Bloniarz, A. et al. (2011) International Worm Meeting "Circuits for Copulation in the C. elegans Male Tail."

Hall, D. H. H., Xu, M., Emmons, S. W., Jarrell, T., Wang, Y., Bloniarz, A.

[International Worm Meeting, 2011]

Reconstruction of the posterior connectome of a C. elegans adult male is now essentially complete. The current dataset is denoted Release 2.2, which should be used for analysis. The total dataset includes processes of 171 neurons and 64 muscles joined by over 4000 chemical and 4000 electrical synapses. One hundred thirty-seven of the 171 neurons lie on short synaptic pathways between sensory input and endorgan (muscles and gonad) output. These neurons presumably control male mating behavior. Five neurons present in both sexes and 3 male-specific neurons (EF[1-3]) receive extensive input from male-specific neurons in the tail but have little output in the tail. They send processes through the ventral nerve cord into the nerve ring where their output may communicate circuit activity in the tail to the head. The remaining 26 neurons in the connectome are present in both sexes with similar connectivity and little or no interaction with male-specific circuits. The 137 neurons of the mating circuits form a highly interconnected neural network. The chemical network has a single giant component. It is a so-called small world network with short average minimum path length of 3.3 and a high clustering coefficient of 0.291 (probability that two neurons connected to a third are connected to each other). A corresponding random graph has a similar average minimum path length but a much smaller clustering coefficient (0.074, P = 0.005). Forty-five percent of the input to muscles comes directly in monosynaptic pathways from sensory neurons. The network is strongly recurrent, with 43% of the output of sensory neurons directed onto other sensory neurons, and 56% of interneuron output directed onto other interneurons. The high degree of cross-connectivity presents a challenge to identification of functional pathways controlling the separate sub-behaviors of mating. To identify such pathways, we employed published mathematical algorithms for optimal partitioning of networks into communities or modules, groups of nodes more highly connected to each other than to nodes in other communities. We found it is possible to partition the male mating network into 5 modules that seem to have biological significance. These modules appear to govern respectively the search for the vulva (backwards locomotion with unique posture), behavior when the vulva is detected (spicule prodding, spicule insertion, and ejaculation), locomotion, and, for two of the modules, body posture. Sensory input is partitioned among these modules into clear receptive fields. These findings provide a basis for reverse engineering the network through analyzing the effects of perturbations of various kinds. (The first three authors made equal contributions.).

Bloniarz A E et al. (2010) Biology of the C. elegans Male, Madison, WI "Numerical methods for analyzing neural connectivity in the C. elegans nervous system"

Bloniarz A E, Emmons S W

[Biology of the C. elegans Male, Madison, WI, 2010]

The connectome of the posterior nervous system of the C. elegans male consists of a complex network of interacting nerve cells. We utilize concepts from the mathematical field of graph theory to describe the properties of the network. The connectome is modeled as a graph in which neurons and synapses are represented by vertices and edges, respectively, and the edge weights are determined by the total number and sizes of presynaptic densities. The pattern of connections is described by an adjacency matrix for the graph. Taken together, the network of chemical and electrical interactions form a mixed graph, consisting of both directed and undirected edges. The degree of a vertex is the number of edges incident to that vertex, representing the number of pre- or postsynaptic partners of a neuron. It has been suggested that many biological neural networks form a scale-free topology, in which the degree distribution follows a power law. Utilizing a most-likelihood estimator, we test the hypothesis that the degree distribution of the male connectome follows a power law. Also, the clustering coefficient is a property of a graph which gives the probability that two vertices are connected to each other by an edge, given that they both have an edge to a common vertex. We calculate the clustering coefficient and average shortest path length of the graph to analyze the small-world properties of the network. We identify neurons with high connectivity – so-called 'hub' neurons – which may synchronize the activity of large groups of neurons. We also calculate the betweenness centrality of neurons, which measures how often a neuron is involved in a shortest path between two other neurons. Neurons with high betweenness centrality may define common pathways of information flow that support specific substeps of male copulating behavior. We also take a more statistical approach to analyzing the male connectome. The connectivity of each neuron can be thought of as a point in high dimensional space, in which each dimension of the data point of a neuron represents the input from or output to another neuron or group of neurons. We can quantify the degree of similarity between neurons by defining a distance metric on this space. We use the cosine distance to capture correlations in the connectivity of two neurons. After grouping left/right pairs in the data points, we see that most pairs have a statistically significant degree of similarity, suggesting an overall symmetry in the wiring of the nervous system. We can also utilize a nearest-neighbor method in this distance metric to classify neuron processes which cannot be traced to a cell body. Finally, this metric is used to create a hierarchical clustering of neurons which may reveal biologically significant groupings in this high dimensional space.

Xu M et al. (2010) Biology of the C. elegans Male, Madison, WI "Digital Alignment of Electron Microscopic Images for Nervous System Reconstruction"

Xu M, Hood G, Bloniarz A, Emmons S, Wetzel A

[Biology of the C. elegans Male, Madison, WI, 2010]

It is important to have electron microscopic images aligned before nervous system reconstruction. Alignment corrects the translation, rotation, magnification and distortion that occur during E.M. acquisition. A well-aligned E.M. dataset facilitates reconstruction using our program ELEGANCE, making it easier to identify neuron profiles and trace their progress through the EM stack. Moreover, alignment smooths the neuron process through sections to get better and more accurate maps. Alignment between high power series and low power series makes it possible to create a global coordinate system for neuron reconstruction across the whole worm. Alignment software created at the Pittsburgh Supercomputing Center is being used in our male nervous system reconstruction project. This software, which utilizes parallel computing algorithms, corrects misalignments including image warping. This program is installed on the Albert Einstein College of Medicine's Rock version computer cluster server with 65 nodes and 520 processors. We are currently using it to montage and align our new EM dataset of the male head, which when completed will consist of tens of thousands electron micrographs necessary to reconstruct the male anterior nervous system across 5000 serial sections. The software can also be applied to our already completed reconstruction of the male posterior nervous system to improve the accuracy of neuron maps.

Emmons S W et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Neural Circuits for Mating in the C. elegans Male Posterior Nervous System"

Wang Y, Jarrell T, Xu M, Bloniarz A, Emmons S W, Hall D H

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

The neurons in the C. elegans male tail form a synaptic network comparable in size to that found in the anterior nerve ring. The complexity of the structure makes reverse engineering of its function a challenge. Neural circuits situated mostly in the pre-anal ganglion receive sensory inputs from over 56 sensory neurons present in 25 different sensilla. In our reconstruction of a single adult animal, about half of the synaptic input to end organs comes directly from sensory neurons. The remainder is channeled in one or two steps through interneurons and neurons that act primarily as motor neurons. By tracing up the network from specific muscle groups or the gonad, and integrating information from experimental analysis of sensory neurons, it is possible to suggest neural pathways for specific sub-behaviors in copulation. The gonad is stimulated by post-cloacal sensilla neurons, spicule neurons, and a subset of the male-specific CP interneurons (CP4-6), presumably for ejaculation. This same set of neurons have output onto body wall muscles and diagonal muscles, suggesting muscle contraction accompanies ejaculation. Spicule Prodding appears to be triggered by direct innervation of oblique and gubernacular muscles by post-cloacal sensilla neurons. The Response step appears to involve the male-specific interneuron PVY. The Turn step may involve male-specific interneurons PVX and CP7-9 along with the motor neuron PDB, one of several examples of a sexually-dimorphic shared neuron with little synaptic activity in the hermaphrodite but extensive involvement in the male. Stopping at the vulva and ejaculation appear to involve male-specific interneuron PVZ, while the shared GABAergic interneuron DVB might be involved in terminating copulation. In a first attempt at reverse engineering, laser ablation of the large interneuron PVX resulted in no discernable effect on male behavior, illustrating the robustness of these circuits. The spectrum of postsynaptic partners in polyadic synapses (58% of chemical synapses) suggests a model in which positive feedback forces the network into one of a small number of discrete stable modes of activity, each with output associated with one of the copulatory sub-behaviors.

Bayer, Emily A et al. (2017) International Worm Meeting "Monoamines control the feeding-dependent emergence of"

Hobert, Oliver, Bayer, Emily A

[International Worm Meeting, 2017]

Sexual dimorphism is a widely acknowledged biological phenomenon, yet the mechanisms underlying specific developmental dimorphisms are largely unknown. As a result of the defined connectome available for both C. elegans males and hermaphrodites, it is clear that there are dimorphic synaptic wiring differences of neurons that are shared between male and hermaphrodites (1). Several of these dimorphisms are found in the phasmid sensory neurons, whose chemical synapses onto the command interneurons are sexually dimorphic, suggesting that they have sex-specific functions. Using GRASP transsynaptic labeling technology we have previously shown that these sexually dimorphic synaptic connections arise from a mixed juvenile state, with many eventually dimorphic adult connections present in both sexes initially and then restricted by sex-specific synaptic pruning (2). I have found that this sex-specific synaptic pruning is compromised in males that have been subjected to periods of starvation during the larval stages, whereas starved hermaphrodites are apparently unaffected. Correspondingly, males that have undergone periods of larval starvation retain their ability to respond to aversive cue, an ability normally lost due to synaptic pruning. I found that this starvation effect on synaptic pruning is mimicked by exogenously added octopamine in the presence of food, and completely rescued by exogeously added serotonin during starvation. The SSRI fluoxetine (Prozac) can also function to modulate the effects of starvation on male synaptic pruning. I will describe an octopamine receptor and a serotonin receptor that are required for this process. Taken together, our results show that experience can impact neuronal patterning in a sex-specific manner. 1. Jarrell TA, Wang Y, Bloniarz AE, Brittin CA, Xu M, Thomson JN, Albertson DG, Hall DH, Emmons SW. 2012. Science 337, 437-444. 2. Oren-Suissa M, Bayer EA, Hobert O. 2016. Nature, 533, 206-211.

Jarrell, T. et al. (2011) International Worm Meeting "Progress Towards a Reconstruction of a C. elegans Male Anterior Nervous System."

Bloniarz, A., Wang, Y., Jarrell, T., Xu, M., Emmons, S. W., Nguyen, K., Hall, D. H. H., Brittin, C.

[International Worm Meeting, 2011]

Having completed the connectivity of the posterior nervous system of a C. elegans male, we are now pursuing reconstruction of the male head. The anterior nervous system of the male contains about 200 neurons common to both sexes and only 4 male-specific CEM neurons as well as the processes of three EF neurons from the tail. This is in stark contrast with the posterior nervous system, which contains 85 male-specific neurons, 55 common neurons whose cell bodies are in the posterior and the processes of 18 common neurons running into the tail from the anterior. Our present connectivity results from the tail demonstrate that some common neurons have sexually dimorphic wiring. Therefore, we anticipate that common neurons in the head may also display different wiring in the male compared to the hermaphrodite. Male-specific EF neuron processes which run into the head will establish further differences. In addition to copulation, male behavior differs from that of the hermaphrodite in several general ways, such as locomotion, chemotaxis, and attraction to food and mates. Coordination of mating behavior with non-mating behavior likely is embedded in the nervous system wiring of the head. In order to compare male connectivity in the head to that of the hermaphrodite, and to complete the male connectome, a reconstruction of the anterior nervous system is necessary. Using traditional fixation, sectioning and staining methods, over 5,000 serial thin sections were obtained from a healthy male and then imaged using two TEMs. A Philips CM10 was used to image the ventral and dorsal nerve cords, and a Philips Tecnai 20 was used to generate montages of over 100 images necessary to cover each section in the nerve ring. Over 110,000 images in all were collected during a 17 month period. Images were digitally aligned using software designed by G. Hood and A. Wetzel at the Pittsburgh Supercomputing Center (see abstract). Aligned images were then entered into the software platform Elegance where reconstruction is under way. A full reconstruction is expected to take six to eight weeks. In addition, work has begun on an Elegance-based reconstruction of the hermaphrodite nerve ring from the Cambridge electron micrographs used for Mind of the Worm. Having the connectivity data in the same format will be necessary in order to make a rigorous comparison of male and hermaphrodite wiring, including relative synaptic strengths.

Wang Y et al. (2010) Biology of the C. elegans Male, Madison, WI "Progress Towards A Reconstruction Of The C. elegans Male Anterior Nervous System"

Bloniarz A, Xu M, Wang Y, Hall D, Emmons S, Nguyen K

[Biology of the C. elegans Male, Madison, WI, 2010]

Having completed the connectivity of the posterior nervous system of the C. elegans male, we are now pursuing reconstruction of the C. elegans male head. Unlike the posterior nervous system, which contains 85 male-specific neurons, 55 common neurons whose cell bodies are in the posterior and 18 processes of common neurons running into the tail from the anterior, the head is occupied by 200 common neurons and four male-specific neurons as well as 4 processes of male-specific neurons running through from the posterior to the anterior. Our present connectivity results demonstrated that some common neurons have sexually dimorphic wiring. They establish input and output synapses with male-specific neurons in the tail and also may be connected differently to each other. Therefore, we suppose that common neurons in the head may also display different wiring in the male compared to the hermaphrodite. Male-specific neuron processes which run into the head will establish further differences. In addition to copulation, male behavior differs from that of the hermaphrodite in several general ways, such as locomotion, chemotaxis, and attraction to food and mates. To compare male connectivity in the head to that of the hermaphrodite, we have begun to collect EM images of the male head. We chose males which were capable of mating and used traditional methods to fix and section them. We have 5000 serial sections of three animals' heads. We are collecting EM data from two available electron microscopes in Albert Einstein College of Medicine. Digital images are computationally aligned (see abstract by Xu et al) and neurons are traced using our reconstruction platform Elegance. If everything is on schedule, we can reconstruct the C. elegans male head within this year.

Bayer, Emily et al. (2015) International Worm Meeting "Investigating the genetic basis of neuronal dimorphisms in C. elegans."

Oren-Suissa, Meital, Hobert, Oliver, Bayer, Emily

[International Worm Meeting, 2015]

Sexual dimorphism is a widely acknowledged biological phenomenon, yet the mechanisms underlying specific developmental dimorphisms are largely unknown. As a result of the defined connectome available for both C. elegans males and hermaphrodites, it is clear that there are dimorphic wiring differences in shared neurons between the adult animals of the two sexes (1). Several of these dimorphisms are found in the phasmid sensory neurons, whose synapses onto the command interneurons are sexually dimorphic, suggesting that they have sex-specific functions. I am confirming the predicted sex-specific functions of the phasmid neurons using behavioral assays. To study how these dimorphic patterns of connectivity (and function) are established, I am using transsynaptic labeling (GRASP technology; 2) to visualize both male-specific and hermaphrodite-specific synaptic connections of phasmid neurons. Sexual determination is regulated across many invertebrate and vertebrate species by the highly-conserved doublesex/mab (DM) domain genes. In C. elegans, the DM domain class contains 11 paralogs, the majority of which have no known function. I analyzed expression patterns for 8 of the dmd genes in both larval and adult stages in the two sexes to identify dimorphisms, and identified dmd-4 as a candidate for dimorphic regulation in the PHA and PHB phasmid neurons. dmd-4 is an embryonic lethal gene, so I am generating a conditional knockout allele and perform mosaic rescue analysis to determine if dmd-4 is part of the genetic regulatory system for dimorphism in the phasmid neurons, and if this function is cell-autonomous. Ultimately, we will seek to elucidate the genetic regulatory logic of dimorphisms in shared neurons.1. Jarrell TA, Wang Y, Bloniarz AE, Brittin CA, Xu M, Thomson JN, Albertson DG, Hall DH, Emmons SW. 2012. Science 337, 437-444.2. Feinberg EH, VanHoven MK, Bendesky A, Wang G, Fetter RD, Shen K, Bargmann CI. 2008. Neuron 57(3), 353-363.

Bayer, E.A. et al. (2019) International Worm Meeting "A highly conserved protein domain directs sex- and neuron type-specific protein degradation of a Doublesex-type transcription factor."

Hobert, O, Bayer, E.A.

[International Worm Meeting, 2019]

Differences between female and male brains have been observed in species across the animal kingdom. In C. elegans, the defined connectomes for both male and hermaphrodite animals give neuron-specific resolution of highly sexually dimorphic synaptic connectivity (1), but the genetic factors underlying this dimorphic connectivity are largely unknown. Genes of the Doublesex/mab domain (dmd) family exist in all animal phyla and have been broadly implicated downstream of sex determination pathways. One of the few highly conserved members of this family in C. elegans, dmd-4, is expressed non-dimorphically in the sex-shared phasmid sensory neurons of juvenile animals, with expression becoming sexually dimorphic in adulthood. In adult animals, the phasmid neurons make sexually dimorphic synaptic connections onto downstream sex-shared interneurons, and we have shown that these synaptic connections facilitate sexually dimorphic behavioral outputs (2). However, the genes underlying these cellular dimorphisms have not been described. Although there is evidence that dimorphic expression of other dmd genes in C. elegans is established transcriptionally by the tra-1/Gli transcription factor (3), we found that dmd-4 is transcribed in both sexes and dimorphic adult expression is established post-translationally via a highly conserved domain, the DMA domain, which we find binds ubiquitin both in C. elegans and in humans. When fused to GFP alone and expressed throughout the entire nervous system, the DMA domain is sufficient to confer protein degradation with temporal, but with no spatial or sexual specificity. Our findings suggest the existence of a novel regulatory mechanism acting in the phasmid neurons to establish sexually dimorphic connectivity and behavior. We have generated a tissue-specific knockout allele to pursue this potential role of dmd-4 in establishing neuron identity, sexually dimorphic synaptic connectivity, and downstream behavioral output. 1. Jarrell TA, Wang Y, Bloniarz AE, Brittin CA, Xu M, Thomson JN, Albertson DG, Hall DH, Emmons SW. 2012. Science 337, 437-444. 2. Oren-Suissa M, Bayer EA, Hobert O. 2016. Nature 533, 206-211. 3. Mason DA, Rabinowitz JS, Portman DS. 2008. Development 135, 2373-2382

Quartey MO et al. (2021) Sci Rep "The A(1-38) peptide is a negative regulator of the A(1-42) peptide implicated in Alzheimer disease progression."

Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO

[Sci Rep, 2021]

The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).