Most trkA positive cells are labelled by BSI. TrkA is also co-localised with CGRP (92%) (Verge et al, 1989; Crowley et al, 1994; Averill et al, 1995; Silos-Santiago et al,
1995) and peripherin (Molliver et al, 1995). 18% of trkA cells also express the large cells marker RT97 (Averill et al, 1995). TrkA expressing neurons however, are not extensively co-expressed the PKC5 isoform, BSI-B4, LA4 (6%) or a-Internexin (3%), suggesting that trkA positive and negative neurons express different complements of cell surface glyco-conjugates (Averill et al, 1995; Molliver et al, 1995).
4.5 I-B4
4.5.1 Identification and structure
Isolectin B4 (I-B4) is the B-subunit of the plant lectin Grijfonia simplicifolia agglutinin I, also called Bandeiraea simplicifolia (GSA/BSI-1). It is thought to recognise the terminal a-D-galactose epitope of a membrane associated glyco-conjugate, and binds to the soma and central terminals of a sub-population of unmyelinated primary sensory neurons (Plenderleith et al, 1992). The a-D-galactose residue is a lactoseries carbohydrate (Dodd & Jessel, 1985) and along with the other membrane associated carbohydrate - the globoseries carbohydrates (Dodd et al, 1984) encompass the two classes of surface antigens that have been identified in separate populations of DRG neurons.
IB4 is a tetrameric plant lectin with a molecular weight of 155 kD and is composed of two subunits, A and B. Lectins are isolated from a wide variety of natural sources and are highly specific carbohydrates, binding glycoproteins of non-immune origin. The carbohydrates which serve as receptors or binding sites for the lectin are virtually ubiquitous in all biological, including neuronal, membranes. The specificity of lectins means that glyco-conjugates can be distinguished by botanical lectins on the basis of their different carbohydrate moieties (Streit et al, 1985; 1986), even with identical sugar compositions but different residues or anomeric structures (a and p), (Liener et al, 1986).
The globoseries carbohydrates are found in laminae I, EH and IV of the spinal cord where low-threshold myelinated afferents terminate and hence form a distinct group from the lactoseries antigens described below (Jessel et al, 4 ^ 0 ) .
4.5.2 Expression
IB4 lectin binding is first observed in the central processes of small diameter primary sensory neurons as they enter the dorsal horn of the rat spinal cord at E l 8/E 19 (Plenderleith et al, 1992). In the DRG, EB4 expression increases from 9% at PO to 40% at P14 (Bennett et al, 1996b). However, EB4 has not been reported in the periphery during development. There is also no data available on the degree of co-expression with other DRG markers except for the transient postnatal co-expression with trkA at P3 and P7 (Bennett et al, 1996b).
In the adult, GSA-IB4 labels terminals in the outer SG of the spinal cord with some labelling in the marginal zone. It also differentially stains a large number of thin axons in testicular and comeal whole-mounts (Silverman & Kruger, 1988b). Further staining is located in the small-calibre smooth contour axons of the mystacial pad, in addition to non-neuronal labelling of the vascular lining, surface of follicular cells, sweat glands and some cells of the sebaceous gland and duct (Rice, 1993).
In the adult DRG there are conflicting reports of the degree of expression. Some of this may be due to the type of antibody used and the criteria for deciding positivity. IB4 is only one subunit of the lectin but many studies use GSA-BSI which is a mixture of five isolectins. In one study, 75% of small neurons in the DRG were labelled. The lectin was mostly co-localised with FRAP (90%) with some CGRP co-localisation (10%). This result is not entirely consistent with the innervation pattern in the SG, as FRAP is usually found in the inner SG while CGRP is located in outer SG (Silverman & Kruger, 1988b).
Confirmation of the small degree of CGRP and I-B4 co-localisation was found in peripheral tissues where these markers labelled virtually separate sets of afferents (Ambalavanar & Morris, 1992). The differential labelling pattern in the spinal cord between EB4 and peptidergic CGRP, indicates that the lectin labels a different population. This assumption is reinforced by findings that although the lectin binds to a sub-population of unmyelinated axons in the rat and cat, these are almost exclusively neurons that innervate the skin (Plenderleith et al, 1992; Plenderleith & Snow, 1993). However, labelling is also produced after injection of tracer into the sciatic and saphenous
nerves. This allows visualisation of synaptic terminals in LE of the dorsal horn, derived from cutaneous C-fibres (Kitchener et al, 1993). This study is also significant because no retrograde labelling of motoneurons occurs establishing I-B4 as a unique marker for sensory neurons. Only one report indicates that I-B4 is also located in lamina I, although the most dense labelling was found in inner lamina E (Wang et al, 1994).
In another study using I-B4 conjugated to HRP, a full analysis of co-localisation and size-distribution within the DRG was undertaken (Wang et al, 1994). 51% of the DRG neurons bound and transported I-B4, but strong labelling was only found in 32% of cells. 35% expression has also been reported in the adult DRG (Bennett et al, 1996). Those strongly labelled cells had areas ranging 150-950jim^, compared to more weakly stained cells that were found in cells as large as 1450pm\ 85% of those EB4+ve cells were FRAP+ve, in agreement with Silverman & Kruger (1988b). 17% of cells co-localised with SP, 9% with SOM and only 3% with RT97. In addition, they determined that approximately 100% of the SOM and FRAP populations were within the I-B4 population. However, there was a discrepancy in the extent of CGRP co-localisation between the Silverman and Kruger (1988a) and Wang (1994) studies. Wang (1994) found 59% of I-B4 neurons were also CGRP+ve in contrast to the former study which reported 10%. The larger overlap is a more predictable representation considering the overlap in lamina II outer of the SG. However, in the adult very few cells express both trkA and IB4 (Bennett et al, 1996a), yet trkA and CGRP show almost parallel expression patterns (Bennett et al, 1996b). This implies that there should be little overlap between IB4 and CGRP.
4.5.3 Plasticity
Capsaicin treatment or a dorsal rhizotomy results in downregulation of binding in the spinal cord which is consistent with synaptic staining of a primary afferent origin (Ambalavanar et al, 1993). Injection of IB4 into the sciatic nerve after axotomy, reveals transport as far as neuronal cell bodies in the DRG, however, it appears that C-fibres are unable to maintain transganglionic transport after injury therefore abolishing transport to
their central terminals in LIE (Kitchener et al, 1994). In animals where the trkA gene has been dismpted the population of IB4 neurons are lost (Silos-Santiago et al, 1995).
4.5.4 Function
The physiological and functional significance of GSA binding sites are unknown. Involvement with nociception is mainly deduced from its localisation, principally at regions where unmyelinated afferents terminate; and its co-localisation with substances which have already been functionally associated with nociception. Nonetheless, the stratified distribution of FRAP and I-B4 to LII inner and II outer respectively led Silverman and Kruger (1990), to hypothesise the existence of two functionally independent C-fibre nociceptor systems.
Cell surface determinants such as this lectin have also been implicated in interactions with second order neurons of the superficial dorsal horn and with target finding in development (Plenderleith et al, 1992). It has been proposed that the glyco-conjugates expressed on subsets of DRG neurons may provide a basis for specific guidance of developing neurites by virtue of glyco-conjugate-mediated adhesive interactions (Dodd & Jessel, 1985; 1986).