Thermosensory Neurons Research Articles

How Caenorhabditis elegans Senses Mechanical Stress, Temperature, and Other Physical Stimuli.

Caenorhabditis elegans lives in a complex habitat in which they routinely experience large fluctuations in temperature, and encounter physical obstacles that vary in size and composition. Their habitat is shared by other nematodes, by beneficial and harmful bacteria, and nematode-trapping fungi. Not surprisingly, these nematodes can detect and discriminate among diverse environmental cues, and exhibit sensory-evoked behaviors that are readily quantifiable in the laboratory at high resolution. Their ability to perform these behaviors depends on <100 sensory neurons, and this compact sensory nervous system together with powerful molecular genetic tools has allowed individual neuron types to be linked to specific sensory responses. Here, we describe the sensory neurons and molecules that enable C. elegans to sense and respond to physical stimuli. We focus primarily on the pathways that allow sensation of mechanical and thermal stimuli, and briefly consider this animal’s ability to sense magnetic and electrical fields, light, and relative humidity. As the study of sensory transduction is critically dependent upon the techniques for stimulus delivery, we also include a section on appropriate laboratory methods for such studies. This chapter summarizes current knowledge about the sensitivity and response dynamics of individual classes of C. elegans mechano- and thermosensory neurons from in vivo calcium imaging and whole-cell patch-clamp electrophysiology studies. We also describe the roles of conserved molecules and signaling pathways in mediating the remarkably sensitive responses of these nematodes to mechanical and thermal cues. These studies have shown that the protein partners that form mechanotransduction channels are drawn from multiple superfamilies of ion channel proteins, and that signal transduction pathways responsible for temperature sensing in C. elegans share many features with those responsible for phototransduction in vertebrates.

A Fly’s Cool Way to Escape the Heat

Accurate sensing of changes in environmental temperature is a fundamental process. Budelli etal. (2019) describe a specific type of phasic thermosensory neurons in the Drosophila arista, dubbed "cooling cells," that rely on ionotropic receptors (IRs) for their function and mediate cold and warm avoidance.

SLO potassium channels antagonize premature decision making in C. elegans

Animals must modify their behavior with appropriate timing to respond to environmental changes. Yet, the molecular and neural mechanisms regulating the timing of behavioral transition remain largely unknown. By performing forward genetics to reveal mechanisms that underlie the plasticity of thermotaxis behavior in C. elegans, we demonstrated that SLO potassium channels and a cyclic nucleotide-gated channel, CNG-3, determine the timing of transition of temperature preference after a shift in cultivation temperature. We further revealed that SLO and CNG-3 channels act in thermosensory neurons and decelerate alteration in the responsiveness of these neurons, which occurs prior to the preference transition after a temperature shift. Our results suggest that regulation of sensory adaptation is a major determinant of latency before animals make decisions to change their behavior.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Thermosensory perception regulates speed of movement in response to temperature changes in Drosophila melanogaster.

Temperature influences the physiology and behavior of all organisms. For ectotherms, which lack central temperature regulation, temperature adaptation requires sheltering from or moving to a heat source. As temperature constrains the rate of metabolic reactions, it can directly affect ectotherm physiology and thus behavioral performance. This direct effect is particularly relevant for insects, as their small bodies readily equilibrate with ambient temperature. In fact, models of enzyme kinetics applied to insect behavior predict performance at different temperatures suggesting that thermal physiology governs behavior. However, insects also possess thermosensory neurons critical for locating preferred temperatures, showing cognitive control. This suggests that temperature-related behavior can emerge directly from a physiological effect, indirectly as a consequence of thermosensory processing, or through a combination of both. To separate the roles of thermal physiology and cognitive control, we developed an arena that allows fast temperature changes in time and space, and in which animals' movements are automatically quantified. We exposed wild-type Drosophila melanogaster and thermosensory receptor mutants to a dynamic temperature environment and tracked their movements. The locomotor speed of wild-type flies closely matched models of enzyme kinetics, but the behavior of thermosensory mutants did not. Mutations in thermosensory receptor gene dTrpA1 (Transient Receptor Potential A1) expressed in the brain resulted in a complete lack of response to temperature changes, while mutations in peripheral thermosensory receptor gene Gr28b(D) resulted in a diminished response. We conclude that flies react to temperature through cognitive control, informed by interactions between various thermosensory neurons, the behavioral output of which resembles models of enzyme kinetics.

Integration of Plasticity Mechanisms Within a Single Sensory Neuron of &lt;i&gt;C. elegans &lt;/i&gt; Actuates a Memory

Neural plasticity, the ability of a neuron to change its cellular properties in response to past experiences, underpins the nervous system9s capacity to form memories and actuate behaviors. How different plasticity mechanisms act together in vivo and at a cellular level to transform sensory information into behavior is not well understood. Here we show that in the nematode C. elegans two plasticity mechanisms, sensory adaptation and presynaptic plasticity, act within a single cell to encode thermosensory information and actuate a temperature-preference memory. Sensory adaptation enables the primary thermosensory neuron, AFD, to adjust the temperature range of its sensitivity to the local environment, thereby optimizing its ability to detect temperature fluctuations associated with migration. Presynaptic plasticity transforms this thermosensory information into a behavioral preference by gating synaptic communication between sensory neuron AFD and its postsynaptic partner, AIY. The gating of synaptic communication is regulated at AFD presynaptic sites by the conserved kinase nPKC epsilon Bypassing or altering AFD presynaptic plasticity predictably changes the learned behavioral preferences without affecting sensory responses. Our findings indicate that two distinct and modular neuroplasticity mechanisms function together within a single sensory neuron to encode multiple components of information required to enact thermotactic behavior. The integration of these plasticity mechanisms result in a single-cell logic system that can both represent sensory stimuli and guide memory-based behavioral preference.

Chapter 4 - Molecular basis of peripheral innocuous warmth sensitivity

The perception of innocuous warmth is a sensory capability that facilitates thermoregulatory, social, hedonic, and even predatory functions. It has long been recognized that innocuous warmth perception is triggered by activation of a subpopulation of specially tuned peripheral thermosensory neurons. In addition, there is growing evidence that thermotransduction by nonneuronal cells, such as skin keratinocytes, might contribute to or modulate our thermosensory experience. Yet, the precise molecular mechanisms underlying warmth transduction are only now being uncovered. Recent molecular genetics approaches have led to the identification of multiple candidate warmth-transducing molecules that appear to confer thermosensitivity upon innocuous warmth afferents and/or neighboring cell types. Most, but not all, of these candidate transducers are members of the transient receptor potential (TRP) ion channel family. Among the latter, evidence supporting a function in innocuous warmth sensation is strongest for TRPV1 and TRPM2 in mammals and for TRPA1 in nonmammalian species.

Microfluidic Platform for Analyzing the Thermotaxis of C. elegans in a Linear Temperature Gradient.

Caenorhabditis elegans (C. elegans), which shares a considerable amount of characteristics with human genes is one of the important model organisms for the study of behavioral responses. Thermotaxis is a representative behavior response of C. elegans; C. elegans stores the cultivation temperature in thermosensory neurons and moves to the cultivation temperature region in a temperature variation. In this study, we developed a microfluidic system for effective thermotaxis analysis of C. elegans. The microfluidic channel was fabricated using polydimethylsiloxane (PDMS) by soft lithography process. The temperature gradient (15 - 20°C) was generated in the microchannel and controlled by Peltier modules attached to the bottom of the channel. The thermotaxis of wild type (N2), tax-4(p678) and ttx-7(nj50) mutants were effectively analyzed using this microfluidic system. We believe that this system can be employed as a basic platform for studying the neural circuit of C. elegans responding to external stimuli.

Biogenic Amines in Insect Antennae.

Insect antenna is a multisensory organ, each modality of which can be modulated by biogenic amines. Octopamine (OA) and its metabolic precursor tyramine (TA) affect activity of antennal olfactory receptor neurons. There is some evidence that dopamine (DA) modulates gustatory neurons. Serotonin can serve as a neurotransmitter in some afferent mechanosensory neurons and both as a neurotransmitter and neurohormone in efferent fibers targeted at the antennal vessel and mechanosensory organs. As a neurohormone, serotonin affects the generation of the transepithelial potential by sensillar accessory cells. Other possible targets of biogenic amines in insect antennae are hygro- and thermosensory neurons and epithelial cells. We suggest that the insect antenna is partially autonomous in the sense that biologically active substances entering its hemolymph may exert their effects and be cleared from this compartment without affecting other body parts.

Caenorhabditis elegans Genes Affecting Interindividual Variation in Life-span Biomarker Gene Expression.

Genetically identical organisms grown in homogenous environments differ in quantitative phenotypes. Differences in one such trait, expression of a single biomarker gene, can identify isogenic cells or organisms that later manifest different fates. For example, in isogenic populations of young adult Caenorhabditis elegans, differences in Green Fluorescent Protein (GFP) expressed from the hsp-16.2 promoter predict differences in life span. Thus, it is of interest to determine how interindividual differences in biomarker gene expression arise. Prior reports showed that the thermosensory neurons and insulin signaling systems controlled the magnitude of the heat shock response, including absolute expression of hsp-16.2. Here, we tested whether these regulatory signals might also influence variation in hsp-16.2 reporter expression. Genetic experiments showed that the action of AFD thermosensory neurons increases interindividual variation in biomarker expression. Further genetic experimentation showed the insulin signaling system acts to decrease interindividual variation in life-span biomarker expression; in other words, insulin signaling canalizes expression of the hsp-16.2-driven life-span biomarker. Our results show that specific signaling systems regulate not only expression level, but also the amount of interindividual expression variation for a life-span biomarker gene. They raise the possibility that manipulation of these systems might offer means to reduce heterogeneity in the aging process.

Feeling Hot and Cold: Thermal Sensation in Drosophila.

Sensing environmental temperature is crucial for animal life. The model animal, Drosophila melanogaster, can be investigated with a large number of genetic tools, which have greatly facilitated studies of the cellular and molecular mechanisms of thermal sensing. At the molecular level, a group of proteins, including Transient Receptor Potential channels and ionotropic receptors, have been characterized as potential thermal sensors in both larval and adult Drosophila. At the cellular and circuit levels, peripheral and central thermosensory neurons have been identified. More interestingly, thermal information has been found to be specifically encoded by specific central neurons. In this short review, we mainly survey the progress in understanding the molecular mechanisms of thermosensation and the neuronal mechanisms of thermal information processing in the brain of Drosophila. Other recent temperature-related findings such as its impact on neurosecretion and thermotactic behavior in Drosophila are also introduced.

Pan-neuronal screening in Caenorhabditis elegans reveals asymmetric dynamics of AWC neurons is critical for thermal avoidance behavior.

Understanding neural functions inevitably involves arguments traversing multiple levels of hierarchy in biological systems. However, finding new components or mechanisms of such systems is extremely time-consuming due to the low efficiency of currently available functional screening techniques. To overcome such obstacles, we utilize pan-neuronal calcium imaging to broadly screen the activity of the C. elegans nervous system in response to thermal stimuli. A single pass of the screening procedure can identify much of the previously reported thermosensory circuitry as well as identify several unreported thermosensory neurons. Among the newly discovered neural functions, we investigated in detail the role of the AWCOFF neuron in thermal nociception. Combining functional calcium imaging and behavioral assays, we show that AWCOFF is essential for avoidance behavior following noxious heat stimulation by modifying the forward-to-reversal behavioral transition rate. We also show that the AWCOFF signals adapt to repeated noxious thermal stimuli and quantify the corresponding behavioral adaptation.

Coding and Plasticity in the Mammalian Thermosensory System

Perception of the thermal environment begins with the activation of peripheral thermosensory neurons innervating the body surface. To understand how temperature is represented invivo, we used genetically encoded calcium indicators to measure temperature-evoked responses in hundreds of neurons across the trigeminal ganglion. Our results show how warm, hot, and cold stimuli are represented by distinct population responses, uncover unique functional classes of thermosensory neurons mediating heat and cold sensing, and reveal the molecular logic for peripheral warmth sensing. Next, we examined how the peripheral somatosensory system is functionally reorganized to produce altered perception of the thermal environment after injury. We identify fundamental transformations in sensory coding, including the silencing and recruitment of large ensembles of neurons, providing a cellular basis for perceptual changes in temperature sensing, including heat hypersensitivity, persistence of heat perception, cold hyperalgesia, and cold analgesia.

A C. elegans Thermosensory Circuit Regulates Longevity through crh-1/CREB-Dependent flp-6 Neuropeptide Signaling

Sensory perception, including thermosensation, shapes longevity in diverse organisms, but longevity-modulating signals from the sensory neurons are largely obscure. Here we show that CRH-1/CREB activation by CMK-1/CaMKI in the AFD thermosensory neuron is a key mechanism that maintains lifespan at warm temperatures in C.elegans. In response to temperature rise and crh-1 activation, the AFD neurons produce and secrete the FMRFamide neuropeptide FLP-6. Both CRH-1 and FLP-6 are necessary and sufficient for longevity at warm temperatures. Our data suggest that FLP-6 targets the AIY interneurons and engages DAF-9 sterol hormone signaling. Moreover, we show that FLP-6 signaling downregulates ins-7/insulin-like peptide and several insulin pathway genes, whose activity compromises lifespan. Our work illustrates how temperature experience is integrated by the thermosensory circuit to generate neuropeptide signals that remodel insulin and sterol hormone signaling and reveals a neuronal-endocrine circuit driven by thermosensation to promote temperature-specific longevity.

Humidity Sensing in Drosophila

Environmental humidity influences the fitness and geographic distribution of all animals [1]. Insects in particular use humidity cues to navigate the environment, and previous work suggests the existence of specific sensory mechanisms to detect favorable humidity ranges [2-5]. Yet, the molecular and cellular basis of humidity sensing (hygrosensation) remains poorly understood. Here we describe genes and neurons necessary for hygrosensation in the vinegar fly Drosophila melanogaster. We find that members of the Drosophila genus display species-specific humidity preferences related to conditions in their native habitats. Using a simple behavioral assay, we find that the ionotropic receptors IR40a, IR93a, and IR25a are all required for humidity preference in D.melanogaster. Yet, whereas IR40a is selectively required for hygrosensory responses, IR93a and IR25a mediate both humidity and temperature preference. Consistent with this, the expression of IR93a and IR25a includes thermosensory neurons of the arista. In contrast, IR40a is excluded from the arista but is expressed (and required) in specialized neurons innervating pore-less sensilla of the sacculus, a unique invagination of the third antennal segment. Indeed, calcium imaging showed that IR40a neurons directly respond to changes in humidity, and IR40a knockdown or IR93a mutation reduced their responses to stimuli. Taken together, our resultssuggest that the preference for a specific humidity range depends on specialized sacculus neurons, and that the processing of environmental humidity can happen largely in parallel to that of temperature.

Status of and Future Research on Thermosensory Processing.

Thermosensation is critically important for survival of all animals. In the cockroach Periplaneta americana, thermoreceptor neurons on antennae and thermosensory interneurons in the antennal lobe have been characterized electrophysiologically, and recent studies using advanced transgenic technologies in the fruit fly Drosophila melanogaster have added much to the knowledge of these neurons, enabling us to discuss common principles of thermosensory processing systems in insects. Cockroaches and many other insects possess only one type of thermoreceptor neurons on antennae that are excited by cooling and inhibited by warming. In contrast, the antennae of fruit flies and other dipterans possess oppositely responding warm and cold receptor neurons. Despite differences in their thermoreceptive equipment, central processing of temperature information is much the same in flies and cockroaches. Axons of thermoreceptor neurons project to the margin of the antennal lobe and form glomeruli, from which cold, warm and cold-warm projection neurons originate, the last neurons being excited by both cooling and warming. Axons of antennal lobe thermosensory projection neurons of the antennal lobe terminate in three distinct areas of the protocerebrum, the mushroom body, lateral horn and posterior lateral protocerebrum, the last area also receiving termination of hygrosensory projection neurons. Such multiple thermosensory pathways may serve to control multiple forms of thermosensory behavior. Electrophysiological studies on cockroaches and transgenic approaches in flies are encouraged to complement each other for further elucidating general principles of thermosensory processing in the insect brain.

A Glial K/Cl Transporter Controls Neuronal Receptive Ending Shape by Chloride Inhibition of an rGC

Neurons receive input from the outside world or from other neurons through neuronal receptive endings (NREs). Glia envelop NREs to create specialized microenvironments; however, glial functions at these sites are poorly understood. Here, we report a molecular mechanism by which glia control NRE shape and associated animal behavior. The C.elegans AMsh glial cell ensheathes the NREs of12 neurons, including the thermosensory neuron AFD. KCC-3, a K/Cl transporter, localizes specifically to a glial microdomain surrounding AFD receptive ending microvilli, where it regulates K(+) and Cl(-) levels. Wefindthat Cl(-) ions function as direct inhibitorsofanNRE-localized receptor-guanylyl-cyclase, GCY-8, which synthesizes cyclic guanosine monophosphate (cGMP). High cGMP mediates the effects of glial KCC-3 on AFD shape by antagonizing the actinregulator WSP-1/NWASP. Components of this pathway are broadly expressed throughout the nervous system, suggesting that ionic regulation of theNRE microenvironment may be a conserved mechanism by which glia control neuron shape and function.

Reconstruction of Spatial Thermal Gradient Encoded in Thermosensory Neuron AFD in Caenorhabditis elegans.

Deciphering how information is encoded in the nervous system is an important challenge for understanding the principles of information processing in neural circuits. During navigation behavior, animals transform spatial information to temporal patterns of neural activity. To elucidate how a sensory system achieves this transformation, we focused on a thermosensory neuron in Caenorhabditis elegans called AFD, which plays a major role in a sensory behavior. Using tracking and calcium imaging system for freely moving animals, we identified the response property of the AFD. The identified response property enabled us to reconstruct both neural activity from a temperature stimulus and a spatial thermal environment from neural activity. These results shed light on how a sensory system encodes the environment.

DTRPA1 in Non-circadian Neurons Modulates Temperature-dependent Rhythmic Activity in Drosophila melanogaster.

In fruit flies Drosophila melanogaster, environmental cycles of light and temperature are known to influence behavioral rhythms through dedicated sensory receptors. But the thermosensory pathways and molecular receptors by which thermal cycles modulate locomotor activity rhythms remain unclear. Here, we report that neurons expressing warmth-activated ion channel Drosophila Transient Receptor Potential-A1 (dTRPA1) modulate distinct aspects of the rhythmic activity/rest rhythm in a light-dependent manner. Under light/dark (LD) cycles paired with constantly warm ambient conditions, flies deficient in dTRPA1 expression are unable to phase morning and evening activity bouts appropriately. Correspondingly, we show that electrical activity of a few neurons targeted by the dTRPA1(SH)-GAL4 driver modulates temperature-dependent phasing of activity/rest rhythm under LD cycles. The expression of dTRPA1 also affects behavior responses to temperature cycles combined with constant dark (DD) or light (LL) conditions. We demonstrate that the mid-day "siesta" exhibited by flies under temperature cycles in DD is dependent on dTRPA1 expression in a small number of neurons that include thermosensory anterior cell neurons. Although a small subset of circadian pacemaker neurons may express dTRPA1, we show that CRY-negative dTRPA1(SH)-GAL4 driven neurons are critical for the suppression of mid-thermophase activity, thus enabling flies to exhibit siesta. In contrast to temperature cycles in DD, under LL, dTRPA1 is not required for exhibiting siesta but is important for phasing of evening peak. Our studies show that activity/rest rhythms are modulated in a temperature-dependent manner via signals from dTRPA1(SH)-GAL4 driven neurons. Taken together, these results emphasize the differential influence of thermoreceptors on rhythmic behavior in fruit flies in coordination with light inputs.

Single-Cell Memory Regulates a Neural Circuit for Sensory Behavior.

Unveiling the molecular and cellular mechanisms underlying memory has been a challenge for the past few decades. Although synaptic plasticity is proven to be essential for memory formation, the significance of "single-cell memory" still remains elusive. Here, we exploited a primary culture system for the analysis of C.elegans neurons and show that a single thermosensory neuron has an ability to form, retain, and reset a temperature memory. Genetic and proteomic analyses found that the expression of the single-cell memory exhibits inter-individual variability, which is controlled by the evolutionarily conserved CaMKI/IV and Raf pathway. The variable responses of a sensory neuron influenced the neural activity of downstream interneurons, suggesting that modulation of the sensory neurons ultimately determines the behavioral output in C.elegans. Our results provide proof of single-cell memory and suggest that the individual differences in neural responses at the single-cell level can confer individuality.