ASE Neurons Research Articles

Probiotics-sensing mechanism in neurons that initiates gut mitochondrial surveillance for pathogen defense

Animals constantly face microbial challenges, and microbe-mediated infection protection is crucial for host survival. Identifying specific bacteria and their interactions with host intracellular surveillance systems is important but challenging. Here, we develop a "probiotics" screening system that identifies Escherichia coli mutants, such as ΔymcB, which protect hosts from Pseudomonas aeruginosa PA14 infection by activating the mitochondrial unfolded protein response (UPRmt). Genetic screening reveals that MDSS-1, a neuronal transmembrane protein, is crucial for sensing ΔymcB and triggering intestinal UPRmt. MDSS-1 functions as a potential receptor in ASE neurons, detecting ΔymcB and transmitting signals through neuropeptides, GPCRs, Wnt signaling, and endopeptidase inhibitors to activate intestinal UPRmt and enhance protection. Constitutive activation of MDSS-1 in ASE neurons is sufficient to induce UPRmt and confer infection resistance. This study uncovers a neuron-intestine communication mechanism, where ASE neurons detect bacteria and modulate the intestinal mitochondrial surveillance system for host adaptation to pathogens.

Evolution of lateralized gustation in nematodes.

Animals with small nervous systems have a limited number of sensory neurons that must encode information from a changing environment. This problem is particularly exacerbated in nematodes that populate a wide variety of distinct ecological niches but only have a few sensory neurons available to encode multiple modalities. How does sensory diversity prevail within this neuronal constraint? To identify the genetic basis for patterning different nervous systems, we demonstrate that sensory neurons in the Pristionchus pacificus respond to various salt sensory cues in a manner that is partially distinct from that of the distantly related nematode C. elegans. By visualizing neuronal activity patterns, we show that contrary to previous expectations based on its genome sequence, the salt responses of P. pacificus are encoded in a left/right asymmetric manner in the bilateral ASE neuron pair. Our study illustrates patterns of evolutionary stability and change in the gustatory system of nematodes.

Gold Nanoparticles Reduce Food Sensation in Caenorhabditis elegans via the Voltage-Gated Channel EGL-19.

The increasing use of gold nanoparticles (Au NPs) in the medical field has raised concerns about the potential adverse effect of Au NPs exposure. However, it is difficult to assess the health risks of Au NPs exposure at the individual organ level using current measurement techniques. The physical and chemical properties of Au NPs were characterized by transmission electron microscope (TEM), Fourier transform infrared (FTIR), and zeta sizer. The RNA-seq data of Au NPs-exposed worms were analyzed. The food intake was measured by liquid culture and Pharyngeal pumping rate. The function of the smell and taste neurons was evaluated by the chemotaxis and avoidance assay. The activation of ASE neurons was analyzed by calcium imaging. The gene expression of ins-22 and egl-19 was obtained from the C. elegans single cell RNA-seq databases. Our data analysis indicated that 62.8% of the significantly altered genes were functional in the nervous system. Notably, developmental stage analysis demonstrated that exposure to Au NPs interfered with animal development by regulating foraging behavior. Also, our chemotaxis results showed that exposure to Au NPs reduced the sensation of C. elegans to NaCl, which was consistent with the decrease in calcium transit of ASEL. Further studies confirmed that the reduced calcium transit was dependent on voltage-gated calcium channel EGL-19. The neuropeptide INS-22 was partially involved in Au NPs-induced NaCl sensation defect. Therefore, we proposed that Au NPs reduced the calcium transit in the ASEL neuron through egl-19-dependent calcium channels. It was partially regulated by the DAF-16 targeting neuropeptide INS-22. Our results demonstrate that Au NPs affect food sensation by reducing the calcium transit in ASEL neurons, which further leads to reduced pharynx pumping and feeding defects. The toxicology studies of Au NPs from worms have great potential to guide the usage of Au NPs in the medical field such as targeted drug delivery.

Glial regulators of ions and solutes required for specific chemosensory functions in Caenorhabditis elegans.

Glia and accessory cells regulate the microenvironment around neurons and primary sensory cells. However, the impact of specific glial regulators of ions and solutes on functionally diverse primary cells is poorly understood. Here, we systemically investigate the requirement of ion channels and transporters enriched in Caenorhabditis elegans Amsh glia for the function of chemosensory neurons. Although Amsh glia ablated worms show reduced function of ASH, AWC, AWA, and ASE neurons, we show that the loss of glial enriched ion channels and transporters impacts these neurons differently, with nociceptor ASH being the most affected. Furthermore, our analysis underscores the importance of K+, Cl-, and nucleoside homeostasis in the Amphid sensory organ and uncovers the contribution of glial genes implicated in neurological disorders. Our findings build a unique fingerprint of each glial enriched ion channel and transporter and may provide insights into the function of supporting cells of mammalian sensory organs.

Insulin/IGF signaling regulates presynaptic glutamate release in aversive olfactory learning.

Insulin/insulin-like growth factor (IGF) receptor signaling (IIS) supports context-dependent learning in vertebrates and invertebrates. Here, we identify cell-specific mechanisms of IIS that integrate sensory information with food context to drive synaptic plasticity and learning. In the nematode Caenorhabditis elegans, pairing food deprivation with an odor such as butanone suppresses attraction to that odor. We find that aversive olfactory learning requires the insulin receptor substrate (IRS) protein IST-1 and atypical signaling through the insulin/IGF-1 receptor DAF-2. Cell-specific knockout and rescue demonstrate that DAF-2 acts in the AWCON sensory neuron, which detects butanone, and that learning preferentially depends upon the axonally localized DAF-2c isoform. Acute food deprivation increases DAF-2 levels in AWCON post-transcriptionally through an insulin- and insulin receptor substrate-1 (ist-1)-dependent process. Aversive learning alters the synaptic output of AWCON by suppressing odor-regulated glutamate release in wild-type animals, but not in ist-1 mutants, suggesting that axonal insulin signaling regulates synaptic transmission to support aversive memory.

Different modes of stimuli delivery elicit changes in glutamate driven, experience-dependent interneuron response in C. elegans

Memory-related neuronal responses are often elicited by sensory stimuli that recapitulate previous experience. Despite the importance of this sensory input processing, its underlying mechanisms remain poorly understood. Caenorhabditis elegans chemotax towards salt concentrations experienced in the presence of food. The amphid sensory neurons ASE-left and ASE-right respond to increases and decreases of ambient salt concentration in opposite manners. AIA, AIB and AIY interneurons are post-synaptic to the ASE pair and are thought to be involved in the processing of salt information transmitted from ASE. However, it remains elusive how the responses of these interneurons are regulated by stimulus patterns. Here we show that AIY interneurons display an experience-dependent response to gradual salt concentration changes but not to abrupt stepwise concentration changes. Animals with AIY intact (but AIA and AIB ablated) chemotax towards low salt concentrations similarly to wild-type animals after cultivation with low salt. ASE neurons transmit salt information about the environment through glutamatergic signaling, directing the activity of the interneurons AIY that promote movement towards favorable conditions.

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations.

Cell fate is maintained over long timescales, yet molecular fluctuations can lead to spontaneous loss of this differentiated state. Our simulations identified a possible mechanism that explains life-long maintenance of ASE neuron fate in Caenorhabditis elegans by the terminal selector transcription factor CHE-1. Here, fluctuations in CHE-1 level are buffered by the reservoir of CHE-1 bound at its target promoters, which ensures continued che-1 expression by preferentially binding the che-1 promoter. We provide experimental evidence for this mechanism by showing that che-1 expression was resilient to induced transient CHE-1 depletion, while both expression of CHE-1 targets and ASE function were lost. We identified a 130 bp che-1 promoter fragment responsible for this resilience, with deletion of a homeodomain binding site in this fragment causing stochastic loss of ASE identity long after its determination. Because network architectures that support this mechanism are highly conserved in cell differentiation, it may explain stable cell fate maintenance in many systems.

Signal Decoding for Glutamate Modulating Egg Laying Oppositely in Caenorhabditis elegans under Varied Environmental Conditions.

SummaryAnimals' ability to sense environmental cues and to integrate this information to control fecundity is vital for continuing the species lineage. In this study, we observed that the sensory neurons Amphid neuron (ASHs and ADLs) differentially regulate egg-laying behavior in Caenorhabditis elegans under varied environmental conditions via distinct neuronal circuits. Under standard culture conditions, ASHs tonically release a small amount of glutamate and inhibit Hermaphrodite specific motor neuron (HSN) activities and egg laying via a highly sensitive Glutamate receptor (GLR)-5 receptor. In contrast, under Cu2+ stimulation, ASHs and ADLs may release a large amount of glutamate and inhibit Amphid interneuron (AIA) interneurons via low-sensitivity Glutamate-gated chloride channel (GLC)-3 receptor, thus removing the inhibitory roles of AIAs on HSN activity and egg laying. However, directly measuring the amount of glutamate released by sensory neurons under different conditions and assaying the binding kinetics of receptors with the neurotransmitter are still required to support this study directly.

Transcription factor autoregulation is required for acquisition and maintenance of neuronal identity.

The expression of transcription factors that initiate the specification of a unique cellular identity in multicellular organisms is often maintained throughout the life of the respective cell type via an autoregulatory mechanism. It is generally assumed that such autoregulation serves to maintain the differentiated state of a cell. To experimentally test this assumption, we used CRISPR/Cas9-mediated genome engineering to delete a transcriptional autoregulatory, cis-acting motif in the che-1 zinc-finger transcription factor locus, a terminal selector required to specify the identity of the ASE neuron pair during embryonic development of the nematode Caenorhabditis elegans. We show that che-1 autoregulation is indeed required to maintain the differentiated state of the ASE neurons but that it is also required to amplify che-1 expression during embryonic development to reach an apparent minimal threshold to initiate the ASE differentiation program. We conclude that transcriptional autoregulation fulfills two intrinsically linked purposes: one in proper initiation, the other in proper maintenance of terminal differentiation programs.This article has an associated 'The people behind the papers' interview.

Loss of CaMKI Function Disrupts Salt Aversive Learning in C. elegans.

The ability to adapt behavior to environmental fluctuations is critical for survival of organisms ranging from invertebrates to mammals. Caenorhabditis elegans can learn to avoid sodium chloride when it is paired with starvation. This behavior may help animals avoid areas without food. Although some genes have been implicated in this salt-aversive learning behavior, critical genetic components, and the neural circuit in which they act, remain elusive. Here, we show that the sole worm ortholog of mammalian CaMKI/IV, CMK-1, is essential for salt-aversive learning behavior in C. elegans hermaphrodites. We find that CMK-1 acts in the primary salt-sensing ASE neurons to regulate this behavior. By characterizing the intracellular calcium dynamics in ASE neurons using microfluidics, we find that loss of cmk-1 has subtle effects on sensory-evoked calcium responses in ASE axons and their modulation by salt conditioning. Our study implicates the expression of the conserved CaMKI/CMK-1 in chemosensory neurons as a regulator of behavioral plasticity to environmental salt in C. elegansSIGNIFICANCE STATEMENT Like other animals, the nematode Caenorhabditis elegans depends on salt for survival and navigates toward high concentrations of this essential mineral. In addition to its role as an essential nutrient, salt also causes osmotic stress at high concentrations. A growing body of evidence indicates that C. elegans balances the requirement for salt with the danger it presents through a process called salt-aversive learning. We show that this behavior depends on expression of a calcium/calmodulin-dependent kinase, CMK-1, in the ASE salt-sensing neurons. Our study identifies CMK-1 and salt-sensitive chemosensory neurons as key factors in this form of behavioral plasticity.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in <em>C. elegans</em>

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Coordinated control of terminal differentiation and restriction of cellular plasticity.

The acquisition of a specific cellular identity is usually paralleled by a restriction of cellular plasticity. Whether and how these two processes are coordinated is poorly understood. Transcription factors called terminal selectors activate identity-specific effector genes during neuronal differentiation to define the structural and functional properties of a neuron. To study restriction of plasticity, we ectopically expressed C. elegans CHE-1, a terminal selector of ASE sensory neuron identity. In undifferentiated cells, ectopic expression of CHE-1 results in activation of ASE neuron type-specific effector genes. Once cells differentiate, their plasticity is restricted and ectopic expression of CHE-1 no longer results in activation of ASE effector genes. In striking contrast, removal of the respective terminal selectors of other sensory, inter-, or motor neuron types now enables ectopically expressed CHE-1 to activate its ASE-specific effector genes, indicating that terminal selectors not only activate effector gene batteries but also control the restriction of cellular plasticity. Terminal selectors mediate this restriction at least partially by organizing chromatin. The chromatin structure of a CHE-1 target locus is less compact in neurons that lack their resident terminal selector and genetic epistasis studies with H3K9 methyltransferases suggest that this chromatin modification acts downstream of a terminal selector to restrict plasticity. Taken together, terminal selectors activate identity-specific genes and make non-identity-defining genes less accessible, thereby serving as a checkpoint to coordinate identity specification with restriction of cellular plasticity.

Pharyngeal pumping inhibition and avoidance by acute exposure to high CO2 levels are both regulated by the BAG neurons via different molecular pathways.

Carbon dioxide (CO2) is a key molecule in many biological processes. Studies in humans, mice, D. melanogaster, C. elegans, unicellular organisms and plants have shed light on the molecular pathways activated by elevated levels of CO2. However, the mechanisms that organisms use to sense and respond to high CO2 levels remain largely unknown. Previous work has shown that C. elegans quickly avoid elevated CO2 levels using mechanisms that involve the BAG, ASE and AFD neurons via cGMP- and calcium- signaling pathways. Here, we discuss our recent finding that exposure of C. elegans to high CO2 levels leads to a very rapid cessation in the contraction of the pharynx muscles. Surprisingly, none of the tested CO2 avoidance mutants affected the rapid pumping inhibition response to elevated CO2 levels. A forward genetic screen identified that the hid-1-mediated pathway of dense core vesicle maturation regulates the pumping inhibition, probably through affecting neuropeptide secretion. Genetic studies and laser ablation experiments showed that the CO2 response of the pharyngeal muscle pumping is regulated by the BAG neurons, the same neurons that mediate CO2 avoidance.

Two distinct types of neuronal asymmetries are controlled by the Caenorhabditis elegans zinc finger transcription factor die-1

Left/right asymmetric features of animals are either randomly distributed on either the left or right side within a population ("antisymmetries") or found stereotypically on one particular side of an animal ("directional asymmetries"). Both types of asymmetries can be found in nervous systems, but whether the regulatory programs that establish these asymmetries share any mechanistic features is not known. We describe here an unprecedented molecular link between these two types of asymmetries in Caenorhabditis elegans. The zinc finger transcription factor die-1 is expressed in a directionally asymmetric manner in the gustatory neuron pair ASE left (ASEL) and ASE right (ASER), while it is expressed in an antisymmetric manner in the olfactory neuron pair AWC left (AWCL) and AWC right (AWCR). Asymmetric die-1 expression is controlled in a fundamentally distinct manner in these two neuron pairs. Importantly, asymmetric die-1 expression controls the directionally asymmetric expression of gustatory receptor proteins in the ASE neurons and the antisymmetric expression of olfactory receptor proteins in the AWC neurons. These asymmetries serve to increase the ability of the animal to discriminate distinct chemosensory inputs.

Defining specificity determinants of cGMP mediated gustatory sensory transduction in Caenorhabditis elegans.

Cyclic guanosine monophosphate (cGMP) is a key secondary messenger used in signal transduction in various types of sensory neurons. The importance of cGMP in the ASE gustatory receptor neurons of the nematode Caenorhabditis elegans was deduced by the observation that multiple receptor-type guanylyl cyclases (rGCs), encoded by the gcy genes, and two presently known cyclic nucleotide-gated ion channel subunits, encoded by the tax-2 and tax-4 genes, are essential for ASE-mediated gustatory behavior. We describe here specific mechanistic features of cGMP-mediated signal transduction in the ASE neurons. First, we assess the specificity of the sensory functions of individual rGC proteins. We have previously shown that multiple rGC proteins are expressed in a left/right asymmetric manner in the functionally lateralized ASE neurons and are required to sense distinct salt cues. Through domain swap experiments among three different rGC proteins, we show here that the specificity of individual rGC proteins lies in their extracellular domains and not in their intracellular, signal-transducing domains. Furthermore, we find that rGC proteins are also sufficient to confer salt sensory responses to other neurons. Both findings support the hypothesis that rGC proteins are salt receptor proteins. Second, we identify a novel, likely downstream effector of the rGC proteins in gustatory signal transduction, a previously uncharacterized cyclic nucleotide-gated (CNG) ion channel, encoded by the che-6 locus. che-6 mutants show defects in gustatory sensory transduction that are similar to defects observed in animals lacking the tax-2 and tax-4 CNG channels. In contrast, thermosensory signal transduction, which also requires tax-2 and tax-4, does not require che-6, but requires another CNG, cng-3. We propose that CHE-6 may form together with two other CNG subunits, TAX-2 and TAX-4, a gustatory neuron-specific heteromeric CNG channel complex.

A model of chemotaxis and associative learning in C. elegans

The nematode C. elegans has attracted a great deal of interest from the neuroscience community due to the simplicity of its nervous system, which in the hermaphrodite is composed of just 302 neurons. C. elegans is known to engage in a number of sophisticated behaviours such as chemo- and thermotaxis. Experimental work has shown that these behaviours can be modified by experience and that C. elegans is capable of associative learning. In this paper, we focus on the chemotactic response of C. elegans to sodium chloride mediated by the ASE sensory neurons. We construct a biophysical model of the ASEL and ASER neurons that captures the time course of the ASE responses in response to up- and down-steps in NaCl concentration. We use this model to show that the time course of the ASE responses provide sufficient temporal resolution to successfully drive chemotaxis in C. elegans via steering, pirouettes and control of final turn angle. We show that these different locomotion strategies are individually capable of driving chemotaxis and that by working together they produce the best chemotactic response. We find that there is a separation into upward and downward drives mediated by the left and right ASE neurons. We show that the connectivity from ASEL and ASER must be of opposite polarity and that ASER, and the concomitant ability to sense when the worm is moving down the gradient, is more important for chemotaxis than ASEL, findings that are consistent with existing modelling studies in the literature. Finally, we examine associative learning in the network and show that experimental data can be explained by changes that occur at either the synaptic or sensory neuron level, the choice of which has distinct consequences for network function.

Neuronal and molecular substrates for optimal foraging in Caenorhabditis elegans.

Variation in food quality and abundance requires animals to decide whether to stay on a poor food patch or leave in search of better food. An important question in behavioral ecology asks when is it optimal for an animal to leave a food patch it is depleting. Although optimal foraging is central to evolutionary success, the neural and molecular mechanisms underlying it are poorly understood. Here we investigate the neuronal basis for adaptive food-leaving behavior in response to resource depletion in Caenorhabditis elegans, and identify several of the signaling pathways involved. The ASE neurons, previously implicated in salt chemoattraction, promote food-leaving behavior via a cGMP pathway as food becomes limited. High ambient O(2) promotes food-leaving via the O(2)-sensing neurons AQR, PQR, and URX. Ectopic activation of these neurons using channelrhodopsin is sufficient to induce high food-leaving behavior. In contrast, the neuropeptide receptor NPR-1, which regulates social behavior on food, acts in the ASE neurons, the nociceptive ASH neurons, and in the RMG interneuron to repress food-leaving. Finally, we show that neuroendocrine signaling by TGF-β/DAF-7 and neuronal insulin signaling are necessary for adaptive food-leaving behavior. We suggest that animals integrate information about their nutritional state with ambient oxygen and gustatory stimuli to formulate optimal foraging strategies.

A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.

Temperature, Oxygen, and Salt-Sensing Neurons in C. elegans Are Carbon Dioxide Sensors that Control Avoidance Behavior

SummaryHomeostatic control of body fluid CO2 is essential in animals but is poorly understood. C. elegans relies on diffusion for gas exchange and avoids environments with elevated CO2. We show that C. elegans temperature, O2, and salt-sensing neurons are also CO2 sensors mediating CO2 avoidance. AFD thermosensors respond to increasing CO2 by a fall and then rise in Ca2+ and show a Ca2+ spike when CO2 decreases. BAG O2 sensors and ASE salt sensors are both activated by CO2 and remain tonically active while high CO2 persists. CO2-evoked Ca2+ responses in AFD and BAG neurons require cGMP-gated ion channels. Atypical soluble guanylate cyclases mediating O2 responses also contribute to BAG CO2 responses. AFD and BAG neurons together stimulate turning when CO2 rises and inhibit turning when CO2 falls. Our results show that C. elegans senses CO2 using functionally diverse sensory neurons acting homeostatically to minimize exposure to elevated CO2.