SUMMARY The basic mechanisms underlying noxious cold perception are not well understood. We developed Drosophila assays for noxious cold responses. Larvae respond to near-freezing temperatures via a mutually exclusive set of singular behaviors–in particular, a full body contraction (CT). Class III (CIII) multidendritic sensory neurons are specifically activated by cold and optogenetic activation of these neurons elicits CT. Blocking synaptic transmission in CIII neurons inhibits CT. Genetically, the transient receptor potential (TRP) channels Trpm, NompC, and Polycystic kidney disease 2 (Pkd2) are expressed in CIII neurons where each is required for CT. Misexpression of Pkd2 is sufficient to confer cold-responsiveness. The optogenetic activation level of multimodal CIII neurons determines behavioral output, and visualization of neuronal activity supports this conclusion. Coactivation of cold and heat responsive sensory neurons suggests that the cold-evoked response circuitry is dominant. Our Drosophila model will enable a sophisticated molecular genetic dissection of cold nociceptive genes and circuits.

Transcription factors (TFs) have emerged as essential cell autonomous mediators of subtype specific dendritogenesis; however, the downstream effectors of these TFs remain largely unknown, as are the cellular events that TFs control to direct morphological change. As dendritic morphology is largely dictated by the organization of the actin and microtubule (MT) cytoskeletons, elucidating TF-mediated cytoskeletal regulatory programs is key to understanding molecular control of diverse dendritic morphologies. Previous studies in have demonstrated that the conserved TFs Cut and Knot exert combinatorial control over aspects of dendritic cytoskeleton development, promoting actin and MT-based arbor morphology, respectively. To investigate transcriptional targets of Cut and/or Knot regulation, we conducted systematic neurogenomic studies, coupled with genetic screens utilizing multi-fluor cytoskeletal and membrane marker reporters. These analyses identified a host of putative Cut and/or Knot effector molecules, and a subset of these putative TF targets converge on modulating dendritic cytoskeletal architecture, which are grouped into three major phenotypic categories, based upon neuromorphometric analyses: complexity enhancer, complexity shifter, and complexity suppressor. Complexity enhancer genes normally function to promote higher order dendritic growth and branching with variable effects on MT stabilization and F-actin organization, whereas complexity shifter and complexity suppressor genes normally function in regulating proximal-distal branching distribution or in restricting higher order branching complexity, respectively, with spatially restricted impacts on the dendritic cytoskeleton. Collectively, we implicate novel genes and cellular programs by which TFs distinctly and combinatorially govern dendritogenesis via cytoskeletal modulation.

Background: Cardiovascular diseases, including heart failure, are the most common cause of death globally. Recent studies support a high degree of comorbidity between heart failure and cognitive and mood disorders resulting in memory loss, depression, and anxiety. While neuroinflammation in the hypothalamic paraventricular nucleus contributes to autonomic and cardiovascular dysregulation in heart failure, mechanisms underlying cognitive and mood disorders in this disease remain elusive. The goal of this study was to quantitatively assess markers of neuroinflammation (glial morphology, cytokines, and A1 astrocyte markers) in the central amygdala, a critical forebrain region involved in emotion and cognition, and to determine its time course and correlation to disease severity during the progression of heart failure. Methods: We developed and implemented a comprehensive microglial/astrocyte profiler for precise threedimensional morphometric analysis of individual microglia and astrocytes in specific brain nuclei at different time points during the progression of heart failure. To this end, we used a well-established ischemic heart failure rat model. Morphometric studies were complemented with quantification of various pro-inflammatory cytokines and A1/A2 astrocyte markers via qPCR. Results: We report structural remodeling of central amygdala microglia and astrocytes during heart failure that affected cell volume, surface area, filament length, and glial branches, resulting overall in somatic swelling and deramification, indicative of a change in glial state. These changes occurred in a time-dependent manner, correlated with the severity of heart failure, and were delayed compared to changes in the hypothalamic paraventricular nucleus. Morphometric changes correlated with elevated mRNA levels of pro-inflammatory cytokines and markers of reactive A1-type astrocytes in the paraventricular nucleus and central amygdala during heart failure.

Summary The precise spatiotemporal characteristics of subcellular calcium (Ca 2+ ) transients are critical for the physiological processes. Here we report a green Ca 2+ sensor called “G-CatchER + ” using a protein design to report rapid local ER Ca 2+ dynamics with significantly improved folding properties. G-CatchER + exhibits a superior Ca 2+ on rate to G-CEPIA1er and has a Ca 2+ -induced fluorescence lifetimes increase. G-CatchER + also reports agonist/antagonist triggered Ca 2+ dynamics in several cell types including primary neurons that are orchestrated by IP 3 Rs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER + -JP45), we report a rapid local Ca 2+ release that is likely due to calsequestrin. Transgenic expression of G-CatchER + in Drosophila muscle demonstrates its utility as an in vivo reporter of stimulus-evoked SR local Ca 2+ dynamics. G-CatchER + will be an invaluable tool to examine local ER/SR Ca 2+ dynamics and facilitate drug development associated with ER dysfunction.

Diabetes-associated nociceptive hypersensitivity affects diabetic patients with hard-to-treat chronic pain. Because multiple tissues are affected by systemic alterations in insulin signaling, the functional locus of insulin signaling in diabetes-associated hypersensitivity remains obscure. Here, we used Drosophila nociception/nociceptive sensitization assays to investigate the role of Insulin receptor (Insulin-like receptor, InR) in nociceptive hypersensitivity. InR mutant larvae exhibited mostly normal baseline thermal nociception (absence of injury) and normal acute thermal hypersensitivity following UV-induced injury. However, their acute thermal hypersensitivity persists and fails to return to baseline, unlike in controls. Remarkably, injury-induced persistent hypersensitivity is also observed in larvae that exhibit either type 1 or type 2 diabetes. Cell type-specific genetic analysis indicates that InR function is required in multidendritic sensory neurons including nociceptive class IV neurons. In these same nociceptive sensory neurons, only modest changes in dendritic morphology were observed in the InRRNAi-expressing and diabetic larvae. At the cellular level, InR-deficient nociceptive sensory neurons show elevated calcium responses after injury. Sensory neuron-specific expression of InR rescues the persistent thermal hypersensitivity of InR mutants and constitutive activation of InR in sensory neurons ameliorates the hypersensitivity observed with a type 2-like diabetic state. Our results suggest that a sensory neuron-specific function of InR regulates the persistence of injury-associated hypersensitivity. It is likely that this new system will be an informative genetically tractable model of diabetes-associated hypersensitivity.

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To investigate cellular, molecular and behavioral mechanisms of noxious cold detection, we developed cold plate behavioral assays and quantitative means for evaluating the predominant noxious cold-evoked contraction behavior. To characterize neural activity in response to noxious cold, we implemented a GCaMP6-based calcium imaging assay enabling in vivo studies of intracellular calcium dynamics in intact Drosophila larvae. We identified Drosophila class III multidendritic (md) sensory neurons as multimodal sensors of innocuous mechanical and noxious cold stimuli and to dissect the mechanistic bases of multimodal sensory processing we developed two independent functional assays. First, we developed an optogenetic dose response assay to assess whether levels of neural activation contributes to the multimodal aspects of cold sensitive sensory neurons. Second, we utilized CaMPARI, a photo-switchable calcium integrator that stably converts fluorescence from green to red in presence of high intracellular calcium and photo-converting light, to assess in vivo functional differences in neural activation levels between innocuous mechanical and noxious cold stimuli. These novel assays enable investigations of behavioral and functional roles of peripheral sensory neurons and multimodal sensory processing in Drosophila larvae.

Nociceptive sensitization involves an increase in responsiveness of pain sensing neurons to sensory stimuli, typically through the lowering of their nociceptive threshold. Nociceptive sensitization is common following tissue damage, inflammation, and disease and serves to protect the affected area while it heals. Organisms can become sensitized to a range of noxious and innocuous stimuli, including thermal stimuli. The basic mechanisms underlying sensitization to warm or painfully hot stimuli have begun to be elucidated, however, sensitization to cold is not well understood. Here, we develop a Drosophila assay to study cold sensitization after UV-induced epidermal damage in larvae. Larvae respond to acute cold stimuli with a set of unique behaviors that include a contraction of the head and tail (CT) or a raising of the head and tail into a U-Shape (US). Under baseline, non-injured conditions larvae primarily produce a CT response to an acute cold (10°C) stimulus, however, we show that cold-evoked responses shift following tissue damage: CT responses decrease, US responses increase and some larvae exhibit a lateral body roll (BR) that is typically only observed in response to high temperature and noxious mechanical stimuli. At the cellular level, class III neurons are required for the decrease in CT, chordotonal neurons are required for the increase in US, and chordotonal and class IV neurons are required for the appearance of BR responses after UV. At the molecular level, we found that the transient receptor potential (TRP) channel brivido-1 (brv1) is required for these behavioral shifts. Our Drosophila model will allow us to precisely identify the genes and circuits involved in cold nociceptive sensitization.