Pergamon 0306-4522(94)E0181-3 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR ,Ve:eerohmrVol. 62, No. 2, pp. 539-551, 1994 Elsevier Science Ltd pyright 0 1994 IBRO Printed in Great Britain. All rights reserved 03064522/94 $l.rm + 0.00 TRANSGANGLIONIC TRANSPORT AND BINDING OF THE ISOLECTIN B4 FROM GRIFFONIA SIMPLICIFOLIA I IN RAT PRIMARY SENSORY NEURONS H. WANG, C. RIVERO-MELIblN, B. ROBERTSON and G. GRANT* Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm, Sweden Abstract-The isolectin B4 from Griffonia zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI sim p lic ifo lia I binds to a subpopulation of rat small-diameter dorsal root ganglion neurons, and to fibres and presumed terminals in laminae I II of the spinal cord dorsal horn. In the present study we investigated B4 and B4 conjugated to horseradish peroxidase as potential transganglionic tracers of somatic primary afferent neurons after injection into a peripheral nerve. We also tried to identify the specific subpopulation of dorsal root ganglion neurons that bind and transport B4. Following injection of B4 or Bhhorseradish peroxidase into the sciatic nerve, labelled presumed terminals that reached peak labelling at two days were found exclusively in regions of the spinal cord gray matter known to receive unmyelinated primary afferent fibres. Almost all dorsal root ganglion cells that transported Bhhorseradish peroxidase also bound B4. Cell counts showed that 51% of the dorsal root ganglion neurons were B4-positive and cell area measurements that these were all in the small size range. An extensive overlap was found between 84 and fluoride-resistant acid phosphatase (85%), and between B4 and calcitonin gene-related peptide (59%). Seventeen per cent of the B6positive cells were substance P-immunoreactive and 9% were immunoreactive to somatostatin. Minimal overlap was seen between B4-positive cells and cells positive for RT97 (3%), a selective marker of primary afferent neurons with myelinated axons. All somatostatin-immunoreactive cells and almost all (95%) of the fluoride-resist- ant acid phosphatase-positive cells were contained within the B4-positive population. This comprised also 58% of the cells immunoreactive to calcitonin gene-related peptide and 42 5’0of those immunoreactive to substance P. The results obtained show that 84 binds to a subpopulation of unmyelinated primary afferent neurons, and that 84 and Bkhorseradish peroxldase can be used as selective transganglionic tracers of this specific cell subpopulation. Dorsal root ganglion (DRG) neurons of the rat and mouse have been divided into two main subpopu- lations on the basis of their staining pattern, distri- bution of Nissl substance and neurofilament content, namely into the small, dark (B) and the large, light (A) neurons.6,‘4.” It has been shown in the rat that these two subpopulations can be distinguished by a monoclonal anti-neurofilament antibody, RT97, which labels specifically and exclusively the large, light cell populatiomXJ Subsets of the small, dark DRG cells have been investigated using techniques such as peptide immunohistochemistry and enzyme histochemistry.‘.g, ‘6~*~22~28~42~56~67 More recently, lectin histochemistry and immunohistochemistry have re- vealed that subpopulations of DRG cells specifically express particular oligosaccharide residues on their *To whom correspondence should be addressed. Abbreoiafions: AMCA, 7-amino-4-methylcoumarin-3-acetic acid; B4, isolectin B4 from Gr@ imia simplicifolia I; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglion; FITC, fluorescein isothiocyanate; FRAP, fluoride-resistant acid phosphatase; GSA, G riffo niu sim - plicqolia agglutinin; HRP, horseradish peroxidase; LI, like immunoreactivity; PBS, phosphate-buffered saline; SBA, soybean agglutinin; SP, substance P; WGA, wheat germ agglutinin. surface.45”9~54,5558,59,65 Moreover, monoclonal anti- bodies that recognize different surface oligosacchar- ides have been shown to be valuable markers of different subpopulations of DRG cells.2~3~‘z~13~26~J5 The isolectin B4 of the plant lectin zyxwvutsrqponmlkjihgfedc GrifSoniusimpii- cifofia I (B4), which recognizes a-D-galactose carbo- hydrate residues, has been found to bind to a subset of small DRG cells and presumed unmyelinated primary afferent fibres and terminals in the dorsal horn laminae I-Il.54~55~58~59~65 The majority of B6posi- tive primary affercnt neurons have been shown to contain fluoride-resistant acid phosphatase (FRAP), whereas few BCpositive neurons were found to con- tain the peptides calcitonin gene-related peptide (CGRP) and substance P (SP).55,66 Streit et ~1.;~ however, found all SP-immunoreactive DRG cells able to bind the soybean agglutinin (SBA) lectin, which they considered recognizing the same popu- lation of cells as B4. Different plant and bacterial lectins, among them wheat germ agglutinin (WGA) and the binding B-subunit of cholera toxin, choleragenoid, have not only been found to bind to different subpopulations of DRG cells, but also to be transported differently in different types of primary afferent neur- ons.7.32.4648,M,5’ The aim of the present study was first 539