Worm Breeder's Gazette 7(1): 70
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Eight pairs of neurons specifically concentrate fluorescein when animals are placed in a solution of dye. These have been identified by as amphid neurons ASE, ADF, ASI, ASJ, ASK, and ADL and phasmid neurons PHA and PHB. Amphid neurons ASG and ASH do not stain nor do the amphid wing (AWA, AWB, AWC) or finger (AFD) neurons. The processes of stained neurons are visible in live animals. Several fluorescein derivatives (aminofluorescein and fluorescein isothiocyanate) also stain these cells but less related dyes ( rhodamines, Hoescht 33258, Lucifer Yellow) are not taken up under the same conditions. J. Sulston, M. Chalfie, and C. Trent have observed that certain of these neurons can be loaded with exogenous serotonin or catecholamines though these are not their natural transmitters. Possibly all of these compounds are entering by a common mechanism. Interestingly, the equivalent neurons in Panagrellus redivivus do not concentrate fluorescein. Different types of evidence suggest that dye uptake occurs through functional sensilla: (1) Laser ablation of the phasmid sheath cell or the T cell ( precursor of the phasmid sockets) abolishes staining of the ipsilateral phasmid neurons (2) Amphid neurons stain in mid L1 larvae and stain brightly by late L1. Phasmid neurons do not stain until mid L2 and not brightly until L4. This suggests that the phasmid neurons may not be functional until after the postembryonic development of the phasmid sensilla is complete (T lineage). (3) Dye microinjected into the pseudocoelom is not concentrated. (4) Mutants in four genes (che- 2, daf-6, and daf-10) that block dye uptake have been examined by electron microscopy (Lewis and Hodgkin (1977), Albert et al. (1981)). All have identified defects in sensory endings. (5) Mercurochrome, a mercury derivative of fluorescein, concentrates ( though poorly) in the amphid and phasmid sheath cells, not in neurons. (6) Additional sensory neurons stain in certain mutants. These include an amphid wing neuron in che-1 (e1034), cephalic and post- derid neurons in e1861, and ray neurons in osm-1 males. In all of these cases the staining is rather stochastic. We have examined the cephalic sensilla in e1861 by electron microscopy and found that they open to the outside in some cases. Similarly, ray neurons in osm-1 males do not stain until after the L4 cuticle is shed. Taken together, these results suggest that dye uptake requires a functional sensillum open to the outside. This is necessary but insufficient since many neurons, open by EM criteria, do not accumulate dye. Some additional feature, perhaps a physiological property of the sensory dendrite, determines which exposed neurons stain. (Apparently the dopaminergic cephalic and post-derid neurons share this second property but are normally closed to the outside. We have not yet established which ray neurons (RnA or RnB) stain in osm-1 males but staining is not limited to rays with dopaminergic neurons.) Mutations in thirteen genes block dye uptake in both amphids and phasmids. These are: [See Figure 1] Incomplete patterns of neuron staining are found in mec-8 (e398), unc-33 (e204), and unc-44 (e362). The amphid and phasmid neurons stain in unc-51 (e369) animals but have abnormal swellings where the processes terminate in the nerve ring and preanal ganglion, respectively. The phasmid processes terminate prematurely in the preanal ganglion in unc-76 (e911) mutants. We are determining which behaviors are affected by each gene and hope to examine mutant sensilla by electron microscopy. Several of the mutations have previously been shown to be pleiotropic in that they affect several behaviors (chemotaxis, osmotic avoidance, dauer formation, male mating, egg-laying) or several types of sensilla. Where applicable, fluorescein uptake provides a convenient common method for isolating, complementing and mapping mutations affecting these behaviors. We hope to identify lineage mutants affecting neurons and support cells and possibly new wiring mutants by direct screening with the fluoresence microscope. Staining Protocol: Add 50 l of 20 mg/ml FITC in dimethylformamide (stored indefinitely at -20 C) to 200 l M9 buffer and apply uniformly to a 10ml seeded NGM plate (final FITC concentration is 0.1 mg/ml). Worms are placed on for two hours or overnight and then transferred to a seeded plate without dye for at least 10 minutes to remove free FITC and labeled bacteria from the intestine. They remain stained for hours and can be viewed by epifluoresence on a 5X agar pad as described by John Sulston for Nomarski. The entire cells, including their processes and nuclei, are uniformly filled. Dye can also be added directly to plates containing live worms. They continue to develop and stained males will mate.