lucifer-yellow and sulforhodamine-101

Compound eyes of insects in 16 orders were tested for the presence of a blood-retina barrier (BRB) by injecting the hemolymph with Procion yellow, which was excluded from the eye in all Neoptera but not in two apterygotes. A primitive apterygote (Petrobius, Machilidae) was investigated further. Epifluorescence observations with small dyes Lucifer yellow (LY) and sulforhodamine 101 (SR) confirmed uptake by the eye within 3 min of injection. LY and SR both penetrated the eye, particularly the cornea, examined in sections. Uptake was quantified by microfluorometry, yielding entry half-times (t(1/2)) of 1-1.4 min, fitting predictions for a model where tracer uptake is limited by passive diffusion. A much larger fluorescent dextran entered at a similar rate (t(1/2) = 1.70 +/- 0.77 min; n = 22), too fast to be diffusion-limited, pointing to an active process, probably flushing of hemolymph through the retina. This is not an artifact associated with tracer injection and may be the natural result of circulatory pressures. Microfluorometry gave a first estimate of hemolymph volume (2.9% of body weight), of hemolymph mixing time (t(0.95) = 77 min); the eyes' receptive fields were also determined. All results point to a primitive crustacean-like condition in Petrobius, with open access of hemolymph to the eye and no BRB. An evolutionary hypothesis is suggested to explain how a primitive central nervous system barrier later extended to cut off the eye in Neoptera, in the face of access problems for respiratory gases and metabolites.

Topics: Animals; Axonal Transport; Axons; Crustacea; Female; Fluorescent Dyes; Insecta; Isoquinolines; Microscopy, Fluorescence; Nervous System Physiological Phenomena; Ocular Physiological Phenomena; Photoreceptor Cells, Invertebrate; Retina; Rhodamines

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.

Topics: Animals; Coloring Agents; Extracellular Space; Female; Fluorescent Dyes; Houseflies; Isoquinolines; Light; Microscopy, Electron; Nerve Degeneration; Neurology; Neurons; Permeability; Photoreceptor Cells; Retina; Rhodamines