotonergic system in rat embryo: an immunocytochemical study, Brain Res. Bull., 10 (1983) 459-479. 50 Westlund, K.N., Bowker, R.M., Ziegler, M.G. et al., Descending noradrenergic projections and their spinal terminations. In Kuypers, H.G.J.M. and Martin, G.F. (Eds.), Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 219-236. 51 Westlund, K.N., Bowker, R.M., Ziegler, M.G. and Coulter, J.D., Noradrenergic projections to the spinal cord of the rat, Brain Res., 263 (1983) 15-31.
52 Westlund, K.N., Bowker, R.M., Ziegler, M.G. and Coulter, J.D., Origins of spinal noradrenergic pathways demonstrated by anterograde transport of antibody to dopamine-fl-hydroxylase, Neurosci. Lett., 25 (1981) 243-249. 53 Westlund, K.N., Bowker, R.M., Ziegler, M.J. and Coulter, J.D., Origins and terminations of descending noradrenergic projections to the spinal cord of monkey, Brain Res., 292 (1984) 1-16. 54 Yakovleff, A., Roby-Brami, A., Guezard, B., Mansour, H., Bussel, B. and Privat, A., Locomotion in rats transplanted with noradrenergic neurons, Brain Res. Bull., 22 (1989) 115-121.
237
BRESD 51457
Pre- and postnatal development of noradrenergic projections to the rat spinal cord: an immunocytochemical study N. Rajaofetra a, P. Poulat a, L. Marlier a, M.
Geffard b and A. Privat a
aI.N.S.E.R.M. U336 (DPVSN)-EPHE (NBD), Montpellier (France) and bI.N.S.E.R.M. CJF 88-13, BP 66, Universit~ de Bordeaux, Bordeaux (France)
(Accepted 4 February 1992) Key words: Noradrenaline; Ontogeay; Spinal cord; Rat
Using immunocytochemistry with a specific antiserum against noradrenaline, the pre- and postnatal development of noradrenergic (NA) projections to the rat spinal cord was studied from embryonic day 16 (E16) to adulthood (the day following nocturnal mating being considered as E0). In this study, pregnant animals were pre-treated with the MAO inhibitor pargyline (200 mg/kg i.p.), whereas postnatal animals received 100 mg/kg. In vibratome sections, noradrenaline-immunoreactive (NA-IR) axons were seen to invade the spinal cord at El6, at cervical and upper thoracic levels, from the ventral funiculus. At El8, small caliber NA-IR fibers were present in the ventral horn at all cord levels, and some fibers were seen in the intermediolateral cell column (IML) at thoracic level. The growth of axons towards the dorsal horn became noticeable by postnatal day 0 (P0). At P3, fine beaded and radially orientated NA-IR fibers were observed at all levels. The pattern of NA innervation of the dorsal horn was similar to that of the adult by P7. The segregation of noradrenaline immunoreactivity in the ventral and dorsal horns, the IML and the periependymal area was more obvious at all levels by P14 and P20. From P30 the NA innervation was similar to that found in the adult spinal cord. Thus, noradrenaline, like serotonin, was present early in the spinal cord before the onset of specific functions. In addition to and prior to its transmitter function, it might play atrophic role in the neurogenesis of the spinal cord. INTRODUCTION The early development of the monoaminergic systems in the central nervous system of the rat was studied by Olson and Seiger as using a histofluorescence technique: noradrenaline was detected in the 11 m m embryo after serotonin and dopamine. However, very few studies had dealt in detail with the development of noradrenergic (NA) projections to the rat spinal cord. Moreover, they were analyzed with histofluorescence methods 9.t°'3s'43. The details of these N A projections had not yet been described with modern and specific immunocytochemical techniques, except for the postnatal study of A r a m a n t et al. 2 who used an antiserum against dopamine-fl-hydroxylase ( D B H ) to detect noradrenaline-like immunoreactire fibers. Moreover, the distributioa of spinal cord N A fibers had been thoroughly studied recently in the adult by immunocytochemistry, thanks to the availability of antibodies against the transmitter molecule 4°. The present report is the first to describe the day-today development of noradrenaline-immunoreactive (NAIR) fibers in the spinal cord from embryonic day 16 (E16) to adulthood, with immunocytochemistry, using an antiserum against noradrenaline ts'35. The findings of
this study are directly related to our strategy of intraspinal transplantation of embryonic N A - I R neurons 54. MATERIALS AND METHODS For this study, Sprague-Dawley OFA rats (lffa-C. ~do, France) at embryonic day (E) 16 to adulthood were used (the day following mating being considered as day 0). Pregnant females received the MAO inhibitor pargyline (200 mg/kg i.p.) 2 h before sacrifice. After laparotomy, fetuses were sacrificed daily from 16 to 20 days by intracardiac perfusion with a fixative composed of 5% glutaraldehyde in 50 mM sodium metabisulfite (SMB)-50 mM cacodylate buffer (pH 7.5). Postnatal animals (after 90 min of pargyline treatment (100 mg/kg i.p.) and under deep anesthesia with sodium pentobarbital (45 mg/kg, i.p.)), were perfused with the same fixative at 0, 3, 4, 7, 14, 20, 30 days, and at 2-3 months, as adults. For each age, at least three animals were used. After perfusion, the spinal cords were removed and post-fixed overnight in fresh fixative. Transverse sections (50 gm thick) were cut with a vibratome in Tris (50 mM)-SMB (50 mM) buffer at pH 7.6. at cervical, thoracic, lumbar and sacral levels. In addition, some longitudinal sections were also made at key stages. Noradrenaline immunocytochemistry was performed according to the general methods of our laboratory 41. In short, after treatment with trypsin-EDTA (0.25%, Gibco) for 30 s (E16-P7) and 5 min (P14-aduits), and sodium borohydride (10 raM) for 10 rain, sections were incubated with primary antiserum against noradrenaline Is at a dilution of 1:15,000 with the addition of 1% non-specific goat serum (NSS) and 0.1% Triton X-100 (P4-adults) for 48 h at 4°C in Tris-SMB buffer. Following rinses in "Iris saline buffer (50 raM) at pH 7.6, sections were processed for immunocytochemistry
Correspondence: N. Rajaofetra, I.N.S.E.R.M. U336 (DPVSN)-EPHE (NBD), Case courrier 106, U.S.T.L. Place Eug6ne Bataillon, 34095 Montpellier Cedex 05, France. Fax: (33) (67) 14 3318.
238 Fig. 3. Longitudinal section of the spinal cord at El6 showing NA-IR fibers (arrows) in the ventral funiculus at cervical level. (R) and (C) = rostral and caudal parts of the cord. Bar = 40 ~m. Fig. 4. At El7, immunoreactive NA-IR varicosities were seen in the medial part of the ventral horn (arrowheads) at cervical level. Arrow points to an NA-IR axon directed towards the central canal (CC). Bar = 40 lzm. Fig. 5. Transverse section of the cord at El8, showing at cervical level the density of NA innervation in the ventral horn (compare with Fig. 4). Bar = 40 l~m. Fig. 6. Transverse section of spinal cord at El9, showing at thoracic level numerous immunoreactive fibers invading the dorsal part of the central canal (CC). Bar = 30 #m. Figs. 7 and 8. Transverse sections of cervical spinal cord at sho~mg thin NA-IR fibers (arrows) in the deep part of the dorsal horn (Fig. 7), whereas in the ventral horn they were concentrated around motoneuron areas (Fig. 8). Bars = 50 l~m.
E20
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drochloride and 0.01% H202 in the same buffer. Finally, sections were transferred to 60 mM phosphate buffer at pH 7.6, mounted on glass slides, dried overnight at 50°C, cleared in xylene and mounted with Gurr-DPX.
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El6 In longitudinal sections, NA-IR fibers were seen running in the basal plate (Fig. 3). In addition, an orthogonal angulation of some fibers was seen occasionally, directed towards the ventral horn at cervical and upper thoracic levels. On transverse sections, at the cervical level, fibers were seen in the entire thickness of the ventral funiculus. At upper thoracic level very few immunoreactive fibers were present and were mostly located in the ventral part of the cord. No immunoreactivity was seen in the lumbar spinal cord.
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In the present study transverse and longitudinal sections were used. Longitudinal sections were made in the horizontal plane. For the sake of clarity, schematic drawings in Figs. I and 2 summarize the results of this immunocytochemical study, showing the distribution of NA projections from E16 to P30.
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2 Figs. 1 and 2. Schematic drawings of the distribution of NA projections at different ages at cervical (C), thoracic (T), lumbar (L) and sacral (S) levels of the spinal cord. Bars -- 0.5 ram.
using the peroxidase-antiperoxidase (PAP) staining method46: they were successively incubated with goat anti-rabbit lgG (Nordica) and PAP (Dako) diluted 1:I00 in Tris saline-buffer added in 1% NSS for 30 min at room temperature. Under visual control, peroxidase deposit was revealed with 0.05% 3,3'-diaminobenzidine hy-
E17 At cervical level, numerous fibers were seen in the ventral funiculi. The ventral horns were invaded by thin immunoreactive fibers (Fig. 4). At the thoracic and lumbar levels, individual pioneering fibers were seen invading the grey matter. ,\t all levels, the lateral and dorsal horns were totally devoid of immunoreactivity. E18
The ventral horns were invaded by many fine and var-
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240 icose NA-IR fibers at all levels. The overall density of the ventral horn innervation increased progressively throughout the cord when compared with El7. Immunoreactive fine fibers were more numerous, particularly at cervical (Fig. 5) and upper thoracic levels where they were seen invading the medial part of the ventral horn. At the lower thoracic level, immunoreactive fibers were less numerous than in 'he upper thoracic cord. They also innervated the medial part of the ventral horn. However, at the lumbar level, immunoreactive fibers appeared much more numerous around the motoaeuron area, contrasting with the individual pioneering fibers detected at the sacral level at this age and in the lumbar horn at El7. In the dorsal and lateral funiculi, very few immunoreactive fibers were seen at the cervical and upper thoracic levels. At the mid-thoracic level, isolated NA-IR fibers were detected in the intermediolateral cell column (IML) and in the deep part of the dorsal horn.
El9 Immunoreactivity was increased around the motoneuron regions of the ventral horn at all levels, and this was particularly striking at the thoracic level. Again, at thoracic level, the dorsal part of the central canal (CC) corresponding to the dorsal autonomic nucleus showed a high concentration of fibers that began to form a commissure which linked the two IMLs horizontally (Fig. 6). E20 Immunoreactive fibers still increased in number in the ventral horn at all levels: they appeared thinner than at El9 except at lumbar level, where thick NA-IR fibers were concentrated in the medial part of the ventral horn (Fig. 8). A rostro-caudal density gradient was still present. At the thoracic level, immunoreactive fibers began to concentrate in the IML. Moreover, fine beaded fibers appeared around the CC, particularly in its dorsal part, to form a dorsal commissure. Finally, thin beaded NA-IR fibers were seen in layers III and IV of the dorsal horn at cervical and upper tho-
racic levels (Fig. 7).
Newborn The only difference from E20 was the further reinforcement of the lateral horn innervation. Indeed, longitudinal sections at thoracic level showed a typical organization of the IML, with parallel streams of fibers (Fig. 9) extending from the medial side of the cord around the CC. Occasionally, in the white matter, immunoreactive fibers with growth cones were still seen running longitudinally and at the cervical level, fine immunoreactive fibers were seen invading the whole ventral horn. At the lumbar and sacral levels, immunoreactive fibers were concentrated in the internal group of the ventral horn motoneurons. Growth cones were still evident at sacral level. In the dorsal horn, very few thin fibers were found in the superficial layers, and only at cervical level. P3 The increased innervation of the dorsal horn was a characteristic feature of this age. At all levels fine beaded and radially oriented NA-IR fibers were more conspicuous than at birth. At the cervical level, numerous immunoreactive fibers were seen innervating the dorsal horn following two patterns: layers III and IV were diffusely invaded from the central part of the cord, whereas fine fibers concentrated in layer I and the outer part of layer II were derived from the dorsal funiculus. At the lumbar level, individual fibers were seen in the external part of the dorsal horn. In the ventral horn, the density of the innervation of the motoneuron areas still increased over the background of the grey matter. A dense fine network of NA-IR fibers was observed all along the cord. /'4 The only difference from P3 was the progression of the dorsal horn innervation at lumbar level. Indeed, more fibers were seen invading layer I, as shown in Fig. 1.
Fig. 9. Longitudinal section of the thoracic spinal cord at PO showing the organi=ation of NA-IR fibers. Bar = 80 l*m. Figs. I0 and 11. Intrinsic NA-IR cell bodies located in the dorsal or dorsolate-al parts of the central canal (CC) of the high cervical spinal cord, at PI4 (Fig. 10) and P30 (Fig, IlL Bars - 80 ~um, Fig. 12. Longitudinal section of the thoracic spinal cord at P14 showing the increase of NA innervation intensity when comparing with that at PO (Fig. 9). Bar - 200 l~m. Figs. 13 and 15. Longitudinal sections of thoracic spinal cord at P30. Fig. 13. In the dorsal horn, NA-IR fibers oriented longitudinally were located in layer II. Bar = 200 ~m. Fig. 14. Longitudinal section of the thoracic spinal cord at P20, The periodicity of transversal orientation and the intensity of NA-IR fibers were more conspicuous than at PI4 (Fig. 12), Bar -- ~m. Fig. 15. Dense noradrenaline immunoreactivity was seen in the ventral horn, as in the adult cord. Bar -- 80/~m.
241
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242 pattern of radially oriented fibers in layers II and III at lower thoracic level. The organization of the IML was most prominent at thoracic levels.
P7 There was still a general progression of dorsal horn innervation a~ all levels with the onset of a characteristic
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Figs. 16 and 17. Transverse sections of adult spinal cord showing the distribution and radial orientation (arrows) of NA-IR fibers in the dorsal horn (Fig. 16). Fig. 17 shows at low magnification NA distribution in the dorsal horn (DH), surrounding the central canal (CC), the intermediolateral cell column (IML), and around the motoneurons in the ventral horn (VH) at mid-thoracic level• Bars = 40/~m in Fig. 16, and 200 #m in Fig. 17.
243
P14 The pattern of innervation was now almost similar to that of the adult, especially for the dorsal horn, with an invasion of layers I and II by dense, thin, immunoreactive fibers at all levels following a rostro-caudal density gradient. For example, at the sacral level, immunoreactive fibers were less conspicuous than at other levels. The increased segregation of immunoreactive fibers in the ventral horn and the IML was conspicuous at all levels. At cervical and thoracic levels, fine immunoreactive fibers and varicosities were seen densely distributed around all groups of motoneurons in the ventral horn. They were particularly concentrated in the external part of the ventral horn at the lumbar level, while they were more prominent in the medial and internal parts at thoracic and sacral levels. Immunoreactive fibers were conspicuous in the IML at both thoracic and sacral levels. The periodic organization of the transversal fibers was more obvious than at P7. NA-IR perikarya were occasionally observed in the dorsal part of the CC (Fig. 10) at the cervical level only, and in the dorsolateral funiculus (in the reticular nucleus of the dorsal horn). However, the innervation did not appear to be stabilized at this age. Indeed, we observed several immunoreactive fibers showing unusual dilatations in the ventral horn at lumbar and sacral levels, suggestive of involutive processes. P20 The difference from P14 was the further overall increase in the density of innervation (compare Figs. 12 and 14). Specifically, the number of fine-beaded NA-IR fibers increased in the ventral horn at all levels when compared with that seen at P14. The IML presented a characteristic concentration of NA-IR profiles which were stained much more intensely than at P14 at thoracic (Fig. 14) and sacral levels. In the dorsal horn, at sacral level, immunoreactive fibers were more numerous and more intensely stained than at P14. P30 At this age, the innervation pattern was now identical to that of the adult. In the ventral horn, immunoreactive profiles appeared concentrated around the motoneurons, as seen in horizontal sections (Fig. 15). In the IML, at the thoracic level, the highest concentration of immunoreactive fibers was identical to that of the adult spinal cord as shown in Fig. 17. Specifically, periodic transverse connections between the dorsal au-
tonomic nucleus and IML were now more conspicuous than at previous stages. In the dorsal horn, at all levels, thin immunoreactive fibers ran longitudinally in layers I and II, as seen in horizontal sections (Fig. 13), whereas radially oriented fibers were seen coursing in the inner part of layer II and layer III, as seen in transverse sections of the adult cord (Fig. 16). Occasionally, a few NA-IR perikarya were found close to the central canal at upper cervical level (Fig. 11). DISCUSSION Direct immunocytochemical detection of noradrenaline was used in the present study to determine the timing of spinal cord innervation and to describe the dayto-day development and distribution of NA fibers. To date, studies of NA innervation of the cord, during development, or in the adult, have been conducted either with histochemical detection using formaldehydeinduced histofluorescence procedures 13 or with the immunocytochemical detection of noradrenaline synthesizing enzymes. In the adult, projections of locus coeruleus (LC) neurons to the spinal cord were first demonstrated by Dahlstr6m and Fuxe ~'~2. They were then confirmed by several studies t6'36'5°-53. Paralleily, neuroanatomical studies using a retrograde cell marker confirmed that numerous LC neurons projected to the spinal cord 4'21'29. The topography of NA spinal innervation was also detailed with histofluorescence by Mizukawa 34 and McLachlan and Oldfield 32. However, this latter technique did not permit unequivocal discrimination between fluorophores derived from NA and dopaminergic fibers6'45, and was not compatible with electron microscopy. Immunocytochemical detection of DBH, the final enzyme in the biosynthetic pathways of noradrenaline also permitted the detection of noradrenaline-containing neurons and of their spinal projections s'37 but DBH immunocytochemistry 2'16't7'5°'5 did not distinguish between dopaminergic, NA and adrenergic systems 24. This is of particular importance since adrenaline is present in the spinal cord. It has been shown unequivocally by immunocytochemical detection of phenylethanolamine-N-methyltransferase (PNMT) and its origin was traced to groups A1 and A2 of monoaminergic neurons, both in rats 5'25'33 and monkeys 7. Worth noting was the massive innervation of the IML, where preceding studies with histofluorescence or DBH immunocytochemistry could have mistaken it for noradrenaline 24. Similarly, during development, the histochemical studies of Olson and Seiger 3s, Seiger and Olson 43, Nygren
244 and Olson 36 and Commissiong 9't° could not discriminate between catecholamines. Moreover, these studies used less sensitive techniques than that ased here, since, for instance, Commissiong t° did not describe IML innervation before birth whereas we detected it as early as El9. In the present study, the use of an anti-noradrenaline antiserum was the method of choice, since cross-reactivity of this antiserum with other monoamines was excluded ts. The source of spinal cord NA fibers appeared essentially supraspinal. During development, NA innervation clearly followed a rostro-caudal gradient, and in addition, we did not detect intrinsic NA-IR perikarya, except at the upper cervical level, where they were continuous with medullary groups. The timing of spinal cord NA innervation is characteristic of monoaminergic systems. As for serotonin 4t, it is an early event in spinal cord ontogeny, as in other CNS areas 26. NA innervation was detected in the ventral horn before the corticospinal tract was established 4s. Similarly, in the dorsal horn NA innervation was present before the sensory system started functioning which was not present until the second postnatal week t4'~5, a period when NA innervation became prominent. Moreover, the sequence of NA spinal cord innervation which we detected, the ventral horn and IML preceding the dorsal horn, is in accordance with the sequence of maturation of motor, visceral and sensory systems 1~'39. When compared with serotonergic innervation, it appears slightly delayed, as 5-HT fibers were present in the cord as early as the 14th embryonic day 4~, This is true for all the specific targets, which were the same for the two amines, with a delay of 2-3 days. The initial pattern of innervation was the same for tile two amines, with an invasion of the grey matter by sharp angulation of fibers coursing in the ventral funiculus 4t' .,9. Similarly, the progressive segregation of fibers around motoneurons in the ventral horn, and the dual innervation of the dorsal horn were reminiscent of 5-HT innerration 4t.
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What is the functional implication of prenatal NA innervation? Is it, as suggested for 5-HT 3°'49, a neurotrophic factor or signal for target areas or, as has been hypothesized for monoamines, a 'differentiation regulator'22'23'27? In the ventral horn, for instance, it can be hypothesizdd that NA innereation could contribute to the stabilization of the motoneuron pool, which decreased after E15, and then remained stable from birth onwards 39. After birth, the basic innervation pattern was progressively completed and this corresponded to the period when the corticospinal aI:d the rubrospinal systems became functional 31'44'48. NA systems had potentially significant functions in somato-motor control and the regulation of autonomic and sensory functions: they may be involved in the modulation of complex motor activities 54, central generation of locomotion 19'2° and the modulation of spinal reflexes 1'36'47. Several studies have implicated the descending NA fibers in central autonomic control 3'2s'42. They were present and organized in the IML from E19 to adulthood at thoracic and sacral levels. These findings suggest that descending NA inputs play an important role in the regulation of sympathetic autonomic function, as well as of parasympathetic function. To summarize, the present study, the accuracy of which was made possible by the use of a specific antiserum, showed the early onset of NA innervation during spinal cord development. Such an early projection may correspond either to a neurotrophic influence on target regions, or to an early influence on spinal cord function. These two possibilities are not mutually exclusive. Further studies will be aimed at elucidating these functions, through early specific lesions and/or agonistic pharmacological manipulations.
Acknowledgemo~ts. The authors acknowledge J.-R. Teilhac for the art work. This work has been supported by D. Heumann Fund, IRME and AFM.
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