Awal MR et al. (2020) Anesthesiology "Collapse of Global Neuronal States in Caenorhabditis elegans under Isoflurane Anesthesia."

Connor CW, Awal MR, Gabel CV, Wirak GS

[Anesthesiology, 2020]

BACKGROUND: A comprehensive understanding of how anesthetics facilitate a reversible collapse of system-wide neuronal function requires measurement of neuronal activity with single-cell resolution. Multineuron recording was performed in Caenorhabditis elegans to measure neuronal activity at varying depths of anesthesia. The authors hypothesized that anesthesia is characterized by dyssynchrony between neurons resulting in a collapse of organized system states. METHODS: Using light-sheet microscopy and transgenic expression of the calcium-sensitive fluorophore GCaMP6s, a majority of neurons (n = 120) in the C. elegans head were simultaneously imaged in vivo and neuronal activity was measured. Neural activity and system-wide dynamics were compared in 10 animals, progressively dosed at 0%, 4%, and 8% isoflurane. System-wide neuronal activity was analyzed using principal component analysis. RESULTS: Unanesthetized animals display distinct global neuronal states that are reflected in a high degree of correlation (R = 0.196 +/- 0.070) between neurons and low-frequency, large-amplitude neuronal dynamics. At 4% isoflurane, the average correlation between neurons is significantly diminished (R = 0.026 +/- 0.010; P < 0.0001 vs. unanesthetized) and neuron dynamics shift toward higher frequencies but with smaller dynamic range. At 8% isoflurane, interneuronal correlations indicate that neuronal activity remains uncoordinated (R = 0.053 +/- 0.029; P < 0.0001 vs. unanesthetized) with high-frequency dynamics that are even further restricted. Principal component analysis of unanesthetized neuronal activity reveals distinct structure corresponding to known behavioral states. At 4% and 8% isoflurane this structure is lost and replaced with randomized dynamics, as quantified by the percentage of total ensemble variance captured by the first three principal components. In unanesthetized worms, this captured variance is high (88.9 +/- 5.4%), reflecting a highly organized system, falling significantly at 4% and 8% isoflurane (57.9 +/- 11.2%, P < 0.0001 vs. unanesthetized, and 76.0 +/- 7.9%, P < 0.001 vs. unanesthetized, respectively) and corresponding to increased randomization and collapse of system-wide organization. CONCLUSIONS: Anesthesia with isoflurane in C. elegans corresponds to high-frequency randomization of individual neuron activity, loss of coordination between neurons, and a collapse of system-wide functional organization. : WHAT WE ALREADY KNOW ABOUT THIS TOPIC: Experimental and human electrophysiologic data suggest that the anesthetic state results from a breakdown in effective communication between neurons in the central nervous systemSystem-wide measurements of neuronal activity with single cell resolution under anesthesia have not been previously reported WHAT THIS ARTICLE TELLS US THAT IS NEW: In vivo imaging of neuronal network activity with single cell resolution in Caenorhabditis elegans reveals that although neurons display highly correlated activity in awake animals, this system-wide organization in neuronal activity is lost under isoflurane anesthesiaThese observations at the single cell level in a complex neuronal network confirm previous electrophysiologic works suggesting functional disintegration of neuronal circuitry as a mechanism of anesthetic-induced unconsciousness.

Awal, M.R. et al. (2017) International Worm Meeting "Multi-neuronal imaging to understand the effects of volatile anesthetics in C. elegans."

Florman, J., Alkema, M., Gabel, C.V., Connor, C., Awal, M.R.

[International Worm Meeting, 2017]

Volatile anesthetics produce all stages of general anesthesia, including unconsciousness, amnesia, analgesia, and muscle relaxation. In this state, the experience, memory, and physical response to pain are all lost, yet patients can be returned to consciousness, making it an essential tool in modern medicine. However, the mechanism by which neuronal systems are disrupted to cause such effects remains a mystery. Mammalian models are limited by their experimental resolution, with fMRI and EEG measurements reporting the mean activity of millions of neurons. Previous research has developed C. elegans as an effective model for volatile anesthetics demonstrating that they display the same behavioral response to increasing levels of anesthesia as humans and identifying numerous genetic mutants that alter anesthetic susceptibility. Taking advantage of C. elegans simple neuro-anatomy and its comprehensive capabilities in multi-neuron imaging, we are defining the mechanism of anesthesia on a circuit level with single neuron resolution. Employing calcium based neuronal reporters (GCaMP) we can measure activity of the command neurons within the well-defined touch circuit with and without anesthetic. In worms under higher levels of anesthesia (~8% isoflurane) that are largely quiescent, we observe a reduction in individual neuronal activity mimicking the reduced activity observed in EEG measurements from humans at a similar level of anesthetic. By contrast, in worms under moderate levels of anesthetics (~4% isoflurane) that are unresponsive to external stimuli (touch), activity of individual neurons is not abolished, but the normal correlation between neurons (i.e. coordinated activity between neurons controlling reversal movement that is anti-correlated with those controlling forward movement) is disrupted. From these observations, we hypothesize that the effects of anesthesia causing analgesia, i.e. the lack of response to external stimuli, are due to the desynchrony of neurons in distinct behavioral circuits. We are currently expanding these measurements to pan-neuronal imaging of C. elegans, in order to understand the relationship between individual neuron activity and measurement of mean activity across entire ganglia (that are akin to EEG measurements in humans). In this way we seek a comprehensive understanding of the system wide effects of volatile anesthetics.

Awal MR et al. (2018) Anesthesiology "Breakdown of Neural Function under Isoflurane Anesthesia: In Vivo, Multineuronal Imaging in Caenorhabditis elegans."

Gabel CV, Florman J, Austin D, Awal MR, Connor CW, Alkema M

[Anesthesiology, 2018]

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Previous work on the action of volatile anesthetics has focused at either the molecular level or bulk neuronal measurement such as electroencephalography or functional magnetic resonance imaging. There is a distinct gulf in resolution at the level of cellular signaling within neuronal systems. We hypothesize that anesthesia is caused by induced dyssynchrony in cellular signaling rather than suppression of individual neuron activity. METHODS: Employing confocal microscopy and Caenorhabditis elegans expressing the calcium-sensitive fluorophore GCaMP6s in specific command neurons, we measure neuronal activity noninvasively and in parallel within the behavioral circuit controlling forward and reverse crawling. We compare neuronal dynamics and coordination in a total of 31 animals under atmospheres of 0, 4, and 8% isoflurane. RESULTS: When not anesthetized, the interneurons controlling forward or reverse crawling occupy two possible states, with the activity of the "reversal" neurons AVA, AVD, AVE, and RIM strongly intercorrelated, and the "forward" neuron AVB anticorrelated. With exposure to 4% isoflurane and onset of physical quiescence, neuron activity wanders rapidly and erratically through indeterminate states. Neuron dynamics shift toward higher frequencies, and neuron pair correlations within the system are reduced. At 8% isoflurane, physical quiescence continues as neuronal signals show diminished amplitude with little correlation between neurons. Neuronal activity was further studied using statistical tools from information theory to quantify the type of disruption caused by isoflurane. Neuronal signals become noisier and more disordered, as measured by an increase in the randomness of their activity (Shannon entropy). The coordination of the system, measured by whether information exhibited in one neuron is also exhibited in other neurons (multiinformation), decreases significantly at 4% isoflurane (P = 0.00015) and 8% isoflurane (P = 0.0028). CONCLUSIONS: The onset of anesthesia corresponds with high-frequency randomization of individual neuron activity coupled with induced dyssynchrony and loss of coordination between neurons that disrupts functional signaling.

Taub, D.G. et al. (2019) International Worm Meeting "O-GlcNAc signaling enhances neuronal regeneration through modulation of cellular metabolism."

Awal, M.R., Taub, D.G., Gabel, C.V.

[International Worm Meeting, 2019]

The lack of neuronal regeneration after damage to the central nervous system remains a critical deficiency in modern medicine. While regrowth of a severed axon is inherently energetically demanding, the mechanisms of metabolic regulation in a damaged neuron remain largely unknown. This represents a critical gap in our understanding of neuronal repair that is fundamental to therapeutic strategies. We have recently discovered that alterations in O-GlcNAc signaling can shift cellular metabolism to dramatically potentiate the regenerative capacity of a damaged neuron in vivo. O-linked ?- N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serines/threonines that functions as a sensor of cellular nutrients. Performing in vivo laser axotomies in C.elegans, we find that neuronal regeneration is substantially increased by disruptions of either the O-GlcNAc Transferase or the O-GlcNAcase that decrease and increase O-GlcNAc levels, respectively. A lack of O-GlcNAc acts through the AKT-1 branch in the insulin-signaling pathway to utilize glycolysis. In contrast, increased O-GlcNAc levels activate an opposing branch of the insulin-signaling pathway whereby SGK-1 modulates the FOXO transcription factor DAF-16 to influence mitochondrial function. Exploiting the effects of O-GlcNAc signaling on metabolism and regeneration, we are determining the metabolic response within a damaged neuron, how cellular metabolism acts as limiting factor in neuronal regeneration and how it might be altered for neuro-therapeutic benefits.

Wirak, G.S. et al. (2019) International Worm Meeting "Imaging the Effects of Volatile Anesthetics on Neuronal Function in C. elegans."

Connor, C.W., Wirak, G.S., Awal, M.R., Gabel, C.V.

[International Worm Meeting, 2019]

Volatile anesthetics are invaluable tools in modern surgical practice, rendering patients immobile, unconscious, and unresponsive to noxious stimuli. While classic EEG measurements generate an averaged picture of neuronal activity under anesthesia, the effects of volatile anesthetics on the function of neuronal circuits remains unknown. Moreover, recent evidence has raised serious concerns regarding the pathological effects of anesthetic exposure on neuronal development. Caenorhabditis elegans has proven to be a powerful model system for the genetic investigation of molecular factors influencing anesthetic susceptibility. The advent of multi-neuron functional imaging in C. elegans presents an opportunity to define, for the first time, the effects of volatile anesthetics on neuronal function with cellular resolution. We have measured the effect of isoflurane anesthetic on the command interneuron circuitry controlling crawling behavior, as well as system-wide activity via pan-neuronal imaging of the majority of neurons in the animal's head. We find that exposure to isoflurane disrupts neuronal function via high-frequency randomization of individual neuron activity and associated loss of coordination between neurons within functional circuits. We conclude that onset of anesthesia corresponds to increased dyssynchrony of neuronal activity. In addition, we observe distinct changes in neuronal dynamics in adult animals following exposure to isoflurane during the L1 developmental stage. Through these efforts we are defining the anesthetic mechanism of action on neuronal function and its pathological effects on neuronal development.

Harris TW et al. (2000) J Cell Biol "Mutations in synaptojanin disrupt synaptic vesicle recycling."

Harris TW, Hartweig EA, Jorgensen EM, Horvitz HR

[J Cell Biol, 2000]

Synaptojanin is a polyphosphoinositide phosphatase that is found at synapses and binds to proteins implicated in endocytosis. For these reasons, it has been proposed that synaptojanin is involved in the recycling of synaptic vesicles. Here, we demonstrate that the unc-26 gene encodes the Caenorhabditis elegans ortholog of synaptojanin. unc-26 mutants exhibit defects in vesicle trafficking in several tissues, but most defects are found at synaptic termini. Specifically, we observed defects in the budding of synaptic vesicles from the plasma membrane, in the uncoating of vesicles after fission, in the recovery of vesicles from endosomes, and in the tethering of vesicles to the cytoskeleton. Thus, these results confirm studies of the mouse synaptojanin 1 mutants, which exhibit defects in the uncoating of synaptic vesicles (Cremona, O., G. Di Paolo, M.R. Wenk, A. Luthi, W.T. Kim, K. Takei, L. Daniell, Y. Nemoto, S.B. Shears, R.A. Flavell, D.A. McCormick, and P. De Camilli. 1999. Cell. 99:179-188), and further demonstrate that synaptojanin facilitates multiple steps of synaptic vesicle recycling.

Tsai MC et al. (2007) J Cell Biol "Microtubules are involved in anterior-posterior axis formation in C. elegans embryos."

Tsai MC, Ahringer J

[J Cell Biol, 2007]

Microtubules deliver positional signals and are required for establishing polarity in many different organisms and cell types. In Caenorhabditis elegans embryos, posterior polarity is induced by an unknown centrosome-dependent signal. Whether microtubules are involved in this signaling process has been the subject of controversy. Although early studies supported such an involvement (O''Connell, K.F., K.N. Maxwell, and J.G. White. 2000. Dev. Biol. 222:55-70; Wallenfang, M.R., and G. Seydoux. 2000. Nature. 408:89-92; Hamill, D.R., A.F. Severson, J.C. Carter, and B. Bowerman. 2002. Dev. Cell. 3:673-684), recent work involving RNA interference knockdown of tubulin led to the conclusion that centrosomes induce polarity independently of microtubules (Cowan, C.R., and A.A. Hyman. 2004. Nature. 431:92-96; Sonneville, R., and P. Gonczy. 2004. Development. 131: 3527-3543). In this study, we investigate the consequences of tubulin knockdown on polarity signaling. We find that tubulin depletion delays polarity induction relative to wild type and that polarity only occurs when a small, late-growing microtubule aster is visible at the centrosome. We also show that the process of a normal meiosis produces a microtubule-dependent polarity signal and that the relative levels of anterior and posterior PAR (partitioning defective) polarity proteins influence the response to polarity signaling. Our results support a role for microtubules in the induction of embryonic polarity in C. elegans.

Michelle Keaney et al. (2001) International C. elegans Meeting "Optimising dietary restriction-mediated lifespan extension"

Michelle Keaney, David Gems

[International C. elegans Meeting, 2001]

A number of reports have shown that dietary restriction (DR) results in increased lifespan in C. elegans, with the magnitude of increase depending upon the method used (1-4). How DR extends lifespan is unknown, but one possibility is that it down-regulates the insulin/IGF (IIS) signalling pathway which regulates ageing. Work to date is both consistent with (5) and contradictory to (4) this hypothesis. We are testing four methods used previously to exert DR: diluted monoxenic liquid culture (1), reduced nutrient NGM (2), axenic culture (3), and the use of eat mutations (4). We are determining which method can be optimised to (a) give the greatest magnitude of lifespan extension; (b) can be made quantitative (so the degree of DR imposed can be varied); and (c) is easy to perform. We are also seeking to maximise DR-mediated lifespan extension such that further reduction of dietary intake would lead to malnutrition and premature death. Our aim is to use an optimised DR protocol to understand the mode of action of DR, and its relationship to IIS, reproduction and oxidative damage. (1) Klass, M.R. Mech. Ageing Dev. 6 : 413 (1977). (2) Hosono, R. et al. Exp. Geront. 24 : 251 (1989). (3) Vanfleteren, J. R. and De Vreese, A. FASEB J. 9 : 1355 (1995). (4) Lakowski, B. and Hekimi, S. PNAS USA 95 : 13091 (1998). (5) Apfeld, J. and Kenyon, C. Nature 402 : 804 (1999).

Sanders, Tom et al. (2015) International Worm Meeting "Nonlinear sensory integration in C. elegans: a computational model."

Cohen, Netta, Ghosh, D.D., Sanders, Tom, Nitabach, M.N.

[International Worm Meeting, 2015]

Animal survival depends on a combination of often conflicting demands such as foraging and evading of dangers. To navigate effectively in such unknown and changing conditions, animals must continuously integrate over a variety of sensory cues, and adapt their decision making strategy in a context dependent manner. Here, we examine the neural control of a sensory integration task in the nematode C. elegans. The task involves an ASH-triggered aversive response to high osmolarity fructose and an AWA-triggered attractive response to diacetyl [1]. In the assay, worms are placed in the center of a ring of fructose; two drops of diacetyl are located outside the ring. We present a computational model, consisting of point worms, situated in a virtual arena that closely mimics this experimental assay, and endowed with a sensory motor pathway of two sensory neurons, a neural integration pathway and two motor programs (pirouettes and steering). A monoamine (PDF-2 and tyramine) modulation circuit involving RIM and ASH is overlaid on the synaptic circuit, in line with molecular data [1]. Model parameters were constrained by behavioral data for wild type and mutant animals for a range of stimulus concentrations. Based on our simulation results, we reject a null hypothesis of a linear sensory integration mechanism in RIM and present results that are consistent with the data for a sensory "coincidence detector" like process in RIM.[1] Ghosh, D.D., Sanders, T., Hong, S., Chase, D.L., Cohen, N., Koelle, M.R., and Nitabach, M.N. "Neuroendocrine reinforcement of a dynamic multisensory decision." International C. elegans meeting.

Masumi Shimada et al. (2003) International Worm Meeting "Possible roles of MOE-1/OMA-1 CCCH-type zinc-finger family proteins in C. elegans oogenesis and embryogenesis"

Hiroyuki Kawahara, Masumi Shimada

[International Worm Meeting, 2003]

Oocyte maturation is an important prerequisite for the production of progeny. Although several germ-line mutations have been reported, the precise mechanism by which the last step of oocyte maturation is controlled remains unclear. In Caenorhabditis elegans, CCCH-type zinc-finger proteins have been shown to be involved in germ cell formation, although their involvement in oocyte maturation had not been fully investigated. Using a multiple RNAi technique, we have identified three redundant CCCH-type zinc-finger genes, named by us moe-1, -2 and moe-3, as a group related by functions and nucleotide sequence1. Similar results were reported by Detwiler et al.(2001), and they gave the names MOE-1 and MOE-2 to OMA-1 and OMA-2, respectively2. We and Detwiler et al. have found that they have overlapping functions that are crucial for oocyte maturation; i.e. simultaneous removal of these proteins by RNAi induces arrest and expansion of growing oocytes. The results of our in situ hybridization have revealed that each of the moe/oma transcripts is expressed from the distal to proximal region of the gonad, while their corresponding proteins accumulate specifically in the cytoplasm of growing oocytes as well as on P granules. Thus, these gene products participate in processes in the final step of the meiotic cell cycle control, a novel function for CCCH-type zinc-finger family proteins thus far discovered. Although MOE-2/OMA-2 protein is rapidly removed from P granules after fertilization, we found that MOE-2/OMA-2 associated with the centrosome-peripheral structure in dividing blastomeres. Our results suggest that moe/oma gene products are unique multifunctional proteins in terms of their redundancy and characteristic behavior during the course of oocyte maturation and subsequent embryogenesis. 1 Shimada, M., Kawahara, H., and Doi, H. (2002). Genes Cells 7, 933-947. 2 Detwiler, M.R., Reuben, M., Li, X., Rogers, E., and Lin, R. (2001). Dev. Cell 1, 187-199.