Sakai, Naoko et al. (2021) International Worm Meeting "Systematic analysis of CAMs expressed in ray neurons in C. elegans."

Sun, Peter, Kim, Byunghyuk, Emmons, Scott, Sakai, Naoko

[International Worm Meeting, 2021]

Although cell adhesion molecules (CAMs) have been studied for many years as essential players in neuronal connectivity, we have yet to break the code by which cell adhesion molecules help neurons find appropriate partners. While some CAMs such as sax-3/ROBO and unc-40/DCC have been heavily researched, many CAMs' functions remain unknown. We recently identified the expression patterns of 100 neural CAMs across 173 neurons and 64 muscles in the C. elegans male tail. This data showed that A type and B type ray neurons, which are adjacent sensory neurons derived from the same progenitor cells, do not share expressions of most CAMs. There are nine pairs of rays in the male tail, with each ray being composed of two different types of sensory neurons (RnA and RnB) and a structural cell. The A- and B-type neurons make different connections, which also differ from ray to ray. To investigate whether cell type-specific CAMs expression is important for ray neuron development and connectivity, we tested all viable mutants of CAMs expressed in RnB neurons. Mutants of unc-40 and sax-3 showed a significant defect in both anterior-posterior and dorsal-ventral axon trajectory, and fmi-1/Flamingo showed a defect in dorsal-ventral and diagonal axon trajectory. In contrast, the other 6 CAMs mutants didn't have any defects in axon pathfinding. We also tested the possibility that CAMs work redundantly by testing multiple mutants and found a deletion in hmr-1B/N-cadherin, which does not lead to any morphological defect in a single mutant, enhanced the defect of the sax-3 mutants. lron-7/OPTC and lron-13 mutations, which do not affect RnB morphology in the single mutants, slightly suppressed the defect of sax-3 and fmi-1. These results suggest that CAMs across gene families function combinatorially to regulate the RnB morphology. We employed fluorescent transgenes for the synaptic vesicle protein RAB-3 driven by a B-type neuron-specific promoter to visualize the synaptic connectivity. The quantification of signal intensity of RAB-3::mCherry showed that unc-40, sax-3, fmi-1, lron-7, lron-13, and nrx-1/neurexin mutants showed significantly low RAB-3 signal intensity. The function of lron-7 and lron-13 in synaptic formation has not been reported before.

Sakai, Naoko et al. (2015) International Worm Meeting "TOR signaling pathway is involved in regulation of salt chemotaxis learning."

Iino, Yuichi, Tomioka, Masahiro, Ohno, Hayao, Sakai, Naoko, Kunitomo, Hirofumi

[International Worm Meeting, 2015]

Previously, we have found that C. elegans changes responses to external salt concentrations depending on food availability. When worms are cultivated on a medium that contains sodium chloride (NaCl) and bacterial food, they are attracted to the NaCl concentration at which they have been previously grown. In contrast, after exposure to NaCl under starvation conditions, they learn to avoid the NaCl concentration. This behavioral change is called "salt chemotaxis learning". We also reported that insulin/PI3 kinase pathway, a well-known molecular pathway regulating dauer formation and aging, is involved in salt chemotaxis learning. However, downstream molecules of the insulin/PI3K pathway are still unknown for salt chemotaxis learning. In this study, we aimed to find new downstream molecules of insulin/PI3 kinase pathway. We employed a candidate screen and found that the TOR signaling pathway is involved in starvation-dependent behavioral changes. TOR, or Target Of Rapamycin is a nutrient-sensitive Ser/Thr kinase controlling cell growth and aging and is highly conserved from yeast to human. TOR-specific inhibitors rapamycin, Torin1 and Torin2 inhibit normal chemotaxis learning, especially after starvation conditioning. Besides, some of TOR signaling mutants also showed defects in chemotaxis learning. TOR forms two structurally and functionally distinct complexes, TORC1 and TORC2 (TOR Complex 1 and 2). Mutants of rsks-1, a downstream molecule of TORC1, prefer higher salt concentrations than wild type N2. On the other hand, mutants of one of the components of TORC2, rict-1, and those of downstream molecules of TORC2, sgk-1 and pkc-2, showed a tendency to be attracted to lower salt concentrations than N2. These results suggest that TORC1 and TORC2 act in opposite directions to control salt chemotaxis learning. .

Ohno, Hayao et al. (2017) International Worm Meeting "Diacylglycerol encodes differences between past and current stimulus intensity."

Ohno, Hayao, Adachi, Takeshi, Iino, Yuichi, Sakai, Naoko

[International Worm Meeting, 2017]

C. elegans can memorize external salt concentrations and is attracted to the salt concentration at which it has been fed, whereas it avoids the previous salt concentration if it has been starved (Kunitomo et al., Nat. Commun., 2013; Ohno et al., Science, 2014). An important question is how worms compare the intensity of the past and current sensory stimuli and execute appropriate chemotactic behaviors. The plasticity of salt chemotaxis is regulated by the diacylglycerol (DAG)/protein kinase C (PKC) pathway and the insulin/PI3K pathway, both of which act in the ASER taste receptor neuron. Genetic manipulations that activate or inactivate the DAG/PKC pathway result in worm migration to higher or lower salt concentrations, respectively. The insulin/PI3K pathway is essential for the avoidance of the salt concentrations associated with starvation. We have monitored the abundance of DAG in the ASER presynaptic regions, where the nPKC-epsilon/eta ortholog TTX-4 (a.k.a. PKC-1) is localized. When the external salt concentration is increased or decreased from the concentration that has been experienced with food, DAG level is reduced or elevated, respectively, in a manner dependent on the TAX-4 CNG channel subunit and the EGL-8 phospholipase C beta . These DAG responses are largely diminished after starvation. An unbiased genetic screen and mutant analyses show that the DAG pathway acts downstream of the insulin/PI3K/Akt pathway. Our results suggest that the abundance of DAG in the ASER presynaptic region can encode differences between past and current salt concentrations and the insulin/PI3K pathway regulates the change of salt concentration preferences through modulation of the DAG dynamics.

Molina-Garcia, L. et al. (2017) International Worm Meeting "Sexy learning in C. elegans."

Lin, L., Molina-Garcia, L., Rashid, S., Barrios, A.

[International Worm Meeting, 2017]

We learn from experience. When an animal repeatedly encounters a signal coupled with either a punishment or a reward, it eventually learns to expect both to occur together in a process called associative learning. A central goal in neuroscience is to understand how neural circuits integrate conflicting (rewarding and aversive) experiences that need to be behaviourally resolved during learning. To shed light into this process at the molecular and cellular level, we are dissecting a neural circuit for associative learning in the C. elegans male. Previously, the Iino lab (Sakai et al., 2013) and our lab (Sammut et al., 2015) have shown that C. elegans males undergo sexual conditioning, a form of associative learning by which a rewarding experience with mates overrides the behavioural consequences of an aversive association with starvation. Sexual conditioning leads to a switch in behavioural responses to an environmental stimulus from avoidance to attraction. We have also identified the MCM interneurons and PDF neuromodulation as regulators of the sexually conditioned behavioural switch (Sammut et al.2015). These studies showed conditioned responses to salt. Here we demonstrate that other stimuli such as benzaldehyde or diacetyl can also be sexually conditioned. In addition, we show that mutants without MCMs and mutants in the neuropeptide pdf-1, which is expressed in the MCMs, fail to undergo sexual conditioning also to odorants. These results indicate that sexual conditioning is a general, broadly employed strategy of the male to effectively search for and locate mates during navigation. Furthermore, the MCM neurons and PDF neuromodulation are common mechanisms underlying sexual conditioning to distinct stimuli. We are now interrogating the circuit for chemotaxis learning to identify the neurons within it that are modulated by the MCMs and PDF neuropeptide.

Martijn Dekkers et al. (2003) International Worm Meeting "Genetic analysis and molecular imaging of olfactory signalling in C. elegans"

Martijn Dekkers, Gert Jansen, Gerard Borst

[International Worm Meeting, 2003]

G-protein signal transduction is one of the most widely used mechanisms for cells to communicate with their environment. In a single cell several different G-protein signalling cascades are present, and recent evidence suggests extensive crosstalk between these individual cascades. How this is organised, while maintaining specificity in signalling is thus far poorly understood. To address this problem we study chemosensory behaviours in C. elegans. The animal is capable of detecting at least 60 odorants and to discriminate between many of them using only two pairs of cells, AWA and AWC. Interestingly, in these cells a specific subset of 6 G subunits is expressed (Jansen ea. 1999). To study the functions of the individual G subunits we use behavioural assays including an odorant discrimination assay (Bargmann ea 1993). Our preliminary data shows that the G subunit ODR-3 is sufficient to discriminate between at least some odorants. Many downstream effectors of G-proteins are known and are being tested for their function in these pathways. However, many of these genes are expressed ubiquitously and some have severe locomotory defects. This renders behavioural assays useless to study the effects of these proteins in signalling. Therefore we are developing imaging tools to visualise responses of individual cells to olfactory or gustatory stimuli. We will use two constructs, the Yellow Cameleon (YC) (Kerr ea. 2000) and the Voltage Sensitive Fluorescent Protein (VSFP) (Sakai ea. 2001). Both constructs utilise the Fluorescence Resonance Energy Transfer (FRET) principle to measure either the calcium fluxes in the cell or changes in the membrane potential, respectively. We will drive expression using cell-specific promotors, which allows us to elucidate the signal processing on a cellular level. To study effects in olfaction we will express the constructs in AWA using the promoter of the odr-10 gene and in AWC with the gpa-13 promoter, for gustation we will use ASE (flp-5 promoter) and ASI (gpa-4 promoter). Together with the data from behavioural assays, this will provide insight in the G-protein signalling pathways in olfaction in C. elegans.

Sammut, Michele et al. (2015) International Worm Meeting "The Mystery Cells of the Male: a novel pair of head interneurons required for sex differences in learning."

Barrios, Arantza, Emmons, Scott W., Cook, Steven J., Felton, Terry, Nguyen, Ken, Hall, David H., Sammut, Michele, Poole, Richard J.

[International Worm Meeting, 2015]

We have discovered that our founding fathers overlooked a bilateral pair of male-specific head interneurons, revealing that the total number of male neurons is 385, not 383. We have termed these neurons the Mystery Cells of the Male (MCMs). Matching the level of characterisation performed by Sulston, White, et al. for all other C. elegans neurons, we have established their identity, lineage origin, connectivity and function. Through reporter gene analysis, and ultrastructural reconstruction, we find that the MCMs are fully differentiated neurons. The cell bodies of the MCMs are located dorsoanterior to the pharyngeal metacorpus, a region devoid of neuronal cell bodies in the hermaphrodite, and send projections posteriorly into the nerve ring and along the ventral cord. The MCMs express neuronal gene batteries for both electrical and chemical communication. They are born at the early L4 larval stage from a male-specific asymmetric division of the glial amphid socket (AMso) cells. This is the first example of glia acting as neural progenitors in an invertebrate and parallels vertebrate neurogenesis. We find that the male AMso cells are fully differentiated, just like the hermaphrodite AMso cells, before they re-enter the cell cycle. This male-specific division results in self-renewal of the AMso, which does not retract its projection during division, and a cell that transdifferentiates into the MCM neurons. Reconstruction of the MCM synaptic connectivity through serial reconstruction of electron micrographs reveals a circuit where these neurons create disynaptic pathways in triplet motifs connecting EF (male-specific), AVF, and RIF to AVB. This suggests that the MCMs may incorporate male-specific sensory inputs, such as mate pheromones, into higher-order processing circuits that control chemotactic behaviours. Consistent with this we find that the MCMs are specifically required for sexual conditioning, a mate-experience dependent plasticity in chemotactic responses that trumps the effect of starvation [1].1. Sakai, N., Iwata, R., Yokoi, S., Butcher, R. A., Clardy, J., Tomioka, M., and Iino, Y. (2013). A sexually conditioned switch of chemosensory behavior in C. elegans. PLoS ONE 8, e68676.

Molina-Garcia, Laura et al. (2021) International Worm Meeting "PDF-1 modulation of aversion and reward during associative learning"

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

[International Worm Meeting, 2021]

A central goal in neuroscience is to understand how neural circuits integrate conflicting (rewarding and aversive) experiences that need to be behaviourally resolved during learning. To shed light into the molecular and cellular mechanisms underlying this process we are dissecting the neuronal circuit that regulates sexual conditioning in C. elegans. Previously, the Iino lab (Sakai et al., 2013) and us (Sammut et al., 2015) have shown that C. elegans males undergo sexual conditioning, a form of associative learning by which a rewarding experience with mates overrides the behavioural consequences of an aversive association with starvation. Thus, sexual conditioning leads to a switch in behavioural responses to an environmental stimulus from avoidance to attraction. Our lab also identified the MCM interneurons and PDF-1 neuromodulation as the cellular and molecular regulators of the sexually conditioned behavioural switch (Sammut et al., 2015). These studies showed conditioned responses to salt. Here we show that other stimuli such as benzaldehyde can also be sexually conditioned and this is also regulated by the MCMs and PDF-1. We find a dual role for PDF-1 in the regulation of aversive learning and sexual conditioning in C. elegans. By using a Cre-Lox intersectional strategy we found that PDF signaling encodes both aversion and sexual conditioning by modulating two partially distinct circuits. Within these circuits, we have identified the interneurons RIA, AIY and RIM as target cells receiving PDF-1 neuromodulation to drive sexual conditioning. Expression of the PDF-1 receptor (pdfr-1) only in these neurons is sufficient to rescue attraction during sexual conditioning, but not repulsion during aversive learning. Furthermore, by removing PDF-1 in specific cells, while keeping the endogenous levels of expression in the rest of the circuit, we have identified the MCMs and AVB neurons, as a source of PDF-1 during sexual conditioning. Importantly, PDF-1 release from the MCMs and AVBs is not required during aversive learning. Thus, highlighting the importance of source specificity versus overall neuropeptide levels during fine tune modulation of specific behaviors. Currently, we are measuring neuronal activity within the chemotaxis circuit to further understand how sensory information is represented after conditioning and PDF neuromodulation to switch odour preferences.

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.