How, J.J. et al. (2019) International Worm Meeting "Graph-theoretic analysis of whole-brain imaging reveals stimulus-specific changes in patterns of neural activity."

How, J.J., Navlakha, S., Chalasani, S.H.

[International Worm Meeting, 2019]

Patterned neural activity has been shown to underlie the perception of olfactory stimuli. However, most work to date has focused on early stages of sensory processing, and thus it is unclear how the rest of the nervous system interprets information transduced at the sensory periphery. Here, we used whole-brain calcium imaging of thirty immobilized Caenorhabditis elegans to study how different chemical stimuli induce unique patterns of neural activity. We exposed groups of three worms to one of ten conditions: benzaldehyde (BZ), diacetyl (DA), isoamyl alcohol (IAA), 2-nonanone (NN), or NaCl, at either high or low concentrations. We generated networks that describe the functional interactions between neurons using mutual information, and extracted 22 graph-theoretic properties that characterize the network's functional integration, segregation, and resilience. We identified a few properties that can distinguish between low and high stimulus concentrations, one between attractants (BZ, DA, IAA, and a low concentration of NaCl) and repellents (NN and a high concentration of NaCl), and a third set of properties that can accurately classify the attractants. High concentrations of chemical stimuli tend to induce a network that is more functionally segregated, while repellents induce a network with a shorter path between any two neurons. Furthermore, attractants can be grouped by three different properties - the centrality of an average neuron, how often a strongly connected neuron is connected to weakly connected neurons, and the distance between the furthest apart pair of neurons. Importantly, all of these properties have values that are different from those obtained by analyzing the structural connectivity between neurons in the head of the worm. Thus, we conclude that the structural connectome provides a rich substrate that is employed in very distinct ways to support perception and, ultimately, behavior. Many of these graph-theoretic features deal with how efficiently information is transmitted throughout the C. elegans nervous system, and may reflect the saliency of the stimulus in question.

Chalasani, Sreekanth H. et al. (2009) International Worm Meeting "AWC sensory neurons release two neurotransmitters to regulate exploratory behavior upon removal from food."

Albrecht, Dirk, Bargmann, Cornelia I., Chalasani, Sreekanth H., Kato, Saul

[International Worm Meeting, 2009]

Animals increase their pirouette frequency in response to removal from food stimulus for a period of 15 min. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY and AIA interneurons inhibit pirouettes (Wakabayashi et al 2004, Gray et al 2005). We have found that AWC sensory neurons become active in response to removal of stimulus, releasing two neurotransmitters (glutamate and a neuropeptide NLP-1). The released glutamate acts to activate AIB and inhibit AIY interneurons, promoting reversals (Chalasani et al 2007). In contrast to glutamate, AWC-released NLP-1 acts on AIA interneurons to suppress reversals, suggesting that reversal frequencies are regulated by at least two opposing signaling systems. AWC calcium responses are modulated in these neurotransmitter mutants, suggesting that feedback pathways affect AWC neuronal activity. References: Chalasani, S. H., Chronis, N., Tsunozaki, M., Gray, J. M., Ramot, D., Goodman, M. B., and Bargmann, C. I. (2007). Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63-70. Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.

Asundi, Aarati et al. (2015) International Worm Meeting "Olfactory Sensory Neuron Regulation of the Germline in Response to Environment."

L'Etoile, Noelle, Suzuki, Atsushi, Goga, Andrei, Asundi, Aarati

[International Worm Meeting, 2015]

The balance between proliferation and differentiation of stem cell populations must be responsive to changing molecular and physiological conditions. However, the mechanism behind this plasticity is not well understood. The C. elegans nematode is a tractable organism to study the influence of food sensation on physiology. Recent studies suggest that the number of proliferating germ cells (PGCs), the only stem cell population in the adult C. elegans body, respond to the environment by communicating with sensory neurons. Sensory neurons are able to relay information about food abundance to the PGCs. Specifically, TGF-b signaling from the ASI gustatory neuron promotes increased PGCs when food is abundant and reduced PGCs when food is scarce [Dalfo et al. 2012]. The olfactory sensory neurons (OSNs), AWA and AWC, are also food-sensing neurons [Chalasani et al. 2007] and thus, may also regulate the PGCs. Indirect evidence suggests that the OSNs along with ASI reduce lifespan, perhaps via the germline [Alcedo et al. 2004 and Hsin et al. 1999]. Studies also suggest that the neurons secrete signals to affect behavioral responses to odor [Chalasani et al. 2010]. We propose that the OSNs may affect the physiology of the C. elegans nematode, as assessed by the PGCs and brood size, via secreted molecules. PGC counts and brood sizes from animals with genetically dysfunctional AWA or AWC neurons are discussed in this poster.A mobile RNA intermediate is an attractive candidate to mediate the communication between the OSNs and the germline. Published data from the L'Etoile lab shows that 22G RNAs synthesized in the AWC show increased levels worm-wide upon odor exposure [Juang et al. 2013] and unpublished data indicate that this increase is dependent on the presence of SID-1 dsRNA channels. We discuss the effect of dysfunctional SID-1 channels on the PGC pool and brood size.Finally, the nuclear RNAi pathway, involving NRDE-1, 2 and 4, is able to regulate gene expression in the germline nuclei [Burton et al. 2011]. Preliminary studies have shown that siRNA knockdown of the human homolog of NRDE-2 decreases the viability of mammalian cells transformed with the oncogene Aurora B kinase, which functions in chromosomal segregation during both mitosis and meiosis. We therefore examined the effect of mutant nrde-2 on the C. elegans PGC pool and brood size. Our results lead us to believe that nrde-2 has a conserved function in maintaining proper cellular proliferation.

Chalasani S H et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Neuropeptide feedback modifies odor induced responses in Caenorhabditis elegans olfactory neurons"

Bargmann C I, Kato S, Chalasani S H, Albrecht D R

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

Neural circuits transform sensory signals to generate behaviors on timescales from seconds to hours. In some C.elegans behaviors, sensory inputs lead to long lasting and complex behavioral outputs. Animals that have been removed from food spend about 15 minutes exploring a local area by interrupting long forward movements with reversals and turns (Wakabayashi et al., 2004, Gray et al 2005). AWC sensory neurons regulate this behavior by releasing two neurotransmitters, glutamate (promoting turns) and NLP-1 (inhibiting turns). AWC sensory neuron released glutamate activates AIB and inhibits AIY and AIA interneurons (Chalasani et al 2007). In contrast to glutamate, AWC neuron released NLP-1 acts on AIA interneurons to suppress reversals, indicating that turn frequencies are regulated by at least two opposing systems. AWC calcium responses are modulated in these neurotransmitter mutants suggesting that multiple pathways can influence AWC dependent behavior and neuronal activity. ReferencesChalasani, S. H., et. al. Dissecting a neural circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63-70 (2007).Gray, J.M., et. al. A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191 (2005).Wakabayashi, T., et. al. Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111 (2004).

Summers P J et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Inhibitory Glutamatergic Signaling is Essential for the Serotonergic Stimulation of Aversive Responses Mediated by ASH Sensory Neurons"

Hapiak V M, Bamber B, Summers P J, Harris G P, Korchnak A C, Komuniecki R W

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

Glutamatergic signaling from sensory neurons that mediate attraction/avoidance is integrated by the AIA, AIB, AIY and AIZ interneurons that coordinately modulate turning rate and locomotory behavior. The AWC sensory neurons direct chemotaxis toward attractive odors by regulating turning behavior and provide both excitatory and inhibitory glutamatergic input into this layer of interneurons, with the excitatory AMPA-like glutamate receptor, GLR-1, required in the AIBs for AWC-dependent increases in AIB Ca++ signaling during arearestricted search off food (Chalasani et al., 2007). To examine the role of glutamatergic signaling associated with aversive behaviors and potential interactions between attraction and avoidance, we have identified the ionotropic glutamate receptors involved in aversive responses mediated by the ASH sensory neurons. ASH-mediated responses to dilute octanol are stimulated by food and 5-HT and this stimulation requires the expression of MOD-1, a 5-HT gated Cl- channel, in the AIBs (Harris et al., 2009). In contrast, aversive responses in animals with null mutations in 1) avr-15 that encodes a glutamate-gated Cl- channel are not stimulated by either food or 5-HT, or 2) glr-1 or glc-4 that encodes a predicted glutamate-gated Cl- channel are wild type on food, but are not stimulated by 5-HT. In addition, wild type animals with glc-4 RNAi in the AIBs also are not stimulated by 5-HT. This data suggests that the overall activity state of the AIBs may be modulated by a balance of excitatory and inhibitory glutamatergic signaling and that increased aversive responses on food or 5-HT may require decreased AIB signaling, either by inhibiting GLR-1, or activating GLC-4 and MOD-1. Indeed, the AIB overexpression of glr-1 inhibits and glc-4 stimulates basal aversive responses off food. This hypothesis might also explain why AIB GLC-4 expression is required on 5-HT, but not food. Food both increases 5-HT levels and inhibits AWC and downstream AIB signaling, potentially reducing the activity state of the AIBs in the absence of GLC-4 mediated inhibitory input from the ASHs. These studies are continuing to examine the role of the AIBs in the food/5-HT stimulation of ASH-mediated aversive responses, using genetics, Ca++ imaging and electrophysiology.

Kim, Kyuhyung et al. (2009) International Worm Meeting "Specification of chemosensory neuron fate by the C. elegans NKX/HMX homolog MLS-2."

Sengupta, Piali, Kim, Rinho, Kim, Kyuhyung

[International Worm Meeting, 2009]

The C. elegans chemosensory nervous system provides an opportunity to investigate development of sensory neuron types at a single cell resolution. The bilateral AWC neurons are members of the amphid sensory organs in the head, and respond to odorants (Bargmann and Horvitz, 1991; Chalasani et al., 2007), as well as temperature (Kuhura et al., 2008; Biron, Wasserman et al., 2008). We previously showed that the CEH-36 OTX/OTD homeodomain transcription factor plays a major role in specification of AWC neuron identity, and is expressed only in the AWC and ASE chemosensory neuron types in adult animals (Lanjuin et al., 2003). To identify additional molecules required for development of the AWC neurons, we screened for mutants in which expression of ceh-36p::gfp was altered. In oy88 mutant animals expression of ceh-36p::gfp was affected in the AWC, but not the ASE neurons. Genetic mapping, genomic rescue, and sequencing of mutant animals showed that oy88 is an allele of the mls-2 NKX class of homeodomain transcription factors. MLS-2 has previously been shown to regulate proliferation, cell division, fate specification and differentiation in the M linage (Jiang et al., 2005). In addition, MLS-2 is required for the development of the CEPsh glia (Yoshimura et al., 2008). Further analyses showed that the expression of multiple terminal differentiation markers was abolished in 50% of AWC neurons in mls-2 mutant animals. mls-2 appears to act upstream of ceh-36 in the genetic pathway regulating AWC neuronal identity. Moreover, 70% of mls-2 mutant animals showed ectopic expression of ASH-specific markers. Loss of AWC-specific expression and ectopic expression of ASH-specific markers are not correlated, suggesting that MLS-2 acts independently in these two lineages. We did not detect any obvious defects in the expression of other amphid neuron-specific markers. Currently we are performing spatial and temporal rescue experiments, mosaic analyses, and lineage analysis to further characterize the roles of MLS-2 in chemosensory neuron development. Furthermore, using promoter analyses and bioinformatics searches, we are attempting to determine whether MLS-2 directly regulates ceh-36 expression, and identify additional targets of MLS-2 regulation in chemosensory neurons.

Colinas Fischer, Susana et al. (2021) International Worm Meeting "Circuit and Molecular Mechanisms of an Associative Learning Task"

Molina-Garcia, Laura, Lin, Lucy, Barrios, Arantza, Colinas Fischer, Susana

[International Worm Meeting, 2021]

The ability of neural circuits to be changed by experience, optimising an organism's behaviour, is key to survival. We are dissecting the circuit underlying aversive olfactory learning to benzaldehyde (BZ), a bacterial metabolite innately attractive to C. elegans. When this odourant is paired with starvation, an aversive experience, the behavioural response to BZ switches from attraction to repulsion (Lee 2010, Lin 2010). Furthermore, this switch in behaviour can be overridden in males if a rewarding experience (mates) is present during aversive conditioning with starvation. This process is termed sexual conditioning and it was first shown to drive flexible learned responses to salt (Sakai et al, 2013). Our lab has previously shown that the neuropeptide PDF-1 is necessary for sexual conditioning. Here we show that both PDF-1 and PDF-2 redundantly mediate aversive learning, and seek to identify the PDF circuit for aversive learning. BZ is sensed primarily by AWC, which synapses onto AIB and AIY to regulate naive chemotaxis (Bargmann 1993, Chalasani 2007). The switch from attraction to repulsion during aversive learning has been proposed to be driven by changes at the AWC-AIB synapse (Cho 2016). We find that AWC-ablated animals have a dampened response to BZ, but we still observe a switch in response from attraction to repulsion after aversive conditioning, indicating that learning can occur elsewhere in the circuit. We are investigating neurons that are involved in PDF signalling to find the circuit that drives this experience-dependent change in behaviour. We are using an intersectional Cre-Lox strategy and a floxed transgene of PDF-1 cDNA integrated in single copy to return physiological levels of PDF-1 to specific neurons, and thus determine which combinations of neurons are the source of PDF-1 in aversive learning. We will also use an approach that exploits the strength of the Cre-Lox system to restore PDFR-1 to certain neurons selectively, to determine which neurons are the target of PDF during aversive learning. Then we will image the activity of source and target neurons to determine what changes occur in the circuit during conditioning that lead to changes in behaviour. Given that associative learning is a highly conserved behaviour, understanding how this simple circuit is capable of supporting aversive learning will provide us with principles that can be applied to further understand how learning occurs in more complex nervous systems.

Schmitt C et al. (2009) European Worm Neurobiology Meeting "Achieving single-cell expression of Channelrhodopsin-2 using the cre-lox system to analyze habituation in neural circuits inducing withdrawal behaviour"

Gottschalk A, Schmitt C

[European Worm Neurobiology Meeting, 2009]

Habituation is a simple form of non-associative learning, in which multiple repetitions of a stimulus cause a reduction in the response .amplitude.. Habituation of the C. elegans withdrawal reflex in response to gentle touch or a tap of the culture dish is well characterized. Yet, only little is known about the (molecular) mechanisms of habituation and the exact point where habituation is effected within the signalling cascade leading to withdrawal. This could occur at the level of the primary sensory receptors, signalling within the sensory neurons, at their output synapses or in the downstream interneuron network. Previously, we could demonstrate that optogenetic, Channelrhodopsin-2 (ChR2) mediated in vivo stimulation of mechanosensory neurons can induce withdrawal behaviour and that multiple optical stimuli also induce habituation [1]. Here we used the mec-4 promoter, which is exclusievely expressed in touch neurons. Expression of ChR2 in the downstream (.backward.) command interneurons also could induce withdrawal behaviour, however, promoters active in these cells are not highly specific for the AVA neuron. Thus the optogenetic stimulation will also affect other cells (e.g. PVC) and so it is difficult to draw firm conclusions. We would like to achieve highly specific expression of ChR2 for the AVA neuron, and thus enable a specific photostimulation, with the assistance of the cre-lox system that was recently introduced for C. elegans [2], or, alternatively, the FLP recombinase system [3]; see poster by Christian Schultheis. This method provides the ability to use two different promotors for gene expression, with expression patterns that include the cell of interest, but no additional overlap in other cells in which each promoter may be active. Thus, expression of ChR2 would occur only at the intersection of the two expression patterns. We like to establish cre-lox driven expression of ChR2 in AVA, then test these transgenic animals for occurrence of photoinduced withdrawal, and, importantly, habituation of the response, just as in mec-neurons. An additional sensory neuron inducing withdrawals is the polymodal aversive neuron ASH, which detects harmful or toxic chemicals. We will establish single-cell expression of ChR2 in ASH, test for photoinduced withdrawal and habituation. If this is also found, potential cross-habituation of the two sensory pathways (mec-neurons and ASH) leading to withdrawal will be tested by combined photo-stimuli and .natural. stimuli, which should occur at the level of the interneurons. This procedure will provide the possibility to find the synapses which are responsible for habituation of the reflex. 1. Nagel, Brauner, Liewald, Adeishvili, Bamberg and Gottschalk (2005) Curr. Biol. 15: 2279-2284 2 Macosko, Pokala, Feinberg, Chalasani, Butcher, Clardy and Bargmann (2009) Nature 58:1171-1175 3. Davis, Morton, Carroll and Jorgensen (2008) PLoS Genetics 4: e1000028

Damien O'Halloran et al. (2008) C.elegans Neuronal Development Meeting "Alpha subunits of cGMP-gated channels CNG-1 and CNG-3 are required for plasticity of the AWC olfactory neurons"

Tsung-Yu Chen, Noelle L'Etoile, Altshuler Svetlana, Zhang Xiao-Dong, Damien O'Halloran

[C.elegans Neuronal Development Meeting, 2008]

Adaptation is a fundamental property of sensory systems that allows a living organism to adjust to ongoing changes in its environment by decreasing its sensitivity to persistent stimuli. In C. elegansAWA and AWC are responsible for sensation of a wide range of attractive volatile odors. Odor exposure for a prolonged time leads to reversible decreases in the animal''s attraction to odor (Colbert and Bargmann, 1995;L''Etoile et al., 2002). The specificity of olfactory adaptation is determined by the feeding status of the animal. If a well-fed worm is exposed to benzaldehyde for 1hr it will ignore both benzaldehyde and isoamyl alcohol. In contrast a starved worm after benzaldehyde exposure will blunt only the response to benzaldehyde leaving the response to isoamyl alcohol intact (Colbert and Bargmann,1997). The TAX-4/TAX-2heterotetrameric cyclic nucleotide-gated channel (cNG) is required for AWC-mediated olfactory responses.TAX-4 is an alpha subunit that can form homomeric channels while TAX-2 is a beta subunit that requires TAX-4 to form a functional channel (Coburn andBargmann, 1996; Komatsu et al., 1999). C. elegansencodes two additional predicted alpha subunits CNG-1 and CNG-3 (Suk-Woo et al., 2004a; 2004b and L''Etoile 2004) and two additional predicted beta subunits Y76B12C.1 and C23H5.7 (L''Etoile, 2004 andCara Coburn, thesis). In mice the olfactory channel is comprised of 3 alpha subunits: 2XCNGA2, 1XCNGA4 and the beta subunit CNGB1b. Like TAX-4,CNGA2 can homomerize but CNGA4 is required for both odor-sensitivity and adaptation to background odors (Munger et al., 2001; Kelliher et al., 2003).The function of CNG-1 and CNG-3 in behavior is unknown. Here we report that though both cng-1 and cng-3 mutants are wild-type in their response to a dilution series of AWC-sensed odors, CNG-3 is required in AWC for short-term adaptation to benzaldehyde and butanone. The cng-1 mutant mimics a starved worm in that benzaldehyde exposure leads to adaptation of only the benzaldehyde response and spares the response to isoamyl alcohol. When we examined the subunits in HEK cells we found that like the mammalian alternate olfactory cNG alpha subunit CNG4A neither CNG-1 nor CNG-3 can homomerize to form a functional channel and yet each modulates the electrophysiological response of TAX-2 and TAX-4 heteromers to cGMP. CNG-1 lengthens the time constant for the channel closing in response to removal of cGMP. Since cGMP levels have been shown to decrease upon odor-exposure (Chalasani et al., 2007), we speculate that cng-3 mutant worms (equivalent to TAX-2/4/CNG-1 expression in HEK cells) would be defective in saturation assays to the same odor. TheTAX-2/4/CNG-3 channel has very similar properties to that of the TAX-2/4channel. We are currently investigating native channel conformations in C.elegans using FRET and in Xenopus oocytes using TIRF microscopy (Ulbrich and Isacoff,2007)