Simultaneous identification of unmyelinated and myelinated primary somatic afferents by co-injection of isolectin B4 and Cholera toxin subunit B into the sciatic nerve of the rat

Abstract

Several studies have used the transganglionic tracers cholera toxin subunit B (CTb) and either Bandeiraea simplicifolia isolectin B4 (IB4) or wheat-germ agglutinin (WGA) to label myelinated and unmyelinated afferent fibres respectively. In this study, we aim to determine whether co-injection of CTb and either IB4 or WGA into the sciatic nerve of rat will selectively label myelinated and unmyelinated simultaneously. A double immunofluorescence approach was used to detect these tracers in dorsal root ganglia (DRGs) and afferent fibre terminals in the spinal cord. CTb- and IB4-labelled neurons were seen mainly in L4 and L5 DRGs, with CTb labelling detected primarily in large sized neurons and IB4 staining seen mainly in smaller cells. Only a minority of CTb labelled DRG neuron profiles (5.1%) were also labelled with IB4. In the spinal cord, IB4-labelling was largely confined to lamina II of spinal segments L3-L5, whereas CTb-labelled terminals were seen in all laminae but sparse in lamina II. Confocal microscopy showed no evidence for colocalisation of CTb and IB4 labelling in any terminals in laminae I-III. Although the central distribution of CTb labelling in laminae I and II inner-IV had the same rostro-caudal and medio-lateral coverage as IB4 labelling in spinal segments L3-L5, CTb labelling in ventral laminae (of putative proprioceptor afferents) extended between T12 and S1. Similar patterns of central labelling were found when CTb and WGA were injected together. We therefore concluded that this co-injection approach provides a reliable method to identify both myelinated and unmyelinated somatic primary afferents simultaneously.