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.

Fluorescein-concentrating Sensory Neurons

E. Hedgecock, N. Thompson, L. Perkins, J. Culotti

Figure 1

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 
[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.

Figure 1