Journal of Neuroscience Methods, 33 (1990) 101-112 101 Elsevier NSM 01096 Dye-induced photopermeabilization and photodegeneration: a lesion technique useful for neuronal tracing Serge Picaud 1, Hansjoerg Wunderer 2 and Nicolas Franceschini 1 t CNRS, Laboratoire de Neurobiologie, F-13009 Marseille (France), and 2 Institut f~r Zoologie der Universitiit, Biologie 1, D-8400 Regensburg (F.R. G.) (Received 16 February 1990) (Accepted 18 April 1990) Key words: Neuronal tracing; Retina; Invertebrate nervous system; Lesion; Neuronal degeneration; Permeabilization; Photosensitization; Dye Using as a neural system the fly retina, which is visually accessible in vivo, we describe a lesion technique that takes advantage of the photodynamic damage produced by extrinsic dyes. Contrary to the photo-inactivation technique described by Miller and Selverston (1979), this technique does not involve intracellular injection, since the dye is applied to the extracellular space of the tissue. This treatment was found to trigger neuronal degeneration and cell permeabilization in fly photoreceptor neurones. We coined the names 'photodegeneration' and 'photopermeabilization' for these two phenomena. While the technique can be used to delete given neurones from the neural circuit after several days' survival time, it was found to produce adequate cytoplasmic labelling for anatomical studies with both light and electron microscopy. Since the area occupied by the degenerating cells is restricted to the light spot imaged onto the nervous tissue, the resolution with this lesion technique can range from single cells to whole neuronal populations. The remarkable precision of the 'photolesions' produced in this way makes this technique a powerful tool for physiological and anatomical investigations on real neural networks, whenever these can be made optically accessible in vivo or in situ. Introduction Throughout the history of the neurosciences, lesion techniques have been providing powerful tools for analysing nervous tissue, as in the case where they serve to delete selected neuronal popu- lations (review: Jonsson, 1981). Lesion techniques have also been widely used in anatomical studies. Indeed when a neurone undergoes the 'dark type' of degeneration (Mugnaini and Friedrich, 1981), the increase in the electron density of the cyto- Correspondence." S. Picaud, CNRS-LNB3, 31 chemin J. Aiguier, F-13009 Marseille, France. Abbreviations: SR 101, Sulforhodamine 101; LY, Lucifer yel- low plasm throughout the cell can be used to trace injured neurones and locate their terminals in order to analyse their connectivities in detail (Heimer, 1970). Moreover, some lesion techniques, such as transection, make it possible to fill injured neurones with extracellularly applied markers such as metallic ions, dyes, proteins (review: Strausfeld, 1983) or coloured microbeads (Katz et al., 1984). A decade ago, a novel lesion technique was reported, in which dye photosensitization was used to photo-inactivate neurons (Miller and Selver- ston, 1979). Cells were first intracellularly filled with a dye via a micropipette and then irradiated with a short pulse of laser light adapted to the absorption band of the dye. Neurones which un- derwent this treatment were photoinactivated and disintegrated at once. More recently, using the fly retina as a model system, we investigated the fate 0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)