Immunocytochemical localization of trkA receptors in chemically identified subgroups of adult rat sensory neurons

Abstract

Immunocytochemistry has been used to examine the location of trkA, the high-affinity receptor for nerve growth factor, in adult rat dorsal root ganglia, trigeminal ganglia and spinal cord. TrkA immunoreactivity was observed in small and medium sized ganglion cells and in the dorsal horn of the spinal cord. In lumbar L4 and L5 ganglia trkA-immunoreactive cells constitute 40% of dorsal root ganglion cells and range in size from 15 to 45 microns in diameter. Double labelling using markers for various dorsal root ganglion subpopulations revealed that virtually all (92%) trkA-immunoreactive cells express calcitonin gene-related peptide (CGRP) immunoreactivity. In contrast only 4 and 13% of trkA-immunoreactive cells are labelled by the monoclonal antibody LA4 or the lectin Griffonia simplicifolia IB4, markers for small non-peptide-containing cells. Eighteen percent of trkA-immunoreactive cells belong to the 'large light' subpopulation, identified by their strong immunostaining by the neurofilament antibody RT97. TrkA immunoreactivity in the dorsal horn is heaviest in laminae I and II outer, has a similar distribution to CGRP, and is depleted by dorsal rhizotomy. Our results show that trkA-expressing cells in dorsal root ganglia correspond almost exactly with the CGRP, peptide-producing population. The receptor is present not only on cell bodies but also on central terminals. Non-peptide-containing small cells, which constitute 30% of dorsal root ganglion cells, are not trkA-immunoreactive and therefore most probably are functionally independent of nerve growth factor.

Fig. 1

Immunofluorescence micrographs showing trkA-immunoreactive cells (A, B) and axons (B, C) in L5 dorsal root ganglia. (A) TrkA-immunoreactive cells are mainly small (arrows) but medium sized cells (arrowheads) are also present. Asterisks indicate neighbouring non-immunoreactive cells. (B) Immunostained cells show diffuse immunostaining plus granular staining (small arrows) which corresponds to Golgi complexes. The open arrowhead indicates a trkA-immunoreactive axon coursing between the cells. (C) Immunostained axons (arrows) in a section of nerve adjoining the ganglion. In some preparations the trkA immunoreactivity had this characteristic speckled appearance. Scale bars = 25 μm.

Fig. 2

Size distribution of trkA-immunostained cells in L4 and L5 dorsal root ganglia. 230 cell profiles were counted in 12 sections taken from three animals. Results from L4 and L5 were pooled.

Fig. 3

Immunofluorescence double labelling for CGRP (A) and trkA (B) in L5 dorsal root ganglia. Most of the cells in this field of view express immunoreactivity for both antigens. Numbers indicate pairs of double-labelled cells. In addition, a few CGRP single-labelled cells are present (asterisks). Arrows in A indicate CGRP-immunoreactive axons. Scale bars = 25 μm.

Fig. 4

Pairs of micrographs showing immunofluorescence double labelling for trkA (A, C, E) and RT97 (B), IB4 (D) and LA4 (F). L5 dorsal root ganglia. (A, B) Several medium to large sized cells are visible which express both trkA and RT97 immunoreactivity (numbers indicate pairs of double-labelled cells) but many small trkA single-labelled cells (arrows) are also present. Open arrows in B indicate large-diameter RT97-immunoreactive axons. (C, D) TrkA single-labelled cells (arrows in C) and IB4 single-labelled cells (open arrow in D) are visible together with a single double-labelled cell (I). (E, F) Three trkA single-labelled cells are visible in E (arrows) and adjoin a group of LA4 single-labelled cells (open arrows in F). The cell indicated by an asterisk is also single-labelled for trkA but in panel F shows some background non-specific staining. Scale bars = 25 μm.

Fig. 5

Correlation between CGRP and trkA immunostaining intensities (mean grey levels, arbitrary units), r² = 0.63.

Fig. 6

Low (A) and high (B) magnification micrographs showing trkA-immunoreactive terminals in the dorsal horn of lumbar spinal cord. Avidin–biotin peroxidase immunostaining with OsO₄ contrasting. (A) TrkA immunostaining is present mainly in the superficial layers (asterisks) of the dorsal horn. Arrows indicate non-immunostained but osmium-contrasted myelinated fibre bundles which penetrate deep dorsal horn. (B) A patch of trkA immunoreactivity in lamina V. The staining consists of fine varicose axons (arrows). Open arrowheads indicate neighbouring non-immunostained but osmium-contrasted myelinated axons. Scale bars = 50 μm (A), 25 μm (B).

Fig. 7

Pairs of micrographs showing immunofluorescence double labelling for CGRP (A, C) and trkA (B, D) in laminae I and II of lumbar dorsal horn. (A. B) Normal animal. Immunostaining for CGRP (A) and trkA (B) is largely coincident in laminae I and II. Arrows indicate double-labelled patches of axons and terminals. (C, D) Animal with a unilateral L5 dorsal rhizotomy. Ipsilateral dorsal horn. Marked depletion of both CGRP (A) and trkA (B) immunofluorescence is apparent in the lateral dorsal horn (asterisks). Arrowheads indicate an isolated spared fibre which is double-labelled for CGRP and trkA. Arrows indicate remaining double-labelled fibre bundles in the adjoining intact medial dorsal horn. Scale bars = 50 μm.

Fig. 8

Pie chart showing neurochemically defined dorsal root ganglion subpopulations.