Cholera toxin B-gold, a retrograde tracer that can be used in light and electron microscopic immunocytochemical studies.
The purpose of this study was to test whether a new retrograde tracer, the B subunit of cholera toxin conjugated to colloidal gold particles (CTB-gold), was taken up and transported by neurons in the central nervous system of the rat. Retrograde transport of CTB-gold was assessed from axon terminals, from damaged nerve fibers, and from axons of passage. For light microscopy, CTB-gold was visualized by silver intensification; for electron microscopy, sections were silver-intensified with or without subsequent gold toning. Retrogradely transported CTB-gold was detected in neurons after survival times of 12 hours to 42 days and appeared as black punctate deposits in perikarya and proximal dendrites at the light microscope level. Ultrastructurally, the deposits were usually associated with lysosomes. Injections of CTB-gold into the caudal ventrolateral medulla or into the lateral horn of the spinal cord gave small well-defined injection sites and resulted in retrograde labelling in medullary neurons in the same locations as similarly placed injections of wheat germ agglutinin-horseradish peroxidase. When injected into the superior cervical ganglion, CTB-gold was transported to nerve cell bodies in the spinal cord, but application of CTB-gold to the cut cervical sympathetic trunk did not label neurons in the spinal cord. Injection of CTB-gold into the nodose ganglion retrogradely labelled neurons in the dorsal motor nucleus of the vagus and the nucleus ambiguus. CTB-gold was not transported anterogradely from injections sites within the medulla. Nerve fibers and cell bodies containing neuropeptides, monoamines, or neurotransmitter-synthesizing enzymes were readily immunostained after silver intensification of retrogradely transported CTB-gold. Immunoreactivity for neuropeptides and enzymes was also demonstrated ultrastructurally after silver intensification and gold toning. These results show that CTB-gold is retrogradely transported from nerve terminals and fibers of passage but not from damaged axons. CTB-gold gives well-localized injection sites and persists in neurons for weeks. Transported CTB-gold is easily visualized and its detection is compatible with light and electron microscopic immunocytochemistry. These properties make CTB-gold a valuable tool for studying the connectivity and neurochemistry of pathways in the central nervous system.
Department of Medicine, School of Medicine, Flinders University, Bedford Park, South Australia.
I J
IJ
Llewellyn-Smith
J B
JB
Minson
A P
AP
Wright
A J
AJ
Hodgson
D000818:Animals; D001370:Axonal Transport; D001931:Brain Mapping; D002490:Central Nervous System / Q000166:cytology* / Q000378:metabolism; D002772:Cholera Toxin / Q000493:pharmacokinetics*; D005260:Female; D006735:Horseradish Peroxidase; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D008854:Microscopy, Electron / Q000379:methods*; D051381:Rats; D011919:Rats, Inbred Strains; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; D014909:Wheat Germ Agglutinins
D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; D014909:Wheat Germ Agglutinins; D002772:Cholera Toxin; D006735:Horseradish Peroxidase