Emmons, Scott W. et al. (2013) International Worm Meeting "Molecular regulators of male sex-drive."

Emmons, Scott W., Barrios, Arantza

[International Worm Meeting, 2013]

Well-fed, mate-deprived adult males make the risky choice to leave a plentiful source of food to explore their environment in search of mates. In contrast, recent experience with a mate while exposed to food produces a durable behavioral switch that restricts exploration within the limits of the food source. From a forward genetic screen for males that do not leave food (leaving assay defective -las), we isolated several mutants. We have recently shown that las-1(bx142) encodes a secretin-like G protein-coupled receptor for the neuropeptide pigment dispersing factor (pdf-1). Male exploratory behavior results from the balance of two physiological needs -feeding and reproduction- that compete for the control of a distributed network for navigation. The phenotype of pdfr-1 males reflects an imbalance in the relative contribution of the circuits that control exploration. pdfr-1 is required in gender-shared sensory neurons PHA, PQR and URY to generate the state of arousal to leave food in search of mates. Thus, pdfr-1 modulates a circuit that senses the internal environment of the animal and antagonizes the food-sensing circuit (Barrios et al., 2012, DOI 10.1038/nn.3253). We are currently identifying the molecular lesion responsible for the phenotype of las-2(bx143). We have mapped bx143 to the distal left arm of chromosome I and through whole genome sequencing we have identified missense mutations in two candidate genes. Rescue experiments and complementation tests with these two candidates are under way. bx143 males display normal locomotion on food but are strongly Las and mate response defective with no apparent morphological defects. These phenotypes suggest a role for bx143 in the regulation of the neural circuits that convey male sex drive.

Emmons, Scott W. et al. (2009) International Worm Meeting "The posterior connectome of the C. elegans male."

Albertson, Donna G., Wang, Yi, Hall, David H., Xu, Meng, Thomson, Nicole, Emmons, Scott W., Jarrell, Travis

[International Worm Meeting, 2009]

For many biological systems knowledge of structure is key to understanding function. It was Sydney Brenner''s insight that the structure of the C. elegans nervous system could be determined and analyzed by means of genetics that provided the inspiration for C. elegans research (Brenner, 1974). For over 20 years, the completed wiring diagram of the C. elegans hermaphrodite has afforded a unique basis for genetic studies of worm behavior. Among C. elegans behaviors, the most complex motor program is the multi-step mating behavior of the male. Reconstruction of the male nervous system was initiated along with that of the hermaphrodite in the 1970''s (Sulston, Albertson and Thomson, 1980), but its completion has awaited development of the modern PC. Using electron micrographs from the MRC we digitized and analyzed using a software platform for annotation of electron micrographs from the computer screen, we have determined the connectivity among the neurons and muscles in the male tail, the posterior connectome. Reconstruction of the anterior nervous system is underway. The male posterior connectome consists of the interconnections among the processes of 175 neurons (85 male-specific and 90 shared with the hermaphrodite) and 65 muscles (41 male-specific and 24 shared). These cells are joined together in a complex network by some 8000 synapses, 4000 chemical and 4000 electrical, more than are contained in the entire hermaphrodite nervous system. The networks of chemical and electrical synapses are largely overlapping, suggesting parallel routes of information transfer and processing. Male-specific and shared neurons and muscles are fully integrated together in the network. Many of the shared neurons are sexually dimorphic, not only in having a more branching architecture and having synapses with male-specific neurons and muscles, but also in lacking some and gaining other synaptic interactions amongst themselves. In spite of its complex network architecture, potential circuits for the various steps of the mating program can be discerned in the connectivity diagram, in some cases revealing previously unsuspected roles for individual neurons or classes of neurons. The results provide an unprecedented opportunity not only to understand how a decision-making, multifunctional neural network processes sensory information in a coherent manner, selecting a choice among alternate behavioral outputs in a goal-oriented behavior, but also an opportunity and a challenge to understand how this incredibly complex structure, the connectome, is specified by the genome.

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.

Kim, Byunghyuk et al. (2015) International Worm Meeting "Gene expression during C. elegans male sexual maturation."

Emmons, Scott W., Kim, Byunghyuk, Suo, Bangxia

[International Worm Meeting, 2015]

Most animals show sexual dimorphism in their body structures as well as behaviors. When compared to the hermaphrodite, the C. elegans male has a distinct gonad system, 40 male-specific muscles, and 85 additional neurons, all of which contribute to the copulatory structures and behavior. The morphological differentiation of the male, including the addition of the male-specific cells and the formation of a large number of synapses, arises mostly at L3 and later stages. To identify genes that are employed during male development, we constructed a time series of the whole-animal transcriptome ranging from L3 to young adult stages for the two sexes. Differential expression analysis of the ten time points (5 male, 5 hermaphrodite) revealed 1,751 genes differentially expressed in the male (>4-fold higher), whereas only 68 genes were upregulated in the hermaphrodite. As expected, the male-enriched set included many genes for known sperm proteins and several transcription factors critical for male development (e.g. egl-5, mab-3, mab-23 and dmd-3). Strikingly, 78% of the male-enriched genes (n=1,366) have no gene name and usually no listed mutant phenotype, probably reflecting the fact that males and male behavior are typically not examined in global RNAi screens. Unbiased gene correlation analysis partitions the 21,143 genes with significant expression into multiple modules, several readily correlated respectively with oogenesis/germline (5,747), ribosomal proteins (1,016), cuticle/hypodermis (1,421), semen (1,148), sperm (2,319), and the nervous system (3,628). Each module contains many uncharacterized genes, suggesting this is a rich source for gene discovery. In preliminary validation experiments, we showed that the most strongly male-expressed gene, F59B2.12, is a component of semen. The large class of clec genes (C-type lectin-like domain genes) partitions between sperm (34), semen (64) and the nervous system (24). Two tested genes selected respectively from the semen and nervous system subsets validated these assignments. The protein product of the strongly male-expressed gene ins-31 is in semen. We have shown this protein is not responsible for the shortened lifespans of mated hermaphrodites (Shi and Murphy, 2014) and are testing the idea that its function is to stimulate release of sperm-attracting prostaglandins by oocytes (Edmonds et al., 2010). By correlation with known synaptic proteins, we identified 47 genes (29 with human orthologs) that are candidates for previously unrecognized synaptic components.

K Omata et al. (1999) International C. elegans Meeting "Characterization of the neural circuit of C. elegans: model analysis of the touch response"

F Yonezawa, K Omata, K Kawamura

[International C. elegans Meeting, 1999]

In order to characterize the neural circuit of C. elagans, we construct a simple model by making use of the data table completed recently by Oshio et al . [1]. We assume that the signal of a neuron is calculated by the product of the signals from the neighboring neurons, and we investigate the touch sensitivity to continuous stimuli described by sinusoidal functions as defined in the rage from 0.0 to 1.0. We calculate the responses of the motor neurons by changing the frequencies of the stimuli. In our calculations, we change only the frequency w PLM for the input signal to the sensory neuron PLM, while the frequency for the other sensory neurons ALM, AVM and PVM is fixed to be a same value w 0 . We show that the output signals from the motor neurons A and B oscillate in time. We measure the minima of the oscillation for each w PLM value. The plot of the minima versus w PLM shows different hehaviors for the case of the neuron A and B. As for the signals from the neuron A, the values of the minima are widely distributed between 0.0 and 1.0 for all w PLM . As for the signals from the neuron B, on the other hand, the features are different for different w PLM values. (a) In the high frequency region of w PLM / w 0 0.4, the oscillation is simple harmonic and there exists only one minimum value (I min = 0.0). (b) As w PLM / w 0 is decreased, another minimum appears at a certain frequency, and the bifurcation takes place discontinuously. This behavior is different from usual continuous bifurcation observed in nonlinear systems. After a few discontinuous branching occur, signals with five periods can be seen in the intermediate frequency region of 0.3 w PLM / w 0 w PLM / w 0 [1] K. Oshio et al. ; C. elegans synaptic connectivity data'', Technical Report, CCEP, Keio Future No.1 (1998).

Kim, Byunghyuk et al. (2015) International Worm Meeting "A gene expression-analysis approach to identify the genetic code for synaptic connections in the C. elegans nervous system."

Ivashkiv, Olha, Lazaro-Pena, Maria, Suo, Bangxia, Kim, Byunghyuk, Emmons, Scott W,

[International Worm Meeting, 2015]

We are constructing an expression map of cell adhesion and recognition genes to identify genes or combinations of genes whose expression patterns correlate with nervous system connectivity. In spite of much effort, how precise patterns of synaptic connectivity are established during development remains to be determined. Our recent reconstruction of the circuits for mating in the C. elegans male tail (Jarrell et al., 2012) points up the complexity of this problem. The male mating network comprises 144 neurons and 64 muscles creating about 3,200 connected cell pairs with over 8,000 synapses. Most of the 81 male-specific neurons located in the tail are born during a short interval at the end of larval development, when, together with the remaining shared neurons, they generate the large number of synapses specific to the male, nearly doubling the size of the nervous system. How these complex and non-random connections are genetically specified in a timely manner is unknown. We are testing the hypothesis that, to direct fasciculation and synapse formation, each neuron and muscle cell expresses on its surface an abstract cell-label comprised of multiple cell-recognition or cell-adhesion proteins. We hypothesize that the probability of adhesion and synapse formation between any pair of cells will depend on the interactions between such molecular cell labels. The C. elegans genome contains over 100 genes encoding transmembrane or secreted proteins with extracellular immunoglobulin, fibronectin, cadherin and other motifs found in proteins known to be involved in the formation of synapses. So far we have examined the expression of 58 of these genes, selected in part by their enriched expression in the male during synaptogenesis (see Kim, Suo, and Emmons Abstract). Forty-three are expressed in the male neural circuits and muscles. Consistent with their function as abstract, combinatorial labels, each gene is typically expressed in multiple neurons and muscles (n=1~43), and each neuron and muscle cell type expresses multiple genes (n=1~9). While a nearly complete expression matrix may be necessary before it becomes possible to discern correlations between connectivity and combinations of genes, we have already found an interesting correlation for the single neuroligin gene nlg-1. nlg-1 is expressed in a small, select subset of neurons which are almost all postsynaptic partners of one sensory neuron pair PCA(L/R). By visualizing specific synapses of PCA in a nlg-1 mutant, we will test the roles of nlg-1 in defining specific synaptic connections in the male mating neural circuits.

Desbois, Muriel et al. (2015) International Worm Meeting "Directional trans-synaptic labeling of specific neuronal connections in live animals."

Bulow, H.nnes E., Desbois, Muriel, Cook, Steven J., Emmons, Scott W.

[International Worm Meeting, 2015]

Understanding animal behavior and development requires visualization and analysis of their synaptic connectivity, but existing methods are laborious or, may not depend on trans-synaptic interactions. Here we describe a transgenic approach for in vivo labeling of specific connections in Caenorhabditis elegans, which we term iBLINC. The method is based on BLINC (Biotin Labeling of INtercellular Contacts) and involves trans-synaptic enzymatic transfer of biotin by the Escherichia coli biotin ligase BirA onto an acceptor peptide. A BirA fusion with the presynaptic cell adhesion molecule NRX-1/neurexin is expressed presynaptically, whereas a fusion between the acceptor peptide and the postsynaptic protein NLG-1/neuroligin is expressed postsynaptically. The biotinylated acceptor peptide::NLG-1/neuroligin fusion is detected by a monomeric streptavidin::fluorescent protein fusion transgenically secreted into the extracellular space. Physical contact between neurons is not sufficient to create a fluorescent signal suggesting that synapse formation is required. The labeling approach captures the directionality of synaptic connections, and quantitative analyses of synapse patterns display excellent concordance with electron micrograph reconstructions. Experiments using photoconvertible fluorescent proteins suggest that the method can be utilized for studies of protein dynamics at the synapse. Applying this technique, we find connectivity patterns of defined connections to vary across a population of wild type animals. In aging animals, specific segments of synaptic connections are more susceptible to decline than others, consistent with dedicated mechanisms of synaptic maintenance. Taken together, we have developed an enzyme-based, trans-synaptic labeling method that allows high-resolution analyses of synaptic connectivity as well as protein dynamics at specific synapses of live animals.

Lazaro-Pena, Maria I. et al. (2011) International Worm Meeting "An approach towards identification of cell recognition molecules involved in synapse specificity in the nematode Caenorhabditis elegans."

Bloniarz, Adam, Lazaro-Pena, Maria I., Jarrell, Travis, Emmons, Scott W.

[International Worm Meeting, 2011]

How neurons recognize their correct synaptic partners remains largely an unsolved problem. Additional synapse-level connectivity from the new field of connectomics reveal the complexity of this process. Neurons in the circuits of the C. elegans male tail that govern mating are joined in a complex network by over 4000 chemical and 4000 electrical synapses. Each neuron makes synapses with many other neurons (average 15-20). We measure the strength of these interactions from the number of serial EM sections over which they occur. Every neuron interacts strongly with some of its partners and weakly with others, forming a smooth distribution from strong to weak. Fifty percent of the total synaptic load is carried by the weaker set of interactions (<20 sections). To determine whether the weaker set was specific or random synaptic noise, we ranked the neurons by the similarity of their connectivity. Ranking neurons according to their weaker connections gave the same result as ranking them by their strong interactions: left/right homologs and other sets of neurons thought to be equivalent ranked as most similar in connectivity. Therefore, at least some of the weaker connections are specific. To determine whether the strength of synaptic interaction is a consequence of the amount of cell contact, we have begun a volumetric reconstruction of the neurons in the adult male pre-anal ganglion. Preliminary results indicate an absence of correlation between the amount of cell contact and the strength of synaptic interaction, implying the presence of cell-specific as well as neighborhood-specific recognition functions. A model to explain these observations hypothesizes that there are a large number of recognition molecules in every neuron mediating synapse formation, the number that match determining the strength of connection. Involvement of a large number of recognition molecules can explain why it has been difficult to find single gene loss-of-function mutations that alter connectivity. To test this hypothesis, we will select a pair of cells that are respectively synaptic and non-synaptic neighbors of the same cell. For example B-type ray neurons strongly synapse onto EF(1-3) interneurons while the A-type ray neurons contact EF(1-3) but do not synapse with them. Using cell-type gene expression profiling, we hope to identify genes expressed in the B-type neurons but not the A-type neurons that are candidates for mediating EF targeting. Their function can be tested by expressing them in the A-type neurons and examining whether this induces synapses with EF(1-3).