The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.
SUMMARY Thermosensation is an indispensable sensory modality. Here, we study temperature coding in Drosophila, and show that temperature is represented by a spatial map of activity in the brain. First, we identify new TRP channels and demonstrate they function in the fly antenna to mediate the detection of cold stimuli. Next, we identify the hot-sensing neurons and show that hot and cold antennal receptors project onto distinct, but adjacent glomeruli in the Proximal-Antennal-Protocerebrum (PAP) forming a thermotopic map in the brain. We use two-photon imaging to reveal the functional segregation of hot and cold responses in the PAP, and show that silencing the hot- or cold-sensing neurons produces animals with distinct and discrete deficits in their behavioral responses to thermal stimuli. Together, these results demonstrate that dedicated populations of cells orchestrate behavioral responses to different temperature stimuli, and reveal a labeled-line logic for the coding of temperature information in the brain.
Garlic's pungent flavor has made it a popular ingredient in cuisines around the world and throughout history. Garlic's health benefits have been elevated from folklore to clinical study. Although there is some controversy as to the efficacy of garlic, garlic products are one of the most popular herbal supplements in the U.S. Chemically complex, garlic contains different assortments of sulfur compounds depending on whether the cloves are intact, crushed, cooked, or raw. Raw garlic, when cut and placed on the tongue or lips, elicits painful burning and prickling sensations through unknown mechanisms. Here, we show that raw but not baked garlic activates TRPA1 and TRPV1, two temperature-activated ion channels that belong to the transient receptor potential (TRP) family. These thermoTRPs are present in the pain-sensing neurons that innervate the mouth. We further show that allicin, an unstable component of fresh garlic, is the chemical responsible for TRPA1 and TRPV1 activation and is therefore likely to cause garlic's pungency.
Members of the transient receptor potential (TRP) ion channel family mediate diverse sensory transduction processes in both vertebrates and invertebrates. In particular, members of the TRPA subfamily have distinct thermosensory roles in Drosophila, and mammalian TRPA1 is postulated to have a function in noxious cold sensation and mechanosensation. Here we show that mutations in trpa-1, the C. elegans ortholog of mouse Trpa1, confer specific defects in mechanosensory behaviors related to nose-touch responses and foraging. trpa-1 is expressed and functions in sensory neurons required for these mechanosensory behaviors, and contributes to neural responses of these cells to touch, particularly after repeated mechanical stimulation. Furthermore, mechanical pressure can activate C. elegans TRPA-1 heterologously expressed in mammalian cells. Collectively, these data demonstrate that trpa-1 encodes an ion channel that can be activated in response to mechanical pressure and is required for mechanosensory neuron function, suggesting a possible role in mechanosensory transduction or modulation.
Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system.
SUMMARY In Drosophila, rapid temperature changes are detected at the periphery by dedicated receptors forming a simple sensory map for hot and cold in the brain1. However, flies show a host of complex innate and learned responses to temperature, indicating that they are able to extract a range of information from this simple input. Here, we define the anatomical and physiological repertoire for temperature representation in the Drosophila brain. First, we use a photolabeling strategy2 to trace the connections that relay peripheral thermosensory information to higher brain centers, and show that they largely converge onto three target regions: the Mushroom Body, Lateral Horn (well-known centers for sensory processing) and the Posterior Lateral Protocerebrum, a region we now define as a major site of thermosensory representation. Then, using in vivo calcium imaging3, we describe the thermosensory projection neurons selectively activated by hot or cold stimuli. Fast-adapting neurons display transient “ON” and “OFF” responses and track rapid temperature shifts remarkably well, while slow-adapting cell responses better reflect the magnitude of simple thermal changes. Unexpectedly, we also find a population of ‘broadly-tuned’ cells that respond to both heating and cooling, and show that they are required for normal behavioral avoidance of both hot and cold in a simple 2-choice temperature preference assay. Taken together, our results uncover a coordinated ensemble of neural responses to temperature in the fly brain, demonstrate that a broadly tuned thermal-line contributes to rapid avoidance behavior, and illustrate how stimulus quality, temporal structure, and intensity can be extracted from a simple glomerular map at a single synaptic station.
SUMMARY Many visual animals have innate preferences for particular wavelengths of light, which can be modified by learning. Drosophila’s preference for UV over visible light requires UV-sensing R7 photoreceptors and specific wide-field amacrine neurons, called Dm8. Here we identify three types of medulla projection neurons downstream of R7 and Dm8, and show that selectively inactivating one of them (Tm5c) abolishes UV preference. Using a modified GRASP method to probe synaptic connections at the single-cell level, we reveal that each Dm8 neuron forms multiple synaptic contacts with Tm5c in the center of Dm8’s dendritic field, but sparse connections in the periphery. By single-cell transcript profiling and RNAi-mediated knockdown, we determine that Tm5c uses the kainate receptor Clumsy to receive excitatory glutamate input from Dm8. We conclude that R7s->Dm8->Tm5c form a hard-wired glutamatergic circuit that mediates UV preference by pooling ~16 R7 signals for transfer to the lobula, a higher visual center.
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