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Individual sensory neurons can be tuned to many stimuli, each driving unique, stimulus-relevant behaviors, and the ability of multimodal nociceptor neurons to discriminate between potentially harmful and innocuous stimuli is broadly important for organismal survival. Moreover, disruptions in the capacity to differentiate between noxious and innocuous stimuli can result in neuropathic pain. Drosophila larval Class III (CIII) neurons are peripheral noxious cold nociceptors and gentle touch mechanosensors; high levels of activation drive cold-evoked contraction (CT) behavior, while low levels of activation result in a suite of touch-associated behaviors. However, it is unknown what molecular factors underlie CIII multimodality. Here, we show that the TMEM16/anoctamins subdued and white walker (wwk; CG15270) are required for cold-evoked CT, but not for touch-associated behavior, indicating a conserved role for anoctamins in nociception. We also evidence that CIII neurons make use of atypical depolarizing chloride currents to encode cold, and that overexpression of ncc69—a fly homologue of NKCC1—results in phenotypes consistent with neuropathic sensitization, including behavioral hypersensitization and spontaneous nociceptor activity, making Drosophila CIII neurons a candidate system for future studies of the basic mechanisms underlying neuropathic pain.

Chronic heart failure (HF) is a serious disorder that afflicts more than 26 million patients worldwide. HF is comorbid with depression, anxiety and memory deficits that have serious implications for quality of life and self-care in patients who have HF. Still, there are few studies that have assessed the effects of severely reduced ejection fraction (≤40 %) on cognition in non-human animal models. Moreover, limited information is available regarding the effects of HF on genetic markers of synaptic plasticity in brain areas critical for memory and mood regulation. We induced HF in male rats and tested mood and anxiety (sucrose preference and elevated plus maze) and memory (spontaneous alternation and inhibitory avoidance) and measured the simultaneous expression of 84 synaptic plasticity-associated genes in dorsal (DH) and ventral hippocampus (VH), basolateral (BLA) and central amygdala (CeA) and prefrontal cortex (PFC). We also included the hypothalamic paraventricular nucleus (PVN), which is implicated in neurohumoral activation in HF. Our results show that rats with severely reduced ejection fraction recapitulate behavioral symptoms seen in patients with chronic HF including, increased anxiety and impaired memory in both tasks. HF also downregulated several synaptic-plasticity genes in PFC and PVN, moderate decreases in DH and CeA and minimal effects in BLA and VH. Collectively, these findings identify candidate brain areas and molecular mechanisms underlying HF-induced disturbances in mood and memory.

Chronic heart failure (HF) is a serious disorder that afflicts more than 26 million patients worldwide. HF is comorbid with depression, anxiety and memory deficits that have serious implications for quality of life and self-care in patients who have HF. Despite evidence that cognitive performance is worse in HF patients with reduced ejection fraction than in HF patients with preserved cardiac function, there are few studies that have assessed the effects of severely reduced ejection fraction (≤40%) on cognition in non-human animal models. Moreover, very limited information is available regarding the effects of HF on genetic markers of synaptic plasticity in brain areas critical for memory and mood regulation. We induced HF in male rats and tested mood and anxiety (sucrose preference and elevated plus maze) and memory (spontaneous alternation and inhibitory avoidance) and measured the simultaneous expression of 84 synaptic plasticity-associated genes in dorsal (DH) and ventral hippocampus (VH), basolateral (BLA) and central amygdala (CeA,) and prefrontal cortex (PFC). We also included the hypothalamic paraventricular nucleus (PVN), which has been implicated in neurohumoral activation in HF. Our results show that rats with severely reduced ejection fraction displayed signs of polydipsia, anhedonia, increased anxiety, and impaired memory in both tasks. HF also produced a drastic downregulation of synaptic-plasticity genes in PFC and PVN, moderate decreases in DH and CeA and minimal effects in BLA and VH. Collectively, these findings identify candidate brain areas and molecular mechanisms underlying HF-induced disturbances in mood and memory.

Summary The precise spatiotemporal characteristics of subcellular calcium (Ca2+) transients are critical for the physiological processes. Here we report a green Ca2+ sensor called “G-CatchER+” using a protein design to report rapid local ER Ca2+ dynamics with significantly improved folding properties. G-CatchER+ exhibits a superior Ca2+ on rate to G-CEPIA1er and has a Ca2+-induced fluorescence lifetimes increase. G-CatchER+ also reports agonist/antagonist triggered Ca2+ dynamics in several cell types including primary neurons that are orchestrated by IP3Rs, 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 Ca2+ 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 Ca2+ dynamics. G-CatchER+ will be an invaluable tool to examine local ER/SR Ca2+ dynamics and facilitate drug development associated with ER dysfunction., Graphical Abstract, Highlights • G-CatchER+ exhibits superior kinetics with 1:1 stoichiometry than G-CEPIA1er • G-CatchER+ captures spatially confined ER Ca2+ dynamics in hippocampal neurons • G-CatchER+-JP45 reports rapid Ca2+ signals adjacent to the junctional SR membrane • G-CatchER+ reports stimulus-evoked SR local Ca2+ dynamics in Drosophila muscle, Biochemistry; cell biology; biophysics

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 Drosophila melanogaster 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 in vivo 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.

Additional file 1:. Figure S1: Heart failure surgery, qPCR and three-dimensional reconstruction (a) representative image showing the echocardiographic assessment left ventricular cardiac function in a sham and HF rats, (b) timetable depicting morphometric assessments and qPCR at 8-, 14- and 16- weeks post-surgery, (c, d) representative confocal images showing IBA1-labeled microglia (red) in the PVN and CeA and the topographic delineation of the nuclei, (e) schematic depiction of the sequential steps used for the 3D morphometric reconstruction and assessment of microglia (IBA1, green), (f) confocal images show the step-by-step process for three-dimensional reconstruction of a single microglia (IBA1, red). For image processing, colors were changed from red to green in IMARIS for better visibility and increased precision during manual editing of reconstructed microglia. Note the merging of red (raw fluorescence) and green (surface reconstruction) channels in the top panel, 3V: third ventricle, AW: anterior wall, CeC: central amygdala capsular division, CeL: central amygdala lateral division, CeM: central amygdala medial division, cst: commissural stria terminalis, LVID, d and LVID, s: left ventricle internal dimensions in diastole and systole, PVN: paraventricular nucleus, PW: posterior wall. Figure S2: Sham surgery per se does not alter the morphology of PVN or CeA microglia (a) brain scheme shows the topographic location of PVN microglia that have been used for the morphometric assessment. The red area within the PVN (heart-shaped nucleus) highlights the fraction of the nucleus where pictures were taken, (b) dot-plot graphs show the individual values of PVN microglia for surface area, cell volume, filament length and IBA 1 intensity for the non-surgery control (N=357 cells from 4 rats), and sham rats at 8, 14 and 16 weeks post-surgery (N=381 cells from 4 rats, N= 365 cells from 4 rats and N=389 cells from 4 rats, respectively), one-way ANOVA was used to analyze the data followed by Tukey post-hoc test, (c) brain scheme shows the topographic location of CeA microglia that have been used for the morphometric assessment. The red area within the CeA highlights the divisions where pictures were taken, (d) dot-plot graphs show the individual values of CeA microglia for surface area, cell volume, filament length and IBA 1 intensity for the non-surgery control (N=297 cells from 4 rats), and sham rats at 8, 14 and 16 weeks post-surgery (N=377 cells from 4 rats, N=339 cells from 4 rats and N=354 cells from 4 rats, respectively). Figure S3: HF does not induce morphometric changes in somatosensory cortex 1 barrel field microglia (a) representative confocal images show IBA1-stained microglia in the S1BF of HF and sham rats 16 weeks post-surgery, (b) brain scheme shows the topographic location of S1BF microglia that have been used for the morphometric assessment. The red area highlights the S1BF, (c) dot-plot graphs show the individual values of S1BF microglia for surface area, cell volume, filament length, microglial branches, microglial segments, filament terminals and IBA 1 intensity for sham rats (n=4) and HF rats (n= 4). One-way ANOVA was used to analyze the data. Figure S4: Morphological changes in microglia are not observed in rats with mild level of HF (a) representative confocal images show IBA1-stained microglia in the PVN of sham and rats that underwent heart failure surgery, but did not develop severe HF (‘mild HF’), indicated by an ejection fraction greater than 50% (EF>50%), (b) brain scheme shows the topographic location of PVN microglia that have been used for the morphometric assessment. The red area highlights the PVN, (c) dot-plot graphs show the individual values of PVN microglia for surface area, cell volume, filament length, microglial branches, microglial segments, filament terminals and IBA 1 intensity for sham rats (n=4) and mild HF rats (n=5). One-way ANOVA was used to analyze the data, (d) plot graphs depicting cell volume, filament length and number of microglial branches as a function of ï values combining sham (n=4), HF rats (EF50%, n=5). R2 and p values were obtained following a Pearson correlation analysis. Figure S5: HF-induced changes in neuroinflammation-associated microglia and astrocyte marker mRNA levels (a-f) bar graphs show the mean fold change in mRNA transcript levels in HF rats compared to sham rats in the PVN (a, b) and the CeA (c, d). All graphs depict the fold changes of mRNA levels in HF group compared to their respective shams. Green bars: cytokines, red bars: a1 astrocyte markers, blue bars: a2 astrocyte markers. Dashed lines indicate the hypothetical mean (+1/-1) for the one-sample t-test. Each dot represents the average value of the pooled samples and the measurements were performed in triplicates. *p

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.

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.

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 aDrosophilaassay 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) channels Polycystic kidney disease gene 2 (Pkd2) andbrivido-1(brv1) are required for these behavioral shifts. OurDrosophilamodel will enable a sophisticated molecular genetic dissection of genes and circuits involved in cold nociceptive sensitization.

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.

Nociception is a protective mechanism that mediates behavioral responses to a range of potentially damaging stimuli, including noxious temperature, chemicals, and mechanical stimulation. Nociceptive mechanisms are found throughout metazoans. Noxious stimuli are transduced by specialized, high-threshold peripheral nociceptors, which fire action potentials to elicit adaptive behavioral responses. Nociception is essential for survival and provides a mechanism for sensory perception of noxious stimuli, which alerts the organism to potential environmental dangers. When coupled with pain sensation and complex behavioral responses, this mechanism protects the organism from incipient damage. Moreover, acute and chronic pain may manifest as altered nociception in neuropathic pain states. Elucidating the neural bases of nociception is therefore important for identifying and implementing novel strategies for the treatment of neuropathic pain, as well as uncovering the mechanistic bases by which the nervous system integrates information to produce specific behaviors in response to a range of noxious stimuli. Invertebrate organisms, such as Drosophila melanogaster and Caenorhabditis elegans, have emerged as powerful, genetically tractable platforms for exploring these questions. Here, we concisely review the current state of knowledge regarding the cells, molecules, neural circuits, and behaviors associated with invertebrate nociception in the fruit fly and nematode worm.

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.

Department of Chemistry, South Gujarat University, Surat-395 007 Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar-388 120 Manuscript received 22 June 1992. revised 23 December 1993, accepted 13 January 1994 Equilibrium Studies on Lanthanone Ions Praseodymium-, Neodymium-, Dysprosium- and Europium(III) with Thio Acids and the corresponding Amino Acids