INTRODUCTION
3.2 RT97 A Selective Marker of Large Light DRG Cells
3.2.1 B a c k g r o u n d
RT97 is a monoclonal antibody against a neurofilament subunit and is used as a selective marker of DRG cells. It’s size-frequency distribution in the adult rat DRG was determined and found to closely resemble that of the large light population of cells with myelinated axons (Lawson et al, 1984). RT97 can therefore be used as a selective label for this population of neurons in the adult rat DRG. However since this analysis has not been conducted in embryonic DRG, it is not known if the same selective labelling occurs during development. The absence of this information has become notable as other markers of the DRG cell population like trk A, IB4 (Bennett et al, 1996b) and peripherin (Troy et al, 1990a), have been found to show different expression patterns and ranges during development in comparison to the adult. In experiments detailed in Chapter 4, I used this antibody to describe the embryonic development of RT97 positive peripheral axons and proposed that these corresponded to the developing large A-fibres. To be
certain of this, however, it was necessary to investigate the size distribution of RT97 in the embryonic DRG to ensure it was labelling the same population as in the adult. E l 8 was chosen as the earliest age when size frequency analysis could be accurately and confidently completed, as at this age maturation of the two sizes of DRG sub-populations has begun.
3.2.2 Identification a n d structure
RT97 antibody is raised against the phosphorylated form of the 200 kDa neurofilament subunit NFH; subsequently also referred to as NFH-P in this thesis. It was discovered in 1982 by Wood and Anderton by raising an antibody against an extract of rat brain. The proof that RT97 does in fact only label the phosphorylated form of NFH came after treatment to dephosphorylate DRG tissue. Normally, non-phosphorylated NFH shows low level staining in the DRG while RT97 is intense in large cells. After dephosphorylation, intense staining is found in the large neurons with NFH but the RT97 label decreases. It was concluded that NFH in large neurons is phosphorylated and that small neurons contain little NFH regardless of phosphorylation state (Perry et al, 1991).
3.2.3 D R G E xpression
In the DRG RT97 expression is expressed almost exclusively in the large cells of rat DRG (Lawson et al, 1984; Goldstein et al, 1991). Phosphorylated N F’s are found in the DRG of all species examined but their distribution differs between species (Klosen et al, 1994). Lawson and colleagues examined the distribution of this NFH-P in the adult rat DRG and found the shape of the size-frequency histogram was the same as that of the large light DRG cell population, therefore linking NF content to cell type (Lawson et al, 1984). The RT97+ve population overlaps with some small diameter dark cells, but does not cover the entire range of neuron sizes within the DRG. In agreement with this, RT97+ve cells were found not to contain tyrosine hydroxylase (TH), substance P (SP) or somatostatin (SOM) (Lawson et al, 1984), markers characteristic of the small dark population but can be found in cells of peptide-containing size (Price, 1985).
The closeness of the RT97 distribution to the normally distributed large light cells suggested that this antibody could be used as a much needed selective label for this population of neurons in rat DRGs. In the adult approximately 40% of DRG neurons express RT97 (Lawson et al, 1984; Robertson et al, 1991; Kitao et al, 1996) but this data has not been obtained for younger animals. In contrast, 96% of human DRG cells express RT97 therefore it cannot be used as a discriminatory marker for the two types of human primary sensory neurons (Suburo et al, 1992; Vega et al, 1994). However, one study in human DRG showed that even though all cells do express NFH/RT97, a definite subpopulation is more intensely stained (Holford et al, 1994).
The non-phosphoylated 200kDa NF labels all rat perikarya, which in motoneurons and dorsal horn neurons is in globular form, indicating it is in a pre-filamentous stage (Perry & Lawson, 1993). In addition to the DRG, the trigeminal and jugular region of the vagal ganglia show comparable cellular labelling but the nodose region of vagal ganglia only have very fine fibres, while the superior cervical ganglion (SCG) containing sympathetic neurons lack RT97-IR (Lawson et al, 1984).
3 .2 .4 C N S E x p ressio n
Nerve fibres throughout the spinal cord produce an overall dense staining pattern with RT97 but it is not found in any spinal cord somata. However there is a significant amount of non-phosphorylated, globular NFH in all neuronal perikarya. Motoneuronal perikarya and processes are innnunoreactive for NFL, NFM and NFH but not RT97. Only the NFL sub-unit is filamentous (Perry & Lawson, 1993). The absence of RT97 in the motoneurons confirms other studies (Lawson et al, 1984; Lee et al, 1987; Mansour et al, 1989). However, there may be some partly phosphorylated NFH in the motoneurons since a polyclonal antibody that detects both phosphorylated and non-phosphorylated forms of NFH shows labelling while nothing is recognised with the antibody to non-phosphorylated NFH. This evidence in addition to the information about filamentous NFL, suggests NF structure in the motoneurons consists of a NFL backbone with poorly phosphorylated sidearms.
In contrast the dorsal horn shows NFH labelling only (Lawson et al, 1984; Perry & Lawson, 1993). This suggests that the NF structure in the dorsal horn departs from the triplet of subunits reported in primary sensory neurons. This triplet is incapable of homopolymeric assembly in vivo (Ching & Liem, 1993) but NFH is able to form short
filament-like structures on its own (Gardner et al, 1984). NFM expression in the spinal cord has been the subject of some debate as it was initially thought to be absent (Dahl et al, 1981; 1983; Lawson et al, 1984), however subsequent IHC studies (Dahl et al, 1988) and in situ hybridisation studies (Muma et al, 1990) have located it in motoneuron cell bodies. All fibres in the white matter and nerve tracts were reactive to NFL, NFM, NFH and RT97 (NFH-P) (Perry & Lawson, 1993).
3.2.5 P eriph eral E xpression
RT97 is present in non-neuronal tissues but is confined to nerve fibres and within the PNS is confined to neurons of neural crest origin as defined by Weston (1970). This proposes that all RT97 positive cells have a common origin despite staining in the nodose ganglion which is possibly of placodal origin (Le Douarin, 1980). A possible explanation for this discrepancy is incomplete separation of placodal and neural crest-derived neurons during development since there are no clear anatomical boundaries between the nodose and jugular regions of the vagal ganglia in the adult (Lawson et al, 1984). In addition to large diameter DRG cells, RT97 has been found in large diameter fibres and end structures of skin. It has been detected in the endings served by the large myelinated fibres, namely Meissner-corpuscles, merkel discs, hair follicle receptors, pacinian and free nerve endings. No labelling was found in unmyelinated endings, intraepidermal fibres or sympathetic fibres (Sann et al, 1995).
3 .2 .6 P h y s io lo g y
Lawson had previously described large light neurons as having a NF rich cytoplasm and myelinated fibres (Lawson et al, 1974). To discover more about the fibres that emanate from RT97+ve somata, an electrophysiological study was undertaken in the
followed by dye injection and immunohistochemistry. The peripheral nerve or dorsal root were electrically stimulated and conduction velocity measured between that site and the cell body. It was found that neurons with A-fibre conduction velocity (CV) have RT97+ve somata, reinforcing the evidence for the presence this NF subunit in A-fibres and cells. More specifically, all Aô fibres and Acx/p fibres were RT97+ve. In addition RT97+ve fibres showed a maximum CV of 31 m/s in 6-8 week old rats, where the greatest CYs measured 40-50m/s (Lawson et al, 1993). These findings linked NF content, state of myelination and conduction velocity. The data associating A-fibres and RT97 is further supported because RT97 innervation in the skin is unaffected by capsaicin, which selectively destroys small unmyelinated fibres (Sann et al, 1995).
3 .2 .7 C o-localisation
RT97 is co-localised with peptide-containing DRG cells. 6% of RT97-kve cells are
SP+ve but none of these are Aa/p cells. 28% of RT97+ve are CGRP-i-ve, 38% of these are A6 with 17% Aoc/p cells (McCarthy & Lawson, 1989; 1990). It is also co-localised with the cell membrane ganglioside receptor, GM1. This was visualised using the binding sub-unit of cholera toxin (CB) and suggested 82% of the RT97 population were also CB positive (Robertson & Grant, 1989).