[
Worm Breeder's Gazette,
1980]
Neurosecretory-like material was investigated in C. elegans L4 and adult hermaphrodites and in males, by histochemical methods in optics microscopy. We tried numerous fixative fluids: Bouin, Dubosq-Brasil, Halmi, Carnoy, Zenker, Osmium tetroxide and several staining techniques : paraldehyde-fuchsin (PF), alcian blue, chrome-hematoxylin, Heidenhain azan. Only the two first dyes react, as usually, with all the cuticular external and internal structures without an oxidizing treatment (because the surface coat external to the cuticular membrane contains mucopolysaccharid acids) and with intestinal microvilli after oxidation. Another positive reaction is obtained on osmium-tetroxide fixed worms, stained by paraldehyde-fuchsin after a hot chromic oxidation. The surprising result was that this positive reaction was observed not in a nerve cell as expected for neurosecretory-like material, but in the two large glandular cells associated to the so- called 'excretory'-system. Paraldehyde-fuchsin positive (PF + ) granules fill the whole cytoplasm of the two secretory gland cells i.e. the nucleus area located ventrally under the terminal bulb of the pharynx, the processes sent anteriorly which fuse with each other under the excretory pore and even the very thin processes sent again more anteriorly ending under the nerve ring. The amount of the PF+ granules is low in L4 hermaphrodites and in males and is mostly found in the anterior processes. It increases during the adult period of the hermaphrodites and is distributed in the whole gland. These granules present the game histochemical properties that neurosecretory- like material and the technical conditions allowing to reveal their presence indicate the nature of a sulfurylated lipo-protein. We will try to bring a proof and a further characterization of a neurosecretory-like nature of the granules content with immunocytological technics using anti-vertebrates neuro-hormones antibodies. A role of this PF + granules in a developmental event is possible since the excretory glands are communicating with the excretory canals : the secreted products can be released here and then in the pseudo-coelomic fluid before to act on target cells.
[
Worm Breeder's Gazette,
1995]
Using simple modifications of existing techniques, I have recentlymade intracellular recordings from neurons and muscles in C. elegans. Larvae (approx. L2) were glued with cyanoacrylate adhesive to acoverslip coated with a moist agarose film.1 The coverslip formed thebottom of the recording chamber, which was filled with a physiologicalsaline and viewed on an inverted microscope using Nomarski optics. Animals were dissected in a two-step procedure. First, internal pressurewas relieved by nicking the cuticle in the mid-gut using a tungstenneedle. Second, either the pharynx or a small bouquet of neurons wasexposed by making a nick in the cuticle of the head. Intracellularmicroelectrodes were used to record from the pharynx and patch electrodeswere used to record from neurons. Microelectrode recordings from muscles of the spontaneously pumpingpharynx revealed rhythmic depolarizations with an amplitude of 50 to 70 mVand a duration of several hundred milliseconds. These events closelyresembled action potentials previously recorded from the pharynx ofAscaris,2 indicating that important aspects of physiological function areretained after gluing and dissection. As a first step in understanding the basic operating principles ofthe C. elegans nervous system, I have concentrated primarily on whole-cellvoltage-clamp recordings. Twenty-one whole-cell or perforated patchrecordings have been made so far. Neuronal input capacitance ranged from0.1 to 2.0 pF. The low end of this range is the capacitance expected ofan isolated L2 soma,3 while the upper end is the capacitance expected ofan L2 neuron with a process about 50 mm long. The apparent neuronal inputresistance ranged from 0.1 to 7.1 G ohms. Patch clamp methodssystematically underestimate capacitance and resistance in small neurons.Nevertheless, these data indicate the membrane time constant, whichdetermines how fast a neuron responds to its inputs, is at least 14 ms. They also suggest the axonal space constant, which determines how far aninput signal propagates passively, is at least 150 mm. This means thatinterneurons confined to the nerve ring should be effectivelyisopotential. Two classes of neurons could be distinguished by differences intheir voltage-dependent currents. Cells of the first class had sustainedoutward currents but no inward currents. Cells of the second class had atransient outward current and also a small, sustained inward current. Because the inward current activates more rapidly and at lower clampvoltages than the outward current, these cells may be capable ofregenerative potentials. Until now, all recordings have been from unidentified neurons. I amhopeful, however, that by using GFP4 labeled worms I will be able torecord from identified neurons. If so, it should be possible to determinewhether different types of neurons have different electrophysiologicalproperties and to correlate these differences with the behavioral rolespredicted by anatomical and laser ablation experiments. Moreover, bycrossing GFP animals with mutant strains, I hope to record from identifiedcells in mutants. Thus, it should be possible to combine theelectrophysiological and the genetic analysis of behavior at the cellularlevel in individual neurons.1. Avery, L., Raizen, D., and Lockery, S.R. (1995) Electrophysiological Methods. In Epstein, H.F. and Shakes, D.C. (eds.) C. Elegans: Modern Biological Analysis of an Organism. Academic Press, Orlando (in press).2. Byerly L., Masuda, M.O. (1979). J. Physiol. 288:263-284.3. David Hall, unpublished data.4. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. and Prasher, D.C. (1994). Science 263:802-5.
[
Worm Breeder's Gazette,
1984]
During the next few years, a major thrust of C. elegans research will be the cloning pf developmentally important genes. The absence of known products means that such genes must be isolated either by the direct insertion of a transposon or by walking along the genome from some genetically identified starting point. Transposition may not be applicable to all loci, and walking is tedious. In the hope of speeding up the cloning process, and of providing information about the genome as a whole, we have begun the task of generating a physical map of the genome - that is to say, of creating an ordered library of cloned DNA fragments which will parallel the genetic map. At the least, such a library will facilitate access to any given region; ultimately, as we identify increasing numbers of transcription units, it may allow immediate isolation of any genetic locus. In order to build up the library we have cloned nematode DNA ( partially digested with Sau3a1) into the cosmid vector pJB8, and have set out some 6000 individual colonies in microtitre wells. In this form, the collection can conveniently be transferred to nitrocellulose (384 clones per 130mm filter) and screened by hybridization. We are now systematically 'fingerprinting' the clones, by the following procedure. The cosmid DNA is cut with a restriction enzyme that recognizes a six base pair sequence, end labelled, and then cut again with a restriction enzyme that recognizes a four base pair sequence. The resulting fragments are separated on a thin polyacrylamide gel, and the positions of the radioactive bands are recorded relative to a set of standards. The pattern derived from each clone is compared by computer with all the others, and significant overlaps are noted. So far we have looked at 1500 clones, and have identified 325 multiple contigs (i.e. groups of overlapping clones); we estimate that about 40% of the genome is now covered. We are continuing at a rate of about 100 clones per week, and expect to finish this phase of the project in 1-2 years. At that time random cloning will have become unproductive, and we shall resort to hybridization to fill the remaining gaps, of which there will be at least several hundred. If there are many sequences in C. elegans that clone poorly or not at all in E. coli, we shall then be in difficulties, (although our contigs will already be useful to genome walkers). In that event, we would hope to continue mapping by a combination of in situ hybridization (Albertson, this newsletter) and genetic markers. It is clear from the outset that the physical map will only become a reality as a communal project. In its final stages it will have to be completed opportunistically, and in any case numerous markers will be required to align it with the genetic map. We therefore invite anyone who has genetically positioned DNA that is >10 Kb in length and available for distribution to collaborate with us, by sending a sample for fingerprinting and comparison with the database. In return, we can send you any flanking cosmids that we find, as well as any others that you need.