Journal of Neuroscience Methods 108 (2001) 19 – 24 Retrograde labeling of primary sensory neurons with fluorescent latex microspheres: a useful tool for long term tagging of neurons Mikael Altun, Esbjo ¨ rn Bergman, Brun Ulfhake * Experimental Neurogerontology, Department of Neuroscience, Karolinska Institutet, Nobels 12 17177 Stockholm, Sweden Received 17 January 2001; received in revised form 17 April 2001; accepted 18 April 2001 Abstract In this study we have used fluorescent microspheres to retrogradely label primary sensory neurons in dorsal root ganglia (DRGs). Following injection into peripheral nerves, the animals were allowed to survive up to 480 days. Simple profile count indicates that there is a substantial retention of the labeling still after at least 480 days, i.e. about two-thirds of a rat’s life span. Moreover, the appearance of the labeling remains quite distinct. Using established markers for axon damage of DRG neurons, we could detect a slight and transient effect of the peripheral nerve injection on the gene expression pattern. It is concluded that fluorescent microspheres represents an attractive means of tagging neurons in experiments covering long time periods. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Neuropeptides; GAP-43; Tracer; Latex microspheres; mRNA; In situ hybridization www.elsevier.com/locate/jneumeth 1. Introduction Tracer substances that are taken up and transported by neurons provide us with a tool to study neurons by their projections and connectivity rather than by the anatomical residency of the cells. Needless to say, this tool has proven indispensable in unraveling the archi- tectonics of the CNS. A range of markers is available for anterograde and/or retrograde transport within neu- rons, and many of them are considered neutral with respect to potential damage to their host neurons (re- viewed by Heimer and Za ´borsky, 1989). A notable shortcoming with most of these tracers is, however, that they are short-lived; usually the labels become fainter after 1–2 weeks and not detectable after several weeks, making them less useful in more complex and time-con- suming experiments. Another problem concerns the injection since, although the tracer molecule may be considered harmless, the pressure, per se, may induce damage to the axons. Reaction to minor axon damage, such as a light crush, is usually evident for a few weeks during which the neuron recovers (Fu and Gordon, 1997; Verge et al., 1996). From this point of view, it would be advisable to have a prolonged post-injection survival time to allow for recovery, however, then the labeling will be gone. Some years ago, it became known that neurons take up and transport latex-microspheres (Katz et al., 1984). The microspheres are transported with the fast axonal transport system to the soma where they are stored in lysosomes. By filling the beads with a fluorophore, they can be visualized by fluorescence microscopy and, in addition, it seems also possible to identify them at the ultrastructural level (Egensperger and Holla ¨nder, 1988; Katz and Iarovici, 1990). More- over, it has been claimed that the labeling is permanent. Another attractive property of latex beads is that they are charged and, thereby, can carry proteins. Thus, latex beads have been used as a delivery system of protein to cells and have at the same time labeled the cells for identification in the microscope (Riddle et al., 1997). Here we have tested the long-term retention of retro- grade labeling of dorsal root ganglion (DRG) neurons in rats with fluorescent latex microspheres. Moreover, established markers for injury such as growth associ- ated protein 43 (GAP-43, Schreyer and Skene, 1991) and the neuropeptides calcitonin gene-related peptide * Corresponding author. Tel.: +46-8-7287888; fax: +46-8-333968. E-mail address: brun.ulfhake@neuro.ki.se (B. Ulfhake). 0165-0270/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S0165-0270(01)00367-3