Scott W Emmons et al. (2001) International C. elegans Meeting "The Mind of a (Male) Worm"

Scott W Emmons, David H Hall, Haftan Eckholdt

[International C. elegans Meeting, 2001]

In 1986, John White and coworkers published their reconstruction of connectivity in the nervous system of the C. elegans hermaphrodite. Since that time, only limited further reconstruction has been carried out. This has included determination of some posterior circuitry governing male mating behavior (Sulston et al., 1980) and outgrowth patterns in the developing embryonic nervous system (Norris et al., 1997 International Meeting). Complete connectivity remains unknown for the adult male and for all larval and embryonic stages. Full knowledge of connectivity is becoming increasingly important as studies of nervous system development and function advance and attempts are made to understand how the nervous system generates and controls complex behaviors. Sexual dimorphism of the nervous system is of particular interest. As part of the efforts in many areas to provide a complete description of the worm, we have undertaken to define additional circuitry within the nervous system. For nervous system reconstruction, the course of neuron processes and the chemical and electrical contacts that they make are determined by following processes through a series of electron micrographs of serial thin sections of fixed worms. In the original work, although a computerized system was developed for analyzing the electron microscopic images and compiling the data (J. White, PhD Thesis, 1974), it did not prove to be greatly superior to hand reconstructions, which were used for much of the analysis. Times have changed computerwise since the early 1970s, and we plan to return to a computer-aided approach and attempt to accelerate the reconstruction process by computerizing as many steps as possible. Serial thin sectioning and electron microscopy will continue to be carried out as before, and identification of neurite profiles and contacts in the images will also be done by visual inspection, but images will be digital and all steps subsequent to process and contact identification will be entirely in digital format. We hope that use of the computer will speed the reconstruction not only by promoting facile data storage, handling, and retrieval, but also by allowing simultaneous reconstruction and data entry that would make it possible for the computer to perform a kind of grammar checking on new data (e.g. no process can have greater than or less than one cell body) and consistency checking (e.g. with known pathways derived from previous reconstructions and GFP reporters). We plan to develop hardware and software and test it by attempting initially to reconstruct the preanal ganglion of the adult male. This ganglion contains circuitry serving to program male mating behavior that is as complex as that found in the nerve ring. The male tail contains 48 sensory neurons with axon processes targeted to this ganglion. Liu and Sternberg (1995) defined the relationship of inputs from these sensory neurons to the sequential steps of copulatory behavior. In our reconstruction, we hope to determine the targets of these sensory neurons and trace neuronal pathways to the copulatory muscles. If we are successful in making extensive additional reconstruction feasible with reasonable time and effort, our long term goal will be to trace male-specific interneurons into the nerve ring and determine how they influence central circuitry, as well as to undertake reconstruction of the L1 larva and other important stages.

Scott W Emmons et al. (2002) East Coast Worm Meeting "Reconstruction of connectivity in the C. elegans male nervous system"

David H Hall, Michael Y Zhang, Haftan Eckholdt, Scott W Emmons

[East Coast Worm Meeting, 2002]

The original reconstruction of connectivity in the C. elegans hermaphrodite nervous system was mostly carried out by marking individual neuron profiles by hand on electron micrographic prints. This effort, undertaken primarily at the MRC Laboratory of Molecular Biology in Cambridge, England, involved a staff of 6-8 people and required 15 years. While some limited analysis of the developing nervous system and of mutants has been performed since that time, no further attempts have been made to define complete connectivity, which remains largely unknown in juvenile stages and in the adult male. To obtain further reconstructions, it is necessary to speed up the process of reconstruction many-fold. To achieve this, we have undertaken to develop computer aided methodology. Embedding, sectioning, and electron microscopy will be carried out as before. Digital analysis begins by scanning electron micrographic negatives. Neuron profiles are identified by an investigator and their positions marked by a single X,Y coordinate or their profiles may be traced. 3D reconstructions are then generated from the aligned series of images. Improved reconstruction methods will be applied to the original photographic images from Cambridge, which are now housed at the Albert Einstein College of Medicine, as well as to new images. On the Cambridge prints, which include a complete set of photographs of the adult male tail (1), as well as many valuable unpublished studies of mutants, corresponding profiles in adjacent sections are already marked by colored pen. For rapid analysis of new series, it will be essential to develop computer algorithms to aid the process of identifying correspondence between sections. As an objective to test our methodology, we have chosen to reconstruct the preanal ganglion of the adult male, which contains the circuitry that processes inputs from a variety of specialized sensillae, including the rays, hook and post-cloacal sensilla (1). Nematode neuron processes are very small (0.1-0.2m diameter), similar in size to the finest terminal branches in mammalian dendrites. They are mostly unbranched, synapses being formed at en passant swellings. In the male, the preanal ganglion is a cylindrical neuropil some 20m in diameter and 60m in length. A typical transverse section contains the profiles of 100-200 neurites, which here, atypically, form a significant number of branches. Our progress in reconstructing this circuitry from the available series of several thousand electron micrographs from the MRC will be reported.

Scott W Emmons et al. (2003) International Worm Meeting "Progress towards a wiring diagram for the C. elegans male: the rays make a complex set of connections in the pre-anal ganglion"

David H Hall, Scott W Emmons, Donna G Albertson, Haftan Eckholdt, Michael Zhang

[International Worm Meeting, 2003]

A major effort was made in the late 1970s at the MRC Laboratory of Molecular Biology, Cambridge, England, to reconstruct the nervous system of the adult C. elegans male. This project has recently been resumed at the Albert Einstein College of Medicine. In the earlier work, a set of complete low-power and high-power prints were generated and partially analyzed (Sulston, J.E., Albertson, D.G., and Thomson, J.N. Dev. Biol. 78, 542-576, 1980). Not included in the earlier results were the postsynaptic targets of the rays. Thirty six ray sensory neurons with sensory endings in the nine bilateral pairs of rays provide major input signaling physical contact with the hermaphrodite during copulation. The ray neurons are of two ultrastructural types, but neurons of each type have different subtype properties in the different rays. For example, the neurotransmitters dopamine, serotonin, and multiple FMRFamides are each expressed in different subsets of the rays (see abstract by Lints and Emmons). This complexity raises the question whether each ray also has a unique set of postsynaptic targets. To examine this question, we compiled the axonal output of the rays from the existing data. Ray cell bodies are located in a bilateral pair of lumbar ganglia and extend axonal processes ventrally through circumferential commissures to the centrally-located, ventral preanal ganglion. There they branch and synapse with multiple postsynaptic target cells. Such branching is unusual for most C. elegans neurons but common for male-specific neurons in the preanal ganglion. Reconstruction of the neurons from most of the rays reveals that each ray neuron contacts from 3 to 8 postsynaptic target cells, and the pattern of targets is different for neurons from different rays. Thus the output of the rays to the circuitry generating copulatory behavior is complex. Computer software is being prepared to aid further reconstruction efforts. Our goal at this stage is to complete the analysis of the existing electron micrographs to provide a wiring diagram for the neurons and circuitry in the male tail.