Cook, Steven J. et al. (2021) MicroPubl Biol

Hobert, Oliver, Vidal, Berta, Cook, Steven J.

[MicroPubl Biol, 2021]

Single neuron-specific drivers are important tools for visualizing neuron anatomy, manipulating neuron activity and gene rescue experiments. We report here that genomic regions upstream of the C. elegans bHLH-PAS gene hlh-34 can be used to drive gene expression exclusively in the AVH interneuron pair and not, as previously reported, the AVJ interneuron pair.

Cook, Steven et al. (2012) Worm Breeder's Gazette "WormWiring: A new online resource for nematode connectivity"

Hall, David, Brittin, Christopher, Cook, Steven, Emmons, Scott

[Worm Breeder's Gazette, 2012]

Cook, Steven et al. (2019) International Worm Meeting "Visualizing structure and development of the C. elegans pharyngeal nervous system."

Hobert, Oliver, Cook, Steven

[International Worm Meeting, 2019]

Despite the central importance of neural circuit development to brain function and behavior, we lack the genetic information required to assemble a complete circuit. The C. elegans pharynx is attractive model for such studies given its modest size (20 neurons, 14 neuronal classes) and isolation from the somatic nervous system (only connection is through RIP). A recent re-analysis of the adult pharyngeal nervous system (wormwiring.org) revealed many new connections in the pharynx, suggesting a more complex synaptic network than previously published (Albertson and Thomson 1976). We are actively pursuing two goals to provide dynamic information of pharyngeal wiring in live animals: 1) confirm novel patterns of synaptic connectivity and 2) dissection of how the pharyngeal nervous system forms during embryogenesis. Using presynaptic fluorescent markers (e.g. CLA-1, RAB-3), we have quantified variability of previously known synapses (e.g. NSM -> M3), as well as confirmed a lack of distinction between axo-dendritic compartments in the pharynx by visualizing synaptic outputs of the I1 and I2 neurons. To visualize connectivity involving pre- and postsynaptic partners, we are also using GFP Reconstitution Across Synaptic Partners (GRASP) labeling for individual and combinations of synapses. We have modified to provide more accurate and reliable labeling of synapses through modification of fluorescent protein, domain deletion, and 3' UTR usage. In addition to new synaptic labeling techniques, we have codon-optimized fluorescent proteins into vectors for strong membrane-localized expression during embryogenesis. Our approach is suitable for simultaneous imaging of up to six fluorescent proteins, allowing us to barcode expression in individual pharyngeal neurons. Together, our approaches and results will be useful in the broad context of understanding the genetic logic of circuit formation.

Cook, Steven J. et al. (2017) International Worm Meeting "Investigating the structure and sexual dimorphisms of the C. elegans connectome."

Nguyen, Ken C.Q., Cook, Steven J., Bayer, Emily A., Hobert, Oliver, Emmons, Scott W., Yakovlev, Maksim, Jarrell, Travis A., Wang, Yi, Hall, David H., Brittin, Christopher A., Tang, Leo T.-H., Bulow, Hannes E.

[International Worm Meeting, 2017]

We present the first whole-animal maps of synaptic connectivity, including anatomical connection strength, of both adult sexes of a species. Our results are based on analyses of legacy and new serial section electron micrographs (EMs) of the C. elegans nervous system and the tissues it innervates. In a graph representation of connectivity, the pathways of information flow can be arranged hierarchically, revealing a largely feed forward structure of shallow (1-5 synapses) depth. Our reconstruction has revealed that muscles and other end-organs are more extensively cross-connected than previously reported. Sensory information converges and diverges widely throughout the fully-connected neural network. The sexes differ not only by the addition of sex-specific neurons and muscles, but also at numerous points in the connectivity of shared neurons. Differences between the hermaphrodite and male reconstructions could be either inter-individual differences or differences due to genetic sex. To distinguish between these possibilities, we examined a subset of 7 synaptic connections that were respectively stronger in the male reconstruction, 4 that were stronger in the hermaphrodite reconstruction, and 4 that were similar, using in vivo trans-synaptic labeling. In each instance, the difference seen in the reconstructions was confirmed in multiple animals. Extrapolating these results to the number of statistically significant differences in the reconstructions, we conclude that there is an unexpectedly large number of sexually dimorphic connections. These connections were mainly located in the nerve ring, and embedded within the connectome at least one synapse away from any sex-specific neuron. Our results showed that AVA receives sex-specific input from ADL, ASH, and AVF in the hermaphrodite, while RIB receives sex-specific input from IL1, IL2, and RIA in the male. AIM, which has been reported to change its neurotransmitter from glutamate in the hermaphrodite to acetylcholine in the male, makes a strong male-specific connection to AIB. These hubs of sex-specific connectivity also maintained the majority of their sex-shared output. Our results suggest that the genetic sex of the nervous system allows for diverse synaptic patterns in a relatively small nervous system.

Cook, Steven et al. (2019) International Worm Meeting "Olfactory circuit structure is reconfigured between divergent nematode species."

Carstensen, Heather, Siebriebriennikov, Bogdan, Bumbarger, Dan, Riebesell, Metta, Cochella, Luisa, Sommer, Ralf, Cook, Steven, Hobert, Oliver, Moreno, Eduardo, Hong, Ray, Castrejon, Jessica, Sarpolaki, Tahmineh

[International Worm Meeting, 2019]

The ability of an organism to process sensory information is dependent upon the structure and connectivity of its neurons. The nematodes P. pacificus and C. elegans have a repertoire of different behaviors and sensory preferences, yet the neural substrate for these differences is unknown. To understand structural differences of the pharyngeal nervous system related to predatory feeding behavior, serial section EM series were generated of P. pacificus. Through a volumetric reconstruction and subsequent analysis, it was shown that an identical set of pharyngeal neurons with similar morphology generated many different synaptic patterns (Bumbarger et al 2013). Toward the goal of better understanding differences in olfactory behavior between P. pacificus and C. elegans we further analyzed these EMs and identified homologous amphid sensory and interneurons between P. pacificus and C. elegans based upon cell body position, dendritic morphology, dye staining, and reporter gene expression (see abstract by R.L. Hong et al). We determined the set of chemical and gap junction connections for the amphid circuit and compared the resultant network to C. elegans. We observed that neurons with the most similar morphology (AFD and AUA) have the most similar connectivity across species, while neurons with different dendritic structure (AWA, AWB, AWC) show a greater number of species-specific connections. Overall, we found that 60% of strong chemical connections and 40% of gap junctions are conserved across species. We next asked whether differences in connectivity are due to changes in neuronal neighborhoods, or synaptic partner choice. We found that in each instance of a discrepant synapse, the pair of neurons in questions are adjacent in both species. We are actively extending our analysis to determine whether sensory neurons are more divergent in their structure and connectivity compared to downstream inter- and motorneurons. Our results suggest evolutionary pressure has kept the overall nervous system plan of nematodes very similar, but has allowed for different synaptic configurations to permit species-specific behavioral differences.

Brittin, Christopher et al. (2015) International Worm Meeting "Spatial aggregation of neurons contributes to network structure in the nematode C. elegans."

Cook, Steven, Cohen, Netta, Brittin, Christopher

[International Worm Meeting, 2015]

Previous studies of real-world networks have suggested that networks motifs, defined as connectivity patterns that are significantly over-represented when compared to random networks with the same degree distribution, may arise due to evolutionary design principles and serve as computational units. For neural networks and other spatially embedded systems, where network connections form between physically proximate nodes, this approach carries a risk of overstating the statistical significance of connectivity patterns. Previous studies have attributed a number of network motifs to the C. elegans neuronal network and suggested that the worm's nervous system may be constructed from these computational modules (Milo et al., 2002; Reigl et al. 2004). However, other groups have conjectured that the high frequency of observed connectivity patterns may simply be a consequence of the organization and localized connectivity of the neuropile (White et al., 1983; Artzy-Randrup et al. 2004). To test these two hypotheses, we measured the spatial aggregation of neurons in the nematode C. elegans and used the data to construct a statistical model with a spatially constrained null-hypothesis. We found that a number of motifs, including the 3-node feed forward loop, are no longer over-represented in our spatially constrained model. Thus, the observed network structure in the C. elegans nervous system may simply be the consequence of how the neuropile is organized.

Lee, Garrett et al. (2021) International Worm Meeting "Understanding the development, plasticity, and function of synaptic asymmetry in C. elegans"

Emmons, Scott, Buelow, Hannes, Cook, Steven, Lee, Garrett, Tang, Leo

[International Worm Meeting, 2021]

Lateralization of the nervous system is conserved across the animal kingdom from invertebrates to humans. The human brain asymmetrically processes faculties across the left and right hemispheres, and impaired functional lateralization has been implicated in depression, schizophrenia, and autism spectrum disorders. However, whether asymmetric connectivity exists in anatomically symmetric structures and what its function may be remains largely unknown. Serial electron micrograph (EM) reconstruction of the C. elegans connectome revealed that the chemosensory ASE neurons exhibit left-right asymmetry in their connection to the odorsensory AWC neurons. While morphologically symmetric, ASEL and ASER exhibit differential gene expression and function. ASEL primarily mediates attraction to increases in Na+ concentration, while ASER triggers avoidance of decreases in Cl- concentration. To confirm the EM data as well as study the connection's development, maintenance, plasticity, and function, we created a fluorescent reporter of this connection using in vivo Biotin Labeling of Intercellular Contacts (iBLINC). iBLINC analysis confirmed that ASEL tends to form more synaptic contacts onto AWC than ASER. Analysis of connectivity over development revealed that the asymmetry is established by L3 and maintained through adulthood. Genetic conversion of both ASE neurons to an ASEL fate reversed the asymmetry. In contrast, neither changing both ASE neurons to the ASER fate nor symmetrizing the postsynaptic AWC neurons displayed altered synaptic lateralization from wildtype. These findings suggest that pre-synaptic identity contributes to establishment of the asymmetric ASE/AWC connection. Interestingly, the left-right lateralization is reversed when C. elegans are exposed to increased NaCl environments. Synaptic number changes progressively over the course of twelve hours and in a dose-dependent manner, suggesting that the lateral connectivity is plastic. Finally, we have identified several genes that are required to establish or maintain the ASE/AWC synaptic asymmetry. Future experiments will characterize the functional significance of left-right asymmetry using Ca2+ imaging. In analyzing this connection, we aim to understand fundamental aspects concerning the formation and function of asymmetric connectivity.

Tang, Leo et al. (2021) International Worm Meeting "Insulin-like signaling regulates left/right asymmetric synaptic connection"

Emmons, Scott, Cook, Steven, Lee, Garrett, Tang, Leo, Buelow, Hannes

[International Worm Meeting, 2021]

Lateral specialization of the central nervous system is a well-established feature across species, yet the underlying mechanism through which functional asymmetry arises is largely unknown. EM reconstruction of the C. elegans connectome found that ASE to AWC synapses were stronger on the left than the right. To corroborate and elucidate this asymmetric connection, we generated a reporter strain labelling ASE->AWC connections using in vivo Biotin Labelling of Intercellular Contact (iBLINC). While we observed the same left-sided bias of this connection, we also discovered that the asymmetric fates of the ASEs are a necessary but not sufficient factor in establishing this left-side bias. Furthermore, we found that ins-6/insulin-like is involved in the establishment of this asymmetric connection. Using a fosmid-based ins-6::GFP reporter, we observed that ins-6 expression in ASJ also exhibits left-sided bias. The asymmetry of ASE to AWC iBLINC signal is abolished in cell-specific knock-out animals of ins-6 in ASJ but not in ASI. Moreover, genetically removing ins-6 in ASJL by use of tbx-37p::Cre reversed the asymmetry of the ASE to AWC connection. Meanwhile, removing the putative ins-6 receptor daf-2 in ASEL but not ASER symmetrized the ASE to AWC connection. Finally, we observed that mutation on an antagonistic insulin, ins-22, partially suppressed the phenotype of ins-6 mutants. These results taken together suggest that the left-side bias of ASE to AWC connection is controlled by insulin signaling, where asymmetrically expressed insulin-like molecules from ASJs act locally to regulate connectivity of the ASE>AWC synaptic connection. We aim to further investigate the effect of insulin signaling on the plasticity of ASE to AWC connection and general synapse dynamics. We also aim to characterize the previously unreported asymmetric gene expression in ASJ

Brittin, Christopher et al. (2015) International Worm Meeting "A 3D Analysis of the Mind of the Worm Connectome."

Hall, David, Emmons, Scott, Cook, Steven, Cohen, Netta, Brittin, Christopher

[International Worm Meeting, 2015]

A connectome is a comprehensive map of all neural connections in an organism's nervous system. The first connectome was published almost 30 years ago by White et al. (1986) and described the structure of the nervous system of the nematode C. elegans adult hermaphrodite. Subsequent network analyses of this data have focused only on the synaptic connectivity of the nervous system, while neglecting much of the spatial information in the data. Initial spatial analyses of the C. elegans connectome reported in (White et al., 1983; Durbin, 1987) used only a sparse sampling of physical neuron contacts. Using the original electron micrographs from (White et al., 1986), we have extended this analysis by performing a 3D reconstruction of every neuron in the C. elegans nerve ring in both the L4 and adult. This represents the first complete volumetric reconstruction of the main neuropil of any animal from multiple developmental stages. With this enriched data set, we have been able to do a comparative analysis of synaptic connectivity, characterize the spatial distribution of synapses for each neuron and analyse the relationship between neuron contact and synapse formation in the C. elegans nerve ring. Similar to (White et al., 1983), we found that ~40% of all possible physical contacts result in a synapse or gap junction. We also found a positive correlation between the frequency of synapse formation and the amount of physical contact between neurons. Specifically, the frequency of synapse formation between two neurons approaches ~0.7 as the amount of physical contact approaches 10% of a neuron's total measured surface area. However, like (Durbin, 1987), we find that synapse probability and synapse number between any pair neurons does not depend strongly on the amount of shared physical contact. Furthermore, synapse volumes appear to be conserved between the L4 and adult, while the number of synapses between any two neurons appear to be, on average, greater in the adult. This could suggest that during late nervous system development, synaptic partnerships are reinforced by creating additional small synapses between neurons rather than enlarging the volume of current synapses. .

Philbrook, Alison et al. (2015) International Worm Meeting "Investigation of spine-like synaptic structures on GABAergic DD neurons."

Cook, Steven J., Philbrook, Alison, Hall, David H., Francis, Michael M.

[International Worm Meeting, 2015]

Synapses are dynamic structures that undergo selective strengthening or weakening to refine their patterns of connectivity. Even after the nervous system has developed, dendritic spines grow and retract, and the placement of synapses can be reengineered to form new neural circuits. In order to investigate molecular mechanisms by which particular synapses are either remodeled or stabilized, we are studying C. elegans GABAergic circuits. We previously identified an ionotropic acetylcholine receptor (ACR-12) that is expressed at synapses onto GABAergic neurons and regulates their activity. In order to investigate mechanisms underlying receptor clustering and synapse dynamics, we sought to establish a system in which we could examine the subcellular localization of ACR-12 receptors in individual neurons in vivo. We generated a transgenic strain expressing GFP-tagged ACR-12 receptors in the GABA DD motor neurons and focused our efforts on a single neuron, DD1. The DD1 cell body and processes are spatially separated from the other DD neurons, enabling in vivo visualization of synapse dynamics on the single DD1 neuronal process. We find that ACR-12 clusters are restricted to the dorsal side in first larval stage (L1) animals. In contrast, punctate ACR-12-GFP fluorescence is localized within a defined spatial domain of the ventral DD1 process in adult animals. These results are consistent with previous studies suggesting that synapses onto the DD neurons undergo developmental remodeling at the end of the L1 stage. In adults, ACR-12 clusters are concentrated at the tips of spine-like dendritic protrusions and are apposed by presynaptic vesicle markers, consistent with a synaptic localization. Spine-like structures are also apparent in volumetric reconstructions of the ventral DD1 dendrite from electron micrographs. Finally, genetic manipulations that reduce cholinergic transmission decrease spine number, suggesting mechanisms for their activity-dependent regulation. Together, our findings raise the interesting possibility that these spine-like structures in DD1 represent an evolutionary precursor to mammalian dendritic spines. We are now working to investigate molecular requirements for the development and maintenance of these synapses and will present our findings.