Developmental Brain Research, 68 (1992) 103-109
103
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00 BRESD 51482
Expression of lectin binding in the superficial dorsal horn of the rat spinal cord during pre- and postnatal development M a r k B. P l e n d e r l e i t h b, L a y n e L. W r i g h t a a n d P e t e r J. S n o w
a
a Cerebraland Sensory Functions Unit, Department of Anatomy, Universityof Queensland, Brisbane, QId. (Australia) and b School of Life Science, Queensland University of Technology, Brisbane, Qld. (Australia) (Accepted 7 April 1992)
Key words: Development; Pain; Lectin; C-fiber; Dorsal horn; Spinal cord
The plant lectin Bandeiraea simplicifolia I-B4 binds to the soma and central terminals of a subpopulation of unmyelinated primary sensory neurones in the adult rat. The binding site of this lectin is thought to be the terminal a-v-galactose residue of a membrane associated glycoconjugate which may be involved ia the development of specific connections between small diameter primary sensory neurones and second order neurones in the superficial dorsal horn of the spinid cord. To begin to investigate this possibility we have examined the development of lectin binding in the dorsal horn of pre- and postnatal rats. Lectin binding first appeared on axon profiles in the superficial dorsal horn of the spinal cord at embryonic days 18/19. Previous studies in the rat have revealed that the central processes of small diameter primary sensory neurones enter the dorsal horn at embryonic days 18/19. Our findings suggest that the glycoconjugate to which this lectin binds, is expressed by the central processes of small diameter primary sensory neurones as they grow into the spinal cord. It is therefore possible that this glycoconjugate is involved in the development of topographically ordered neural connections within the dorsal horn of the spinal cord.
INTRODUCTION It is now well established that in the mammalian spinal cord, neurones within particular laminae of the dorsal horn respond to different peripheral stimuli (e.g. muscle stretch, hair movement, changes in skin temperature, etc.) 4. In addition, many dorsal horn neurones have restricted, well defined receptive fields which are somatotopically organised. These features are a direct consequence of the precise projections of modality and somatotopically appropriate primary sensory neurones onto dorsal horn neurones 26. The temporal sequencing of axon growth into the spinal cord during development may contribute to the general organisation of primary afferent projections into the dorsal horn s'25'33. However, more subtle mechanisms are likely to be involved in guiding developing axons to their appropriate target neurones. It has been postulated that this process is mediated by complementary receptor molecules present on growing axons and their intended target neurones 27. Possible candidates for
such receptors include neuronal membrane glycoconjugates. These molecules have been widely implicated in intercellular recognition or guidance within the developing nervous system ~°'!2a3. Plant lectins are characterised by their differing affinities for the sugar residues of complex carbohydrates ~s. Plant lectins have therefore be used to distinguish between glycoconjugates on the basis of their differing carbohydrate moieties. Of particular interest are the lectins Soybean agglutinin and Bandeiraea simplicifolia I isolectin B 4 (BSI-B4) both of which exhibit a n affinity for terminal a-D-galactose carbohydrate residues 6al. Recently it has been shown that these lectins bind to a subpopulation of unmyelinated primary sensory neurones in the rat and cat 19-21,24'29'30'32. This population is thought to include those neurones that respond to peripheral tissue damage and are referred to as nociceptors ~9-2L24'3°. Recently we have found that BSI-B4 binds almost exclusively to those neurones which innervate the skin (M.B. Plenderleith and P.J. Snow, in preparation). These
Correspondence: M.B. Plenderleith, School of Life Science, Queensland University of Technology, GPO Box 2434, Brisbane, Qld. 4001, Australia. Fax: (61) (7) 864-1510.
104 results suggest that a galactose containing glycoconjugate may be expressed selectively by a functional subpopulation of small diameter primary sensory neurones. The possibility exists that this glycoconjugate is involved in the formation of connections between this population of small diameter primary sensory neurones and their target neurones in the superficial dorsal horn of the spinal cord during development. If this were the case one would expect the BSI-B 4 binding sites to be present upon the central processes of primary sensory neurones as they grow into the spinal cord. Thus the aim of this study was to determine at what stage in development BSI-B4 binding appears in the dorsal horn of the spinal cord of the rat.
MATERIALS AND METHODS Experimental animals in the course of this study, the spinal cords from 72 embryonic and 20 postnatal rats were screened for lectin binding. These were obtained from a total of 24 pregnant Wistar strain rats and 4 litters respecti~,eIy. RAtswere assumed to h..~veconceived at midnight of the day preceding the detection of a vaginal spermatic plug. The day on which the plug was detected was designated embryonic day (E) 0 and the first 24 h post partum designated postnatal day (P) 0. Tissue preparation At developmental stages between El5 and E21 foetuses were removed from the uterus of the pregnant rats under deep sodium pentobarbitone anaesthesia (Nembutal 50 mg/kg i.p.) and perfused transcardially. The perfusion consisted of 10-20 ml of saline (containing 5 l.U./ml Heparin) followed by the same volume of each of 4% paraformaldehyde (in acetate buffer at pH 6.5) and then 4% paraformaldehyde and 0.05% glutaraldehyde (in borate buffer; pH 9.5), The spinal cord was then exposed by laminectomy and the whole embryos were post-fixed for 4 h in the second fixative prior to washing in 30% sucrose in 0.1 M phosphate buffer (PB; pH 7.4) for several days at 4°(2. Postnatal rats were anaesthetised with 4% Halothane in air and perfused using the same fixation protocol. Transverse sections (10-20 pm thick) of mid-cervical spinal cord were then cut on a cryostat, mounted on subbed slides and stored at 4°(2. Histochemistry Sections were incubated overnight (at room temperature) in 6.25 / z g / m l o f a BSI-B4-horseradish peroxidase (BSI-B4-HRP) coltjugate (Sigma Chemical Company) in 0.1 M phosphate buffered saline (pH 7.4) containing 1% bovine serum albumin and 0.3% Triton X. Unbound conjugate was removed by three 5-min washes in PB and the sections were then incubated in a solution consisting of 0.5 mg/ml diaminobenzidine and 0.015% hydrogen peroxide in PB. Sections were subsequently washed, dehy,ira~.,~d, cleared and coverslipped, in some sections endogenous peroxidase was quenched by a 20-min preincubation in 1% hydrogen peroxide in methanol prior to the lectin incubation. Three histochemical controls were used to assess the specificit) of the histochemistry. Firstly, the BSI-B4-HRP conjugate was omitted from the incubation. Secondly sections were incubated in conjugate that had been preabsorbed with between 100 mM and 400 mM N-acetylgalactosamine or D-galactose for 4 h. Finally a section of adult rat spinal cord (in which BSI-B4 binding has been well characteris~;d) was included in every incubation to ensure that any negative result was not due to failure of the histochemistry.
RESULTS In the spinal cord of embryonic, postnatal and adult rats, lectin binding was identified by the d a r k brown reaction product produced by the oxidation of diaminobenzidine in the presence of hydrogen peroxide. Omission of the B S I - B 4 - H R P conjugate from the incubation, or incubation with conjugate that had been preabsorbed with 200-400 m M N-acetylgalactosamine or 100-400 m M D-galactose resulted in no reaction product. Lectin binding to non-neuronal elements At all stages between E l 5 and P l 0 reaction product was found associated with the endothelial surface of blood vessels and with glial cells throughout the grey and white matter of the spinal cord (Figs. 1, 2 and 3). The quenching of endogenous peroxidase prior to the lectin incubation did not affect the reaction product associated with either the blood vessels or glial cells suggesting that this was indeed due to lectin binding sites being present upon these elements. In the course of this study it was our intention to also determine the developmental stage at which lectin binding appeared on neurones within the dorsal root ganglia. However, the small diameter of embryonic dorsal root ganglion
TABLE ! Developmental expression of lectin binding to neuronal elements in the dorsal horn of embryonic (£) and postnatal (P) rats The presence or absence of lectin binding in the superficial dorsal horn o f o n e rat is indicated by the symbols ' + ' and ' - '
respectively.
During the prenatal period (upper panel), 3 embryos were screened from each of 3 or 4 mothers at each embryonic day (total of 24 mothers). For example at El8 a total of 9 embryos were screened from 3 different mothers. Three of the 9 embryos exhibited ]ectin binding in the superficial dorsal horn. During the postnatal period (lower panel) one or two pups from up to 4 litters were sc~'cened.
Eday
Pup
Pup
Pup
Pup
123
123
123
123
.
- - . . . . . . . . . . .
El5 El6 El7 El8 El9 E20 E21
- + +
- - +
+++ +++ +++
+++ +++ +++
+++ +++ +++
+++ +++
P day
Litter I
Litter 2
Litter 3
Litter 4
P0 Pl P2 P3 P6 Pl0
+ ++ + + +
+ + + + + ++
+ + + +
+ +
.
.
.
+
105 cells together with the confusion caused by lectin binding to the blood vessels made this difficult and was subsequently abandoned. Lectin binding to neuronal elements Between El5 and El7 the spinal cords of 30 embryonic rats (from a total of 10 mothers) were screened for BSI-B4 binding (Table I). We found no trace of lectin binding in the dorsal horn of these rats except for that associated with blood vessels and glial cells referred to above. The absence of lectin binding in these rats cannot be attributed to failure of the histochemical
procedure because the adult control sections were positive. Lectin binding to presumed axonal elements in the dorsal horn was first detected in the spinal cords of 3 of the 9 rats screened at El8 (Table I). In these 3 rats a bundle of moderately intense labelled fibres were observed coursing dorso-ventrally through the dorsolateral funiculus. In addition, in each of these 3 El8 rats a very low level of lectin binding was apparent throughout the superficial dorsal horn (laminae I and II). This pattern of labelling was found in the spinal cords of all 9 rats tested at E19 (Table I) but in general the
Fig. 1. A: low power micrograph showing the pattern of BSI-B 4 binding in a transverse section of spinal cord from an embryonic day 17 rat. Lectin binding is associated with the lumen of blood vessels (open arrows) and glial cells (closed arrows). However, terminal labelling in the superficial dorsal horn is absent (compare with Figs. 2 and 3). Bar - 200 ~m. B,C: higher magnification micrograph of the dorsal horn of the same section shown in A to confirm the absence of terminal labelling in the superficial laminae at this developmental stage (compare with Figs. 2 and 3). However, both glial cells (closed arrows) and blood vessels (open arrows) exhibit labelling. Bar -- 100 gm.
106 labelling within the superficial dorsal horn was more intense (Fig. 2). Under a x 100 objective and using oil immersion, the labelling in the superficial dorsal horn at E l 9 appeared to consist of a loose plexus of very fine fibres. At this stage terminal-like varicosities were rare. Between E20 and P6 there was a progressive increase b~,~h in the intensity of lectin binding within tl": superficial dorsal horn and in the density of terminal-like varicosities (Fig. 3). By P6 the intensity of labelling observed in the superficial dorsal horn was approaching that observed in the adult. At no stage were neu-
rones within the dorsal horn found to exhibit BSI-B 4 binding. DISCUSSION In the course of this study we found that the plant lectin BSI-B 4 binds to the endothelial surface of blood vessels and to glial cells in the spinal cord of rats between El5 and PI0. This pattern of lectin binding persists, but is less prominent, in the adult spinal cord. Plant lectins 'specific' for terminal a-D-galactose residues have been previously shown to bind to the
Fig. 2. A: low power micrograph of a transverse section of spinal cord from an embryonic day 19 rat. Leetin binding is found associated with blood vessels (open arrows), glial cells (closed arrows) and with a loose plexus of fine axons capping the dorsal horn. Bar = 200 p.m. B,C: higher powered micrograph of the same section shown in A to illustrate BSI-B 4 binding associated with fine axonal profiles restricted to the superficial laminae of the dorsal horn. Glial cells (open arrow) and blood vessels (closed arrow) are also labelled. Neurones within the dorsal horn are not labelled. Bar = 100 p.m.
107 lumen of blood vessels 17'a4 and to glial cells 28'29'31 in the central nervous system of rodents. These findings suggest galactose containing glycoconjugates are associated with vascular endothelium and glial cells in the rodent. The function of these glial and vascular glycoconjugates is not known. As they are present as early as El5 it is possible that these molecules play a role in the vascularisation of the central nervous system and in glial cell function during development. The major finding of this study is that binding of the plant lectin BSI-B 4 in the superficial dorsal horn of the spinal cord first appears at E18/19. The significance of this result must be considered in light of what is
already known about the development of the projections of primary sensory neurones into the spinal cord of the rat. Primary sensory neurones are born between E l l and E14 TM and by E13 axons can be observed growing into the limb buds and towards the spinal cord. The time at which the axon collaterals of primary sensory neurones enter the grey matter of the spinal cord and form terminal arborizations within particular laminae appears to depend upon the modalitT of the primary sensory neurone. Thus the collaterals of large diameter axons (thought to be muscle afferents) penetrate the grey matter of the spinal cord at E15.5 and subsequently form terminals within the motoneurone
Fig. 3. A: micrograph of a transverse section of spinal cord from a postnatal day 6 rat. Lectin binding associated with blood vessels and gliai cells is less prominent whilst binding associated with the superficial dorsal horn has reached adult-like intensities. Bar -- 200 gin. B,C: higher powered micrographs of the lateral edge of the superficial dorsal horn from a ~ection immediately serial to that shown in A. Note that BSI-B4 binding is associated with terminal-like varicosities while dorsal horn neurones are not labelled. Bar = 100 gm.
108 pools of the ventral horn 25'33. These are followed a day later by the collaterals of other large diameter axons which terminate in the deeper laminae of the dorsal horn and are therefore thought to be low threshold cutaneous afferents 25. Small diameter primary sensory neurones are the last afferents to enter the grey matter of the spinal cord. Until recently it was thought that they did not enter the dorsal horn until 24-48 h post partum 25. However, Regan et al. 22 have identified a carbohydrate binding protein (RL-29) which is expressed selectively by small diameter primary sensory neurones and their central processes in the superficial dorsal horn of the spinal cord. This protein is expressed by dorsal root ganglion cells at El6 and on their central processes in the superficial dorsal horn at El8. Clearly this implies that small diameter primary sensory neurones project into the dorsal horn prenatally. Direct evidence in support of this has come from the work of Fitzgerald 9 who used the transganglionic transport of wheatgerm agglutinin-horseradish peroxidase conjugate to label the central processes of small diameter primary sensory neurones during embryonic development. Labelling in the superficial dorsal horn was first observed at E19 9. Further evidence in support of the prenatal projection of small diameter afferents into the spinal cord has been provided by PignateUi et al. ~s. These workers have identified what appear to be the morphological precursors ef type CI synaptic glomeruli (known to be C-fibre terminals in the adult) in the dorsal horn of tats at P0. Collectively these studies suggest that the central processes of unmyelinated primary sensory neurones begin to grow into the dorsal horn of the spinal cord at E18/19, In this study we have shown that BSI-B4 binding in the superficial dorsal horn appears at exactly the same time. This suggests that the BSI-B 4 binding site is expressed by the central processes of small diameter primary sensory neurones as they grow into the dorsal horn. Recently Scott et al. 23 have shown that in the chick, the plant lectin Dolichos biflorus agglutinin binds to a subpopulation of small diameter primary sensory neurones and their central processes in the dorsal horn. Binding of this lectin was first detected in the dorsal root ganglion at El2 (stage 38) and on processes in the dorsal horn at El6 (stage 42). However in the chick Dolichos biflorus agglutinin binding sites are expressed several days after the central terminals of small diameter primary sensory neurones have reached the superficial dorsal hoa~ ~'7.t~'. Consequently it is unlikely that this lectin binding site plays a critical role in the formation of specific neural connections during the developmental process in this species.
In the rat the precise role of this lectin binding site in developmental process requires further clarification. The observation that plant lectins with an affinity for terminal a-D-galactose residues bind to the plasma membrane and Golgi apparatus of primary sensory neurones has led to the proposal that the ligand is a membrane associated glycoconjugate 2°'29'3°. Membrane associated glycoconjugates have been widely implicated in intercellular recognition and adhesion during development aa°,:2,~3. Evidence in support of a galactose containing glycoconjugate being involved in the formation of orderly connections between primary sensory neurones and neurones in the dorsal horn of the spinal cord has come from in vitro studies. Baker et al. 2'3 have shown that in spinal cord-dorsal root ganglion explants from fetal mice, the addition of galactose to the culture medium potentiates the formation of monosynaptic connections between primary sensory neurones and neurones in the dorsal horn. In this study we have shown that the same carbohydrate moiety is expressed by the central processes of unmye!inated primary sensory neurones as they grow into the spinal cord. This finding is consistent with these binding sites being involved in the formation (or maintenance) of specific connections between unmyelinated primary sensory neurones and neurones in the superficial dorsal horn of the spinal cord during development. This proposal must, however, remain tentative until blockage of this binding site can be shown to affect the pattern of primary sensory neurone projections. Acknowledgements. We would like to thank Drs. Peter Wilson and Bruce Oynther for their helpful comments on earlier versions of the manuscript. This work was supported N.H. & M.R.C.
by the
Australian
REFERENCES 1 Altman, J. and Bayer, S.A., The development of the rat spinal cord. In Advances in Anatomy, Embryology and Cell Biology, VoL 85, Springer, Berlin, 1984. 2 Baker, R., The effects of gangliosides on the development of selective afferent connections within fetal mouse spinal cord explants, Neurosci. Lett., 41 (1983)81-84. 3 Baker, R., Corner, M.L. and Kleiss, M., Effects of chemical additives on functional innervation patterns in mouse spinal cord-ganglion explants in serum-free medium, NeuroscL Lett., 41 (1983) 321-324. 4 Brown, A.G., Organization of the Spinal Cord, Springer, Berlin, 1981, 5 Davis, B.M., Frank, F., Johnson, F.A. and Scott, S.A., Development of central projections of lumbosacral sensory neurons in the chick, J. Comp. Neurol., 279 (1989)556-566. 6 Debray, H., Decout, D., Strecker, G., Spik, G. and Montreuil, J., Specificity of twelve lectins towards oligosaccharides and glycopeptides related to N-glycosyiproteins, Fur. J. Biochem., 117 (1981) 41-55. 7 Du, F., Charnay, Y. and Dubois, P., Development and distribution of substance P in the spinal cord and ganglia of embryonic and newly hatched chicks: An immunofluorescence study, J. Comp. NeuroL, 263 (1987) 436-454.
109 8 Edelman, G.M., Modulation of cell adhesion during induction histogenesis and perinatal development of the nervous system, Annu. Rev. Neurosci., 7 (1984)339-377. 9 Fitzgerald, M., Prenatal growth of fine diameter primary afferents into the rat spinal cord: a transganglionic tracer study, J. Comp. Neural., 261 (1987) 98-104. 10 Gurd, J.W., Giycoproteins of the synapse. In R.U. Margolis and R.K. Margolis (Eds.), Neurobiology of Glycoconjugates, Plenum, New York, 1989, pp. 219-242. 11 Hayes, C.E. and Goldstein, I.J., An a-galactosyl-binding lectin from Bandeiraea simplicifolia seeds, J. Biol. Chem., 249 (1974) 1904-1914. 12 Hynes, M.A., Dodd, J.A. and Jesseli, T.M., Carbohydrate recognition, cell interactions and vertebrate neural development. In R.U. Margolis and R.K. Margolis (Eds.), Neurobiology of Glycoconjugates, Plenum, New York, 1989, pp. 337-365. 13 Kelly, P.T., Nervous system glycoproteins. In R.J. Ivatt (Ed.), The Biology of Glycoproteins, Plenum, New York, 1984, pp. 323-369. 14 Lawson, S.N., Caddy, K.W.T. and Biscoe, TJ., Development of dorsal root ganglion neurones, Cell Tissue Res., 153 (1974) 399413. 15 Lis, H. and Sharon, N., Lectins as molecules and as tools, Annu. Rev. Biochem., 55 (1986) 35-67. 16 New, H.V. and Mudge, A.W., Distribution and ontogeny of SP, CGRP, SOM and VIP in chick sensory and sympathetic ganglia, Dev. Biol., 116 (1986) 337-346. 17 Peters, B.P. and Goldstein, IJ., The use of fluorescein-conjugated Bandeiraea simpliafolia B4-isolectin as a histochemical reagent for the detection of a-D-galactopyranosyl groups, Exp. Cell Res., 120 (1979) 321-334. 18 Pignatelli, D., Ribeiro-da-Silw, A. and Coimbra, A., Postnatal maturation of primary afferent terminations in the substantia gelatinosa of the rat spinal cord. An electron microscopic study, Brain Res., 491 (1989) 33-44. 19 Plenderleith, M.B., Cameron, A.A., Key, B. and Snow, PJ., Soybean agglutinin binds to a subpopulation of primary sensory neurones in the cat, Neurosci. Lett., 86 (1988) 257-262. 20 Plenderleith, M.B., Cameron, A.A., Key, B. and Snow, P.J., The plant lectin soybean agglutinin binds to the soma, axon and central terminals of a subpopulation of small diameter primary sensory neurones in the rat and cat, Neuroscience, 31 (1989) 683-695, 21 Plenderleith, M.B. and Snow, PJ., The effect of peripheral nerve section on lectin binding in the superficial dorsal horn of the rat spinal cord, Brabi Res., 507 (1990) 146-150. 22 Regan, LJ., Dodd, J., Barondes, S.H. and Jesseil, T.M., Selective expression of endogenous lactose-binding lectins and lactoseries
glycoconjugates in subsets of rat sensory neuronL=s, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 2248-2252. 23 Scott, S.A., Patel, N. and Levine, J.M., Lectin binding identifies a subpopulation of neurons in chick dorsal root ganglia, J. Neurosci., 10 (1990) 336-345. 24 Silverman, J.D. and Kruger, L., Lectin ~,d neuropeptide labelling of separate populations of dorsal root ganglion neurones and associated 'nociceptor' thin axons in rat testis and cornea whole-mount preparations, Somatosens. Res., 5 (1988) 259-267. 25 3mith, C.L., The development and postnatal organization of primary afferent projections to the rat thoracic spinal cord, J. Comp. Neurol., 220 (1983) 29-43. 26 Snow, P.J. and Wilson, P., Plasticity in the somatosensory system of developing and mature mammals: the effects of injury to the central and peripheral nervous system. In H. Autrum, D. Ottoson, E.R. Perl, R.F. Schmidt, H. Shimazu and W.D. Willis (Eds.), Progress in Sensory Physiology, Vol. 11, Springer, Berlin, 1991, pp. 1-482. 27 Sperry, R.W., Chemoaffinity in the orderly growth of nerve fiber patterns and connections, Proc. Natl. Acad. Sci. U.S.A., 50 (1963) 703-710. 28 Streit, W.J. and Kreutzberg, G.W., Lectin binding by resting and reactive microglia, J. NeurocytoL, 16 (1987) 249-260. 29 Streit, W.J., Schulte, B.A., Balentine, J.D. and Spicer, S.S., Histochemical localization of galactose-containing glycoconjugates in sensory neurons and their processes in the central and peripheral nervous system of the rat, J. Histochem. Cytochem., 33 (1985) 1042-1052. 30 Streit, W.J., Schulte, B.A., Balentine, J.D. and Spicer, S.S., Evidence for glycoconjugate in nociceptive primary sensory neurons and its origin from the Golgi complex, Brain Res., 377 (1986) 1-17. 31 Suzuki, H., Franz, H., Yamamoto, T., lwasaki, Y. and Konno, H., Identification of the normal microglial population in human and rodent nervous tissue using lectin-histochemistry, Neuropathol. Appl. Neurobiol., 14 (1988) 221-227. 32 Tajti, J., Fischer, J., Knyihar-Csillik, E. and Csillik, B., Transgan#ionic regulation and fine structural localization of iectin-reactire carbohydrate epitopes in primary sensory neurons of the rat, Histochemistry, 88 (1988) 213-218. 33 Vaughn, J.E. and Grieshaber, J.A., A morphological investigation of an early reflex pathway in developing rat spinal cord, J. Comp. Neuroi., 148 (1973) 177-210. 34 Welim, H.B., Thies, M. and Herken, R., Appearance of lectinbinding sites during vascularization of the primordium of the central nervous system in 10- to 12-day-old mouse embryos, Cell Tissue Res., 255 (1989) 627-630.
103
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00 BRESD 51482
Expression of lectin binding in the superficial dorsal horn of the rat spinal cord during pre- and postnatal development M a r k B. P l e n d e r l e i t h b, L a y n e L. W r i g h t a a n d P e t e r J. S n o w
a
a Cerebraland Sensory Functions Unit, Department of Anatomy, Universityof Queensland, Brisbane, QId. (Australia) and b School of Life Science, Queensland University of Technology, Brisbane, Qld. (Australia) (Accepted 7 April 1992)
Key words: Development; Pain; Lectin; C-fiber; Dorsal horn; Spinal cord
The plant lectin Bandeiraea simplicifolia I-B4 binds to the soma and central terminals of a subpopulation of unmyelinated primary sensory neurones in the adult rat. The binding site of this lectin is thought to be the terminal a-v-galactose residue of a membrane associated glycoconjugate which may be involved ia the development of specific connections between small diameter primary sensory neurones and second order neurones in the superficial dorsal horn of the spinid cord. To begin to investigate this possibility we have examined the development of lectin binding in the dorsal horn of pre- and postnatal rats. Lectin binding first appeared on axon profiles in the superficial dorsal horn of the spinal cord at embryonic days 18/19. Previous studies in the rat have revealed that the central processes of small diameter primary sensory neurones enter the dorsal horn at embryonic days 18/19. Our findings suggest that the glycoconjugate to which this lectin binds, is expressed by the central processes of small diameter primary sensory neurones as they grow into the spinal cord. It is therefore possible that this glycoconjugate is involved in the development of topographically ordered neural connections within the dorsal horn of the spinal cord.
INTRODUCTION It is now well established that in the mammalian spinal cord, neurones within particular laminae of the dorsal horn respond to different peripheral stimuli (e.g. muscle stretch, hair movement, changes in skin temperature, etc.) 4. In addition, many dorsal horn neurones have restricted, well defined receptive fields which are somatotopically organised. These features are a direct consequence of the precise projections of modality and somatotopically appropriate primary sensory neurones onto dorsal horn neurones 26. The temporal sequencing of axon growth into the spinal cord during development may contribute to the general organisation of primary afferent projections into the dorsal horn s'25'33. However, more subtle mechanisms are likely to be involved in guiding developing axons to their appropriate target neurones. It has been postulated that this process is mediated by complementary receptor molecules present on growing axons and their intended target neurones 27. Possible candidates for
such receptors include neuronal membrane glycoconjugates. These molecules have been widely implicated in intercellular recognition or guidance within the developing nervous system ~°'!2a3. Plant lectins are characterised by their differing affinities for the sugar residues of complex carbohydrates ~s. Plant lectins have therefore be used to distinguish between glycoconjugates on the basis of their differing carbohydrate moieties. Of particular interest are the lectins Soybean agglutinin and Bandeiraea simplicifolia I isolectin B 4 (BSI-B4) both of which exhibit a n affinity for terminal a-D-galactose carbohydrate residues 6al. Recently it has been shown that these lectins bind to a subpopulation of unmyelinated primary sensory neurones in the rat and cat 19-21,24'29'30'32. This population is thought to include those neurones that respond to peripheral tissue damage and are referred to as nociceptors ~9-2L24'3°. Recently we have found that BSI-B4 binds almost exclusively to those neurones which innervate the skin (M.B. Plenderleith and P.J. Snow, in preparation). These
Correspondence: M.B. Plenderleith, School of Life Science, Queensland University of Technology, GPO Box 2434, Brisbane, Qld. 4001, Australia. Fax: (61) (7) 864-1510.
104 results suggest that a galactose containing glycoconjugate may be expressed selectively by a functional subpopulation of small diameter primary sensory neurones. The possibility exists that this glycoconjugate is involved in the formation of connections between this population of small diameter primary sensory neurones and their target neurones in the superficial dorsal horn of the spinal cord during development. If this were the case one would expect the BSI-B 4 binding sites to be present upon the central processes of primary sensory neurones as they grow into the spinal cord. Thus the aim of this study was to determine at what stage in development BSI-B4 binding appears in the dorsal horn of the spinal cord of the rat.
MATERIALS AND METHODS Experimental animals in the course of this study, the spinal cords from 72 embryonic and 20 postnatal rats were screened for lectin binding. These were obtained from a total of 24 pregnant Wistar strain rats and 4 litters respecti~,eIy. RAtswere assumed to h..~veconceived at midnight of the day preceding the detection of a vaginal spermatic plug. The day on which the plug was detected was designated embryonic day (E) 0 and the first 24 h post partum designated postnatal day (P) 0. Tissue preparation At developmental stages between El5 and E21 foetuses were removed from the uterus of the pregnant rats under deep sodium pentobarbitone anaesthesia (Nembutal 50 mg/kg i.p.) and perfused transcardially. The perfusion consisted of 10-20 ml of saline (containing 5 l.U./ml Heparin) followed by the same volume of each of 4% paraformaldehyde (in acetate buffer at pH 6.5) and then 4% paraformaldehyde and 0.05% glutaraldehyde (in borate buffer; pH 9.5), The spinal cord was then exposed by laminectomy and the whole embryos were post-fixed for 4 h in the second fixative prior to washing in 30% sucrose in 0.1 M phosphate buffer (PB; pH 7.4) for several days at 4°(2. Postnatal rats were anaesthetised with 4% Halothane in air and perfused using the same fixation protocol. Transverse sections (10-20 pm thick) of mid-cervical spinal cord were then cut on a cryostat, mounted on subbed slides and stored at 4°(2. Histochemistry Sections were incubated overnight (at room temperature) in 6.25 / z g / m l o f a BSI-B4-horseradish peroxidase (BSI-B4-HRP) coltjugate (Sigma Chemical Company) in 0.1 M phosphate buffered saline (pH 7.4) containing 1% bovine serum albumin and 0.3% Triton X. Unbound conjugate was removed by three 5-min washes in PB and the sections were then incubated in a solution consisting of 0.5 mg/ml diaminobenzidine and 0.015% hydrogen peroxide in PB. Sections were subsequently washed, dehy,ira~.,~d, cleared and coverslipped, in some sections endogenous peroxidase was quenched by a 20-min preincubation in 1% hydrogen peroxide in methanol prior to the lectin incubation. Three histochemical controls were used to assess the specificit) of the histochemistry. Firstly, the BSI-B4-HRP conjugate was omitted from the incubation. Secondly sections were incubated in conjugate that had been preabsorbed with between 100 mM and 400 mM N-acetylgalactosamine or D-galactose for 4 h. Finally a section of adult rat spinal cord (in which BSI-B4 binding has been well characteris~;d) was included in every incubation to ensure that any negative result was not due to failure of the histochemistry.
RESULTS In the spinal cord of embryonic, postnatal and adult rats, lectin binding was identified by the d a r k brown reaction product produced by the oxidation of diaminobenzidine in the presence of hydrogen peroxide. Omission of the B S I - B 4 - H R P conjugate from the incubation, or incubation with conjugate that had been preabsorbed with 200-400 m M N-acetylgalactosamine or 100-400 m M D-galactose resulted in no reaction product. Lectin binding to non-neuronal elements At all stages between E l 5 and P l 0 reaction product was found associated with the endothelial surface of blood vessels and with glial cells throughout the grey and white matter of the spinal cord (Figs. 1, 2 and 3). The quenching of endogenous peroxidase prior to the lectin incubation did not affect the reaction product associated with either the blood vessels or glial cells suggesting that this was indeed due to lectin binding sites being present upon these elements. In the course of this study it was our intention to also determine the developmental stage at which lectin binding appeared on neurones within the dorsal root ganglia. However, the small diameter of embryonic dorsal root ganglion
TABLE ! Developmental expression of lectin binding to neuronal elements in the dorsal horn of embryonic (£) and postnatal (P) rats The presence or absence of lectin binding in the superficial dorsal horn o f o n e rat is indicated by the symbols ' + ' and ' - '
respectively.
During the prenatal period (upper panel), 3 embryos were screened from each of 3 or 4 mothers at each embryonic day (total of 24 mothers). For example at El8 a total of 9 embryos were screened from 3 different mothers. Three of the 9 embryos exhibited ]ectin binding in the superficial dorsal horn. During the postnatal period (lower panel) one or two pups from up to 4 litters were sc~'cened.
Eday
Pup
Pup
Pup
Pup
123
123
123
123
.
- - . . . . . . . . . . .
El5 El6 El7 El8 El9 E20 E21
- + +
- - +
+++ +++ +++
+++ +++ +++
+++ +++ +++
+++ +++
P day
Litter I
Litter 2
Litter 3
Litter 4
P0 Pl P2 P3 P6 Pl0
+ ++ + + +
+ + + + + ++
+ + + +
+ +
.
.
.
+
105 cells together with the confusion caused by lectin binding to the blood vessels made this difficult and was subsequently abandoned. Lectin binding to neuronal elements Between El5 and El7 the spinal cords of 30 embryonic rats (from a total of 10 mothers) were screened for BSI-B4 binding (Table I). We found no trace of lectin binding in the dorsal horn of these rats except for that associated with blood vessels and glial cells referred to above. The absence of lectin binding in these rats cannot be attributed to failure of the histochemical
procedure because the adult control sections were positive. Lectin binding to presumed axonal elements in the dorsal horn was first detected in the spinal cords of 3 of the 9 rats screened at El8 (Table I). In these 3 rats a bundle of moderately intense labelled fibres were observed coursing dorso-ventrally through the dorsolateral funiculus. In addition, in each of these 3 El8 rats a very low level of lectin binding was apparent throughout the superficial dorsal horn (laminae I and II). This pattern of labelling was found in the spinal cords of all 9 rats tested at E19 (Table I) but in general the
Fig. 1. A: low power micrograph showing the pattern of BSI-B 4 binding in a transverse section of spinal cord from an embryonic day 17 rat. Lectin binding is associated with the lumen of blood vessels (open arrows) and glial cells (closed arrows). However, terminal labelling in the superficial dorsal horn is absent (compare with Figs. 2 and 3). Bar - 200 ~m. B,C: higher magnification micrograph of the dorsal horn of the same section shown in A to confirm the absence of terminal labelling in the superficial laminae at this developmental stage (compare with Figs. 2 and 3). However, both glial cells (closed arrows) and blood vessels (open arrows) exhibit labelling. Bar -- 100 gm.
106 labelling within the superficial dorsal horn was more intense (Fig. 2). Under a x 100 objective and using oil immersion, the labelling in the superficial dorsal horn at E l 9 appeared to consist of a loose plexus of very fine fibres. At this stage terminal-like varicosities were rare. Between E20 and P6 there was a progressive increase b~,~h in the intensity of lectin binding within tl": superficial dorsal horn and in the density of terminal-like varicosities (Fig. 3). By P6 the intensity of labelling observed in the superficial dorsal horn was approaching that observed in the adult. At no stage were neu-
rones within the dorsal horn found to exhibit BSI-B 4 binding. DISCUSSION In the course of this study we found that the plant lectin BSI-B 4 binds to the endothelial surface of blood vessels and to glial cells in the spinal cord of rats between El5 and PI0. This pattern of lectin binding persists, but is less prominent, in the adult spinal cord. Plant lectins 'specific' for terminal a-D-galactose residues have been previously shown to bind to the
Fig. 2. A: low power micrograph of a transverse section of spinal cord from an embryonic day 19 rat. Leetin binding is found associated with blood vessels (open arrows), glial cells (closed arrows) and with a loose plexus of fine axons capping the dorsal horn. Bar = 200 p.m. B,C: higher powered micrograph of the same section shown in A to illustrate BSI-B 4 binding associated with fine axonal profiles restricted to the superficial laminae of the dorsal horn. Glial cells (open arrow) and blood vessels (closed arrow) are also labelled. Neurones within the dorsal horn are not labelled. Bar = 100 p.m.
107 lumen of blood vessels 17'a4 and to glial cells 28'29'31 in the central nervous system of rodents. These findings suggest galactose containing glycoconjugates are associated with vascular endothelium and glial cells in the rodent. The function of these glial and vascular glycoconjugates is not known. As they are present as early as El5 it is possible that these molecules play a role in the vascularisation of the central nervous system and in glial cell function during development. The major finding of this study is that binding of the plant lectin BSI-B 4 in the superficial dorsal horn of the spinal cord first appears at E18/19. The significance of this result must be considered in light of what is
already known about the development of the projections of primary sensory neurones into the spinal cord of the rat. Primary sensory neurones are born between E l l and E14 TM and by E13 axons can be observed growing into the limb buds and towards the spinal cord. The time at which the axon collaterals of primary sensory neurones enter the grey matter of the spinal cord and form terminal arborizations within particular laminae appears to depend upon the modalitT of the primary sensory neurone. Thus the collaterals of large diameter axons (thought to be muscle afferents) penetrate the grey matter of the spinal cord at E15.5 and subsequently form terminals within the motoneurone
Fig. 3. A: micrograph of a transverse section of spinal cord from a postnatal day 6 rat. Lectin binding associated with blood vessels and gliai cells is less prominent whilst binding associated with the superficial dorsal horn has reached adult-like intensities. Bar -- 200 gin. B,C: higher powered micrographs of the lateral edge of the superficial dorsal horn from a ~ection immediately serial to that shown in A. Note that BSI-B4 binding is associated with terminal-like varicosities while dorsal horn neurones are not labelled. Bar = 100 gm.
108 pools of the ventral horn 25'33. These are followed a day later by the collaterals of other large diameter axons which terminate in the deeper laminae of the dorsal horn and are therefore thought to be low threshold cutaneous afferents 25. Small diameter primary sensory neurones are the last afferents to enter the grey matter of the spinal cord. Until recently it was thought that they did not enter the dorsal horn until 24-48 h post partum 25. However, Regan et al. 22 have identified a carbohydrate binding protein (RL-29) which is expressed selectively by small diameter primary sensory neurones and their central processes in the superficial dorsal horn of the spinal cord. This protein is expressed by dorsal root ganglion cells at El6 and on their central processes in the superficial dorsal horn at El8. Clearly this implies that small diameter primary sensory neurones project into the dorsal horn prenatally. Direct evidence in support of this has come from the work of Fitzgerald 9 who used the transganglionic transport of wheatgerm agglutinin-horseradish peroxidase conjugate to label the central processes of small diameter primary sensory neurones during embryonic development. Labelling in the superficial dorsal horn was first observed at E19 9. Further evidence in support of the prenatal projection of small diameter afferents into the spinal cord has been provided by PignateUi et al. ~s. These workers have identified what appear to be the morphological precursors ef type CI synaptic glomeruli (known to be C-fibre terminals in the adult) in the dorsal horn of tats at P0. Collectively these studies suggest that the central processes of unmyelinated primary sensory neurones begin to grow into the dorsal horn of the spinal cord at E18/19, In this study we have shown that BSI-B4 binding in the superficial dorsal horn appears at exactly the same time. This suggests that the BSI-B 4 binding site is expressed by the central processes of small diameter primary sensory neurones as they grow into the dorsal horn. Recently Scott et al. 23 have shown that in the chick, the plant lectin Dolichos biflorus agglutinin binds to a subpopulation of small diameter primary sensory neurones and their central processes in the dorsal horn. Binding of this lectin was first detected in the dorsal root ganglion at El2 (stage 38) and on processes in the dorsal horn at El6 (stage 42). However in the chick Dolichos biflorus agglutinin binding sites are expressed several days after the central terminals of small diameter primary sensory neurones have reached the superficial dorsal hoa~ ~'7.t~'. Consequently it is unlikely that this lectin binding site plays a critical role in the formation of specific neural connections during the developmental process in this species.
In the rat the precise role of this lectin binding site in developmental process requires further clarification. The observation that plant lectins with an affinity for terminal a-D-galactose residues bind to the plasma membrane and Golgi apparatus of primary sensory neurones has led to the proposal that the ligand is a membrane associated glycoconjugate 2°'29'3°. Membrane associated glycoconjugates have been widely implicated in intercellular recognition and adhesion during development aa°,:2,~3. Evidence in support of a galactose containing glycoconjugate being involved in the formation of orderly connections between primary sensory neurones and neurones in the dorsal horn of the spinal cord has come from in vitro studies. Baker et al. 2'3 have shown that in spinal cord-dorsal root ganglion explants from fetal mice, the addition of galactose to the culture medium potentiates the formation of monosynaptic connections between primary sensory neurones and neurones in the dorsal horn. In this study we have shown that the same carbohydrate moiety is expressed by the central processes of unmye!inated primary sensory neurones as they grow into the spinal cord. This finding is consistent with these binding sites being involved in the formation (or maintenance) of specific connections between unmyelinated primary sensory neurones and neurones in the superficial dorsal horn of the spinal cord during development. This proposal must, however, remain tentative until blockage of this binding site can be shown to affect the pattern of primary sensory neurone projections. Acknowledgements. We would like to thank Drs. Peter Wilson and Bruce Oynther for their helpful comments on earlier versions of the manuscript. This work was supported N.H. & M.R.C.
by the
Australian
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