Nociceptors are a subset of small primary afferent neurons that respond to noxious chemical, thermal and mechanical stimuli. Ion channels in nociceptors respond differently to noxious stimuli and generate electrical signals in different ways. Anoctamin 1 (ANO1 also known as TMEM16A) is a Ca(2+)-activated chloride channel that is essential for numerous physiological functions. We found that ANO1 was activated by temperatures over 44 °C with steep heat sensitivity. ANO1 was expressed in small sensory neurons and was highly colocalized with nociceptor markers, which suggests that it may be involved in nociception. Application of heat ramps to dorsal root ganglion (DRG) neurons elicited robust ANO1-dependent depolarization. Furthermore, knockdown or deletion of ANO1 in DRG neurons substantially reduced nociceptive behavior in thermal pain models. These results indicate that ANO1 is a heat sensor that detects nociceptive thermal stimuli in sensory neurons and possibly mediates nociception.

Retinal circuits detect salient features of the visual world and report them to the brain through spike trains of retinal ganglion cells. The most abundant ganglion cell type in mice, the so-called W3 ganglion cell, selectively responds to movements of small objects. Where and how object motion sensitivity arises in the retina is incompletely understood. In this study, we use 2-photon-guided patch-clamp recordings to characterize responses of vesicular glutamate transporter 3 (VGluT3)-expressing amacrine cells (ACs) to a broad set of visual stimuli. We find that these ACs are object motion sensitive and analyze the synaptic mechanisms underlying this computation. Anatomical circuit reconstructions suggest that VGluT3-expressing ACs form glutamatergic synapses with W3 ganglion cells, and targeted recordings show that the tuning of W3 ganglion cells' excitatory input matches that of VGluT3-expressing ACs' responses. Synaptic excitation of W3 ganglion cells is diminished, and responses to object motion are suppressed in mice lacking VGluT3. Object motion, thus, is first detected by VGluT3-expressing ACs, which provide feature-selective excitatory input to W3 ganglion cells.DOI: http://dx.doi.org/10.7554/eLife.08025.001

Summary Neurons that release more than one transmitter exist throughout the CNS. Yet, how these neurons deploy multiple transmitters and shape the function of specific circuits is not well understood. VGluT3-expressing amacrine cells (VG3-ACs) provide glutamatergic input to ganglion cells activated by contrast and motion. Using optogenetics, we find that VG3-ACs provide selective glycinergic input to a retinal ganglion cell type suppressed by contrast and motion (SbC-RGCs). Firing of SbC-RGCs is suppressed at light ON and OFF over a broad range of stimulus sizes. Anatomical circuit reconstructions reveal that VG3-ACs form inhibitory synapses preferentially on processes that link ON and OFF arbors of SbC-RGC dendrites. Removal of VG3-ACs from mature circuits reduces inhibition and attenuates spike suppression of SbC-RGCs in a contrast- and size-selective manner, illustrating the modularity of retinal circuits. VG3-ACs thus use dual transmitters in a target-specific manner, and shape suppressive contrast responses in the retina by glycinergic transmission.

Approaching predators cast expanding shadows (i.e., looming) that elicit innate defensive responses in most animals. Where looming is first detected and how critical parameters of predatory approaches are extracted are unclear. In mice, we identify a retinal interneuron (the VG3 amacrine cell) that responds robustly to looming, but not to related forms of motion. Looming-sensitive calcium transients are restricted to a specific layer of the VG3 dendrite arbor, which provides glutamatergic input to two ganglion cells (W3 and OFFα). These projection neurons combine shared excitation with dissimilar inhibition to signal approach onset and speed, respectively. Removal of VG3 amacrine cells reduces the excitation of W3 and OFFα ganglion cells and diminishes defensive responses of mice to looming without affecting other visual behaviors. Thus, the dendrites of a retinal interneuron detect visual threats, divergent circuits downstream extract critical threat parameters, and these retinal computations initiate an innate survival behavior.

1Transient protein-protein interactions (PPIs), such as those between posttranslational 2 modifying enzymes and their substrates, play key roles in cellular regulation, but are difficult 3 to identify. Here we demonstrate the application of enzyme-catalyzed proximity labeling (PL), 4 using the engineered promiscuous biotin ligase TurboID, as a sensitive method for 5 characterizing PPIs in signaling networks. We show that TurboID fused with the GSK3-like 6 kinase BIN2 or a PP2A phosphatase biotinylates their known substrate, the BZR1 transcription 7 factor, with high specificity and efficiency. We optimized the protocol of biotin labeling and 8 affinity purification in transgenic Arabidopsis expressing a BIN2-TurboID fusion protein. 9Subsequent quantitative mass spectrometry (MS) analysis identified about three hundred 10 proteins biotinylated by BIN2-TurboID more efficiently than the YFP-TurboID control. These 11 include a significant subset of previously proven BIN2 interactors and a large number of new 12 BIN2-proximal proteins that uncover a broad BIN2 signaling network. Our study illustrates 13 that PL-MS using TurboID is a powerful tool for mapping signaling networks, and reveals 14 broad roles of BIN2 kinase in cellular signaling and regulation in plants. 16Protein-protein interactions (PPIs) organize proteins into cellular structures, metabolic 18

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It has been shown that inhibition of GTPase-activating protein of ADP-ribosylation factor (Arf), ArfGAP, with a small molecule (QS11) results in synergistic activation of Wnt/β-catenin signaling. However, the role of Arf in Wnt/β-catenin signaling has not yet been elucidated. Here, we show that activation of Arf is essential for Wnt/β-catenin signaling. The level of the active form of Arf (Arf-GTP) transiently increased in the presence of Wnt, and this induction event was abrogated by blocking the interaction between Wnt and Frizzled (Fzd). In addition, knockdown of Fzds, Dvls or LRP6 blocked the Wnt-mediated activation of Arf. Consistently, depletion of Arf led to inhibition of Wnt-mediated membrane PtdIns (4,5)P2 (phosphatidylinositol 4, 5-bisphosphate) synthesis and LRP6 phosphorylation. Overall, our data suggest that transient activation of Arf modulates LRP6 phosphorylation for the transduction of Wnt/β-catenin signaling.

Physiological need states direct decision-making towards re-establishing homeostasis. Using a two-alternative-forced-choice task for mice that models elements of human decisions, we found that varying hunger and thirst states caused need-inappropriate choices, such as food-seeking when thirsty. These results show limits on interoceptive knowledge of hunger and thirst states to guide decision-making. Instead, need states were identified after food and water consumption by outcome evaluation, which depended on medial prefrontal cortex.