CCT and Cullin1 Regulate the TORC1 Pathway to Promote Dendritic Arborization in Health and Disease.

The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, we confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, we examine CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.

Neural substrates of cold nociception in Drosophila larva.

Metazoans detect and differentiate between innocuous (non-painful) and/or noxious (harmful) environmental cues using primary sensory neurons, which serve as the first node in a neural network that computes stimulus specific behaviors to either navigate away from injury-causing conditions or to perform protective behaviors that mitigate extensive injury. The ability of an animal to detect and respond to various sensory stimuli depends upon molecular diversity in the primary sensors and the underlying neural circuitry responsible for the relevant behavioral action selection. Recent studies in Drosophila larvae have revealed that somatosensory class III multidendritic (CIII md) neurons function as multimodal sensors regulating distinct behavioral responses to innocuous mechanical and nociceptive thermal stimuli. Recent advances in circuit bases of behavior have identified and functionally validated Drosophila larval somatosensory circuitry involved in innocuous (mechanical) and noxious (heat and mechanical) cues. However, central processing of cold nociceptive cues remained unexplored. We implicate multisensory integrators (Basins), premotor (Down-and-Back) and projection (A09e and TePns) neurons as neural substrates required for cold-evoked behavioral and calcium responses. Neural silencing of cell types downstream of CIII md neurons led to significant reductions in cold-evoked behaviors and neural co-activation of CIII md neurons plus additional cell types facilitated larval contraction (CT) responses. We further demonstrate that optogenetic activation of CIII md neurons evokes calcium increases in these neurons. Collectively, we demonstrate how Drosophila larvae process cold stimuli through functionally diverse somatosensory circuitry responsible for generating stimulus specific behaviors.

CCT and Cullin1 regulate the TORC1 pathway to promote dendritic arborization in health and disease.

The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in homeostatic dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multidendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1), in vitro. Through targeted genetic manipulations, we have confirmed that LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases dendritic complexity in CIV neurons. Both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus we examined CIV dendritic development in disease conditions as well. Expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by TORC1 disinhibition via Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through regulation of TORC1 in both health and disease.

Chloride-dependent mechanisms of multimodal sensory discrimination and nociceptive sensitization in Drosophila.

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 innocuous 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 sensitization and neuronal hyperexcitability, making Drosophila CIII neurons a candidate system for future studies of the basic mechanisms underlying neuropathic pain.

PP2A phosphatase regulates cell-type specific cytoskeletal organization to drive dendrite diversity.

Uncovering molecular mechanisms regulating dendritic diversification is essential to understanding the formation and modulation of functional neural circuitry. Transcription factors play critical roles in promoting dendritic diversity and here, we identify PP2A phosphatase function as a downstream effector of Cut-mediated transcriptional regulation of dendrite development. Mutant analyses of the PP2A catalytic subunit (mts) or the scaffolding subunit (PP2A-29B) reveal cell-type specific regulatory effects with the PP2A complex required to promote dendritic growth and branching in Drosophila Class IV (CIV) multidendritic (md) neurons, whereas in Class I (CI) md neurons, PP2A functions in restricting dendritic arborization. Cytoskeletal analyses reveal requirements for Mts in regulating microtubule stability/polarity and F-actin organization/dynamics. In CIV neurons, mts knockdown leads to reductions in dendritic localization of organelles including mitochondria and satellite Golgi outposts, while CI neurons show increased Golgi outpost trafficking along the dendritic arbor. Further, mts mutant neurons exhibit defects in neuronal polarity/compartmentalization. Finally, genetic interaction analyses suggest ß-tubulin subunit 85D is a common PP2A target in CI and CIV neurons, while FoxO is a putative target in CI neurons.

Modality specific roles for metabotropic GABAergic signaling and calcium induced calcium release mechanisms in regulating cold nociception.

Calcium (Ca2+) plays a pivotal role in modulating neuronal-mediated responses to modality-specific sensory stimuli. Recent studies in Drosophila reveal class III (CIII) multidendritic (md) sensory neurons function as multimodal sensors regulating distinct behavioral responses to innocuous mechanical and nociceptive thermal stimuli. Functional analyses revealed CIII-mediated multimodal behavioral output is dependent upon activation levels with stimulus-evoked Ca2+ displaying relatively low vs. high intracellular levels in response to gentle touch vs. noxious cold, respectively. However, the mechanistic bases underlying modality-specific differential Ca2+ responses in CIII neurons remain incompletely understood. We hypothesized that noxious cold-evoked high intracellular Ca2+ responses in CIII neurons may rely upon Ca2+ induced Ca2+ release (CICR) mechanisms involving transient receptor potential (TRP) channels and/or metabotropic G protein coupled receptor (GPCR) activation to promote cold nociceptive behaviors. Mutant and/or CIII-specific knockdown of GPCR and CICR signaling molecules [GABA B -R2, Gαq, phospholipase C, ryanodine receptor (RyR) and Inositol trisphosphate receptor (IP3R)] led to impaired cold-evoked nociceptive behavior. GPCR mediated signaling, through GABA B -R2 and IP3R, is not required in CIII neurons for innocuous touch evoked behaviors. However, CICR via RyR is required for innocuous touch-evoked behaviors. Disruptions in GABA B -R2, IP3R, and RyR in CIII neurons leads to significantly lower levels of cold-evoked Ca2+ responses indicating GPCR and CICR signaling mechanisms function in regulating Ca2+ release. CIII neurons exhibit bipartite cold-evoked firing patterns, where CIII neurons burst during rapid temperature change and tonically fire during steady state cold temperatures. GABA B -R2 knockdown in CIII neurons resulted in disorganized firing patterns during cold exposure. We further demonstrate that application of GABA or the GABA B specific agonist baclofen potentiates cold-evoked CIII neuron activity. Upon ryanodine application, CIII neurons exhibit increased bursting activity and with CIII-specific RyR knockdown, there is an increase in cold-evoked tonic firing and decrease in bursting. Lastly, our previous studies implicated the TRPP channel Pkd2 in cold nociception, and here, we show that Pkd2 and IP3R genetically interact to specifically regulate cold-evoked behavior, but not innocuous mechanosensation. Collectively, these analyses support novel, modality-specific roles for metabotropic GABAergic signaling and CICR mechanisms in regulating intracellular Ca2+ levels and cold-evoked behavioral output from multimodal CIII neurons.

Heart failure impairs mood and memory in male rats and down-regulates the expression of numerous genes important for synaptic plasticity in related brain regions.

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.

Rapid subcellular calcium responses and dynamics by calcium sensor G-CatchER.

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.

Correction to: Three-dimensional morphometric analysisreveals time-dependent structural changesin microglia and astrocytes in the centralamygdala and hypothalamicparaventricular nucleus of heart failure rats.

An amendment to this paper has been published and can be accessed via the original article.

Three-dimensional morphometric analysis reveals time-dependent structural changes in microglia and astrocytes in the central amygdala and hypothalamic paraventricular nucleus of heart failure rats.

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 three-dimensional 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. CONCLUSION: We provide evidence that in addition to the previously described hypothalamic neuroinflammation implicated in sympathohumoral activation during heart failure, microglia, and astrocytes within the central amygdala also undergo structural remodeling indicative of glial shifts towards pro-inflammatory phenotypes. Thus, our studies suggest that neuroinflammation in the amygdala stands as a novel pathophysiological mechanism and potential therapeutic target that could be associated with emotional and cognitive deficits commonly observed at later stages during the course of heart failure.

Injury-induced cold sensitization in Drosophila larvae involves behavioral shifts that require the TRP channel Brv1.

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.

Drosophila Insulin receptor regulates the persistence of injury-induced nociceptive sensitization.

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.

Dendritic Cytoskeletal Architecture Is Modulated by Combinatorial Transcriptional Regulation in Drosophila melanogaster.

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.

Behavioral and Functional Assays for Investigating Mechanisms of Noxious Cold Detection and Multimodal Sensory Processing in Drosophila Larvae.

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.

The TRP Channels Pkd2, NompC, and Trpm Act in Cold-Sensing Neurons to Mediate Unique Aversive Behaviors to Noxious Cold in Drosophila.

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.