InteractiveFly: GeneBrief

Ryanodine receptor: Biological Overview | References

Gene name - Ryanodine receptor

Synonyms -

Cytological map position - 44F1-44F2

Function - calcium channel

Keywords - muscle, CNS, synapse, presynaptic vesiclar mobility, promotion of calcium homeostasis and phagocytosis

Symbol - RyR

FlyBase ID: FBgn0011286

Genetic map position - 2R:4,747,896..4,775,602

Classification - ryanodine-sensitive calcium-release channel activity

Cellular location - endoplasmic reticulum - transmembrane

NCBI links: EntrezGene

RyR orthologs: Biolitmine

Recent literature
Murmu, M. S. and Martin, J. R. (2016). Interaction between cAMP and intracellular Ca-signaling pathways during odor-perception and adaptation in Drosophila. Biochim Biophys Acta 1863: 2156-2174. PubMed ID: 27212269
Binding of an odorant to olfactory receptors triggers cascades of second messenger systems in olfactory receptor neurons (ORNs). The transduction mechanism at ORNs is mediated by cAMP and/or inositol,1,4,5-triphosphate (InsP3)-signaling pathways in an odorant-dependent manner. This study used interfering-RNAi to disrupt the level of cAMP alone or in combination with the InsP3-signaling pathway cellular targets, InsP3 receptor (InsP3R) or ryanodine receptor (RyR) in ORNs, and quantify at ORN axon terminals in the antennal lobe, the odor-induced Ca2+-response. In-vivo functional bioluminescence Ca2+-imaging indicates that a single 5s application of an odor increased Ca2+-transients at ORN axon terminals. However, compared to wild-type controls, the magnitude and duration of ORN Ca2+-response was significantly diminished in cAMP-defective flies. In a behavioral assay, perception of odorants was defective in flies with a disrupted cAMP level suggesting that the ability of flies to correctly detect an odor depends on cAMP. Simultaneous disruption of cAMP level and InsP3R or RyR further diminished the magnitude and duration of ORN response to odorants and affected the flies' ability to detect an odor. In conclusion, this study provides functional evidence that cAMP and InsP3-signaling pathways act in synergy to mediate odor processing within the ORN axon terminals, which is encoded in the magnitude and duration of ORN response.
Feng, R. et al. (2019). Dynamics expression of DmFKBP12/Calstabin during embryonic early development of Drosophila melanogaster. Cell Biosci 9: 8. PubMed ID: 30637096
Lack of calcium signaling protein FK506-binding protein 12 (FKBP12/Calstabin) is known to result in lethal cardiac dysfunction in mouse. This study identified both temporal and localization changes in expression of DmFKBP12, a translational and transcriptional regulator of Drosophila RyR and FKBP12, through embryonic development. DmFKBP12 is first expressed at the syncytial blastoderm stage and undergoes increased expression during the cellular blastoderm and early gastrulation stages. At late gastrulation, DmFKBP12 expression begins to decline until it reaches homeostasis, which it then maintains throughout the rest of development. DmFKBP12 mRNA remain stable, which indicates that protein dynamics are attributed to regulation at the mRNA to protein translation level. Although DmFKBP12 is distributed evenly between the anterior to posterior poles of the blastoderm egg, the protein is expressed more strongly in the cortex of the early Drosophila gastrula with the highest concentration found in the basement membrane of the cellular blastoderm. By late gastrulation, DmFKBP12 is no longer identified in the yolk or lumen of duct structures and has relocated to the future brain (suboesophageal and supraesophageal ganglions), ventral nervous system, and muscular system. Throughout these changes in distribution, in situ DmFKBP12 mRNA monitoring detected equal distribution of DmFKBP12 mRNA. As a critical regulator of the DmRyR-FKBP complex, DmFKBP12 expression in Drosophila fluctuates temporally and spatially with the formation of organ systems. These finding indicate that DmFKBP12 and RyR associated calcium signaling plays an essential role in the successful development of Drosophila melanogaster. Further study on the differences between mammalian RyR-FKBP12 and Drosophila DmRyR-FKBP12 can be exploited to develop safe pesticides.
Delrio-Lorenzo, A., Rojo-Ruiz, J., Alonso, M. T. and Garcia-Sancho, J. (2020). Sarcoplasmic reticulum Ca(2+) decreases with age and correlates with the decline in muscle function in Drosophila. J Cell Sci. PubMed ID: 32005702
Sarcopenia, the loss of muscle mass and strength associated to age, has been linked to impairment of the cytosolic Ca(2+) peak that triggers muscle contraction, but mechanistic details remain unknown. This study explored the hypothesis that a reduction in sarcoplasmic reticulum Ca(2+) concentration ([Ca(2+)]SR) is at the origin of this loss of Ca(2+) homeostasis. Drosophila melanogaster was engineered to express the Ca(2+) indicator GAP3 targeted to muscle SR, and a new method was developed to calibrate the signal into [Ca(2+)]SR. In vivo [Ca(2+)]SR dropped with age from approximately 600 microM down to 50 microM in close correlation to muscle function, which declined monotonically when [Ca(2+)]SR was <400 microM. [Ca(2+)]SR results from the pump-leak steady-state at the SR membrane. However, changes in expression of the SERCA pump and of the ryanodine receptor leak, were too modest to explain the large changes seen in [Ca(2+)]SR. Instead, these changes are compatible with increased leakiness through the ryanodine receptor as the main determinant of the [Ca(2+)]SR decline in aging muscle. In contrast, there were no changes in endoplasmic reticulum [Ca(2+)] with age in brain neurons.
Weiss, S., Clamon, L. C., Manoim, J. E., Ormerod, K. G., Parnas, M. and Littleton, J. T. (2021). Glial ER and GAP junction mediated Ca(2+) waves are crucial to maintain normal brain excitability. Glia. PubMed ID: 34528727
Astrocytes play key roles in regulating multiple aspects of neuronal function from invertebrates to humans and display Ca(2+) fluctuations that are heterogeneously distributed throughout different cellular microdomains. Changes in Ca(2+) dynamics represent a key mechanism for how astrocytes modulate neuronal activity. An unresolved issue is the origin and contribution of specific glial Ca(2+) signaling components at distinct astrocytic domains to neuronal physiology and brain function. The Drosophila model system offers a simple nervous system that is highly amenable to cell-specific genetic manipulations to characterize the role of glial Ca(2+) signaling. This study has identified a role for ER store-operated Ca(2+) entry (SOCE) pathway in perineurial glia (PG), a glial population that contributes to the Drosophila blood-brain barrier. PG cells display diverse Ca(2+) activity that varies based on their locale within the brain. Ca(2+) signaling in PG cells does not require extracellular Ca(2+) and is blocked by inhibition of SOCE, Ryanodine receptors, or gap junctions. Disruption of these components triggers stimuli-induced seizure-like episodes. These findings indicate that Ca(2+) release from internal stores and its propagation between neighboring glial cells via gap junctions are essential for maintaining normal nervous system function.
Ma, R., Haji-Ghassemi, O., Ma, D., Jiang, H., Lin, L., Xu, T., Murayama, T., Moussian, B., Van Petegem, F. and Yuchi, Z. (2022). Structural basis for diamide modulation of ryanodine receptor. J Gen Physiol 154(9). PubMed ID: 34766989
Diamide insecticides target insect ryanodine receptors (RYRs) and cause dysregulation of calcium signaling in insect muscles and neurons, generating worldwide sales over 2 billion US dollars annually. Several resistance mutations have been reported to reduce the efficacy of the diamides, but the exact binding sites and mechanism of resistance mutations were not clear. Recently, the cryo-electron microscopy (cryo-EM) structure of RYR was solved in complex with the anthranilic diamide chlorantraniliprole (CHL). CHL binds to the pseudo-voltage-sensor domain (pVSD) of RYR, a site in proximity to the previously identified resistance mutations. Mutagenesis studies in silico, in mutant cell lines, and in transgenic Drosophila strains revealed the key residues involved in diamide coordination and the molecular mechanism under species-selectivity and resistance mutations. It was also proposed that CHL may alleviate the loss-of-function effects of some central core disease (CCD) mutations by increasing the opening probability (Po) of RYR1. In addition, the crystal structures of several RYR domains were solved from the diamondback moth and the bee, revealing insect-specific structural features which could be potentially targeted by novel insecticides. Interestingly, it was found that the phosphorylation of insect RYR is temperature dependent, facilitated by the low thermal stability and dynamic structure of the insect RYR. These structures provide a foundation for developing novel pesticides to overcome the resistance crisis.
Zhang, W., Lei, X., Zhou, X., He, B., Xiao, L., Yue, H., Wang, S., Sun, Y., Wu, Y., Wang, L., Ghartey-Kwansah, G., Jones, O. D., Bryant, J. L., Xu, M., Ma, J. and Xu, X. (2022). A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo. J Vis Exp(183). PubMed ID: 35604165
Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. This work introduceS a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster, was studied. DmFKBP12 was identified in as early as the syncytial blastoderm stage, and the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.
Zhao, J., Lin, L., Hadiatullah, H., Chen, W., Huang, J., Wu, S., Murayama, T. and Yuchi, Z. (2023). Characterization of Six Diamide Insecticides on Ryanodine Receptor: Resistance and Species Selectivity. J Agric Food Chem 71(29): 11001-11007. PubMed ID: 37462137
Ryanodine receptor (RyR) has been used as an insecticide target to control many destructive agricultural pests. The effectiveness of these insecticides has been limited by the spread of resistance mutations identified in pest RyRs, but the detailed molecular impacts of the individual mutations on the activity of different diamide compounds have not been fully explored. This study created five HEK293 cell lines stably expressing wild type rabbit RyR1, wild type Spodoptera frugiperda RyR (Sf RyR), or Sf RyR carrying different resistance mutations, including G4891E, G4891E/I4734M, and Y4867F, respectively. R-CEPIA1er, a genetically encoded fluorescent protein, was also introduced in these cell lines to report the Ca(2+) concentration in the endoplasmic reticulum. The activities of six commercial diamide insecticides were systematically characterized against different RyRs using the time-lapse fluorescence assay. Among them, cyantraniliprole (CYAN) displayed the highest activity against all three resistant Sf RyRs. The good performance of CYAN was confirmed by the toxicity assay using gene-edited Drosophila expressing the mutant RyRs, in which CYAN showed the lowest LD(50) value for the double resistant mutant. In addition, their acitivty were compared between mammalian and insect RyRs and found that flubendiamide has the best insect-selectivity. The mechanism of the anti-resistance property and selectivity of the compounds was proposed based on the structural models generated by homology modeling and molecular docking. These findings provide insights into the mechanism of insect resistance and guidance for developing effective RyR agonists that can selectively target resistant pests.


Although it has been postulated that vesicle mobility is increased to enhance release of transmitters and neuropeptides, the mechanism responsible for increasing vesicle motion in nerve terminals and the effect of perturbing this mobilization on synaptic plasticity are unknown. In this study, green fluorescent protein-tagged dense-core vesicles (DCVs) are imaged in Drosophila motor neuron terminals, where DCV mobility is increased for minutes after seconds of activity. Ca2+-induced Ca2+ release from presynaptic endoplasmic reticulum (ER) is shown to be necessary and sufficient for sustained DCV mobilization. However, this ryanodine receptor (RyR)-mediated effect is short-lived and only initiates signaling. Calmodulin kinase II (CaMKII), which is not activated directly by external Ca2+ influx, then acts as a downstream effector of released ER Ca2+. RyR and CaMKII are essential for post-tetanic potentiation of neuropeptide secretion. Therefore, the presynaptic signaling pathway for increasing DCV mobility is identified and shown to be required for synaptic plasticity (Shakiryanova, 2007).

It has long been speculated that vesicle motion at synapses could be controlled to influence neurotransmission, but activity-dependent increases in vesicle mobility (i.e., mobilization) in nerve terminals were detected directly only recently. This was first accomplished with neuropeptide-containing dense-core vesicles (DCVs) in Drosophila neuromuscular junction (NMJ) by using two methods, fluorescence recovery after photobleaching (FRAP) and image correlation analysis of time-lapse movies (Shakiryanova, 2005). Subsequently, stimulus-induced vesicle motion was detected with mammalian neuroendocrine DCVs and frog NMJ small synaptic vesicles (SSVs) (Allersma, 2006; Gaffield, 2006). The increase in vesicle mobility in these diverse preparations shares common features. First, mobilized vesicles move randomly, in accordance with single particle-tracking studies that showed that releasable and reserve secretory vesicles move by diffusion, albeit at different rates Second, stimulation-induced mobility is unaffected by depolymerizing F-actin. Along with other experiments, this finding suggests that the long-standing question of whether F-actin is a barrier for vesicle immobilization or a track for motor-mediated translocation is not central to understanding stimulation-induced vesicle motion. Finally, the stimuli in each case are associated with Ca2+ influx, which was explicitly demonstrated to be required for enhancing vesicle mobility in two of the studies (Shakiryanova, 2005; Allersma, 2006). Although SSV mobilization is not seen in all neurons, these shared properties indicate that a conserved mechanism may underlie mobilization of both SSVs and DCVs at sites that are capable of this regulation (Shakiryanova, 2007).

Mobilization of neuropeptide-containing DCVs likely involves sustained signal transduction, because seconds of activity in Drosophila NMJ type Ib boutons induce many minutes of increased DCV mobility even after release is inhibited (Shakiryanova, 2005). However, the specific pathway activated by Ca2+ influx has not been identified. Thus, the role of this signaling pathway in synaptic function and plasticity is not known. Indeed, although pharmacological manipulations have been shown to produce DCV mobility increases that can account for increased release in vitro, inhibiting physiological vesicle mobilization has never been shown to block potentiation of release in vivo (Shakiryanova, 2007).

In this study, in vivo imaging of a green fluorescent protein (GFP)-labeled neuropeptide, which reports native neuropeptide secretion, shows that ryanodine receptor (RyR)-mediated Ca2+ release from presynaptic endoplasmic reticulum (ER) is necessary and sufficient for triggering DCV mobilization in the larval NMJ. Calmodulin kinase II (CaMKII) then acts as an effector of released ER Ca2+. Finally, RyR-mediated Ca2+ release and CaMKII are shown to be required for post-tetanic potentiation (PTP) of neuropeptide secretion (Shakiryanova, 2007).

It is thought that vesicle mobility is increased to facilitate secretion. In favor of this model, in vitro studies showed that DCV mobility limits neuropeptide release, and pharmacologically increasing DCV mobility enhances neuropeptide release. However, native signaling mechanisms that induce such DCV mobilization in a nerve terminal in vivo and the impact of this signaling on release have not been determined. Indeed, the importance of vesicle mobility has been obscured because the term mobilization has been co-opted to include almost any process that increases release. The recent direct detection of regulated vesicle mobility in motor neuron terminals has opened the door to probing the signaling involved in bona fide vesicle mobilization and its connection to synaptic plasticity. Specifically, it was reasoned that identifying the signaling that controls vesicle motion would serve two purposes: (1) to delineate regulatory mechanisms in the nerve terminal and (2) to produce the opportunity to test whether inhibiting mobilization affects release for the first time. In this study, synaptic DCV motion and neuropeptide release were imaged in vivo to show that both activity-dependent increases in DCV mobility and PTP require presynaptic ER Ca2+ release via RyRs leading to CaMKII activation. The use of a single signaling pathway for controlling DCV motion and release provides the first empirical indication of a mechanistic connection between mobilization and synaptic plasticity. Baseline DCV mobility and secretion evoked by low-frequency activity are not markedly affected by inhibiting RyRs or CaMKII. Therefore, this signaling pathway has a minor role until it is recruited by tetanic activity. The activity requirement for recruitment of RyR-CaMKII-activated mobilization and PTP is well matched to the native activity found at the larval NMJ [i.e., rhythmic bursting (Klose, 2005)] and the activity patterns known to be optimal for neuropeptide release. Given past in vitro studies of DCV mobility, the in vivo results presented in this study are consistent with the conclusion that physiological activity triggers RyR-activated CaMKII to increase DCV mobility, which in turn contributes to PTP of neuropeptide secretion (Shakiryanova, 2007).

The necessity for RyR-mediated Ca2+ release for mobilization and PTP is surprising because bulk Ca2+ is effectively elevated in Drosophila motor neuron terminals by Ca2+ influx through plasma membrane voltage-gated channels even when ER Ca2+ is depleted. Indeed, plasma membrane voltage-gated Ca2+ channels are sufficient for triggering some neuropeptide release at low levels of activity but apparently cannot induce sustained mobilization without the participation of RyRs. Thus, as far as mobilization is concerned, CaMKII is apparently more sensitive to Ca2+ released from intracellular stores than extracellular Ca2+ influx. Perhaps this specificity arises because of positioning of CaMKII closer to RyRs than voltage-gated channels in the presynaptic plasma membrane. Alternatively, a small amount of Ca2+ influx through a voltage-gated channel may trigger a much larger Ca2+ spark by opening multiple RyRs. This could be amplified by production of RyR-mediated Ca2+ waves. Thus, local Ca2+ elevation at the surface, which is sufficient for triggering exocytosis of docked vesicles, could give rise to a propagating Ca2+-induced Ca2+ release (CICR) from the ER to activate CaMKII in the cytoplasm where most DCVs reside. This positive feedback might also set the threshold activity required for activating CaMKII-induced mobilization. Hence, RyRs could control the activity dependence and spatial propagation of vesicle mobilization. Regardless of the underlying mechanism, the requirement for ER Ca2+ stores delineated in this study implies that the Ca2+ sources for triggering exocytosis (i.e., plasma membrane voltage-gated Ca2+ channels) and sustained mobilization (i.e., intracellular RyRs) are distinct (Shakiryanova, 2007).

These studies establish that RyR-CaMKII-dependent DCV mobilization is initiated in the presynaptic terminal. First, mobilization is blocked by disabling neuronal Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) function, while leaving muscle SERCA intact. Second, the activity-induced increase in presynaptic DCV motion does not depend on standard synaptic transmission between nerve and muscle: mobilization persists after inhibiting exocytosis (Shakiryanova, 2005) or postsynaptic glutamate receptors with desensitizing doses of glutamate (Shakiryanova, 2005) or an antagonist. RyR and CaMKII are present in muscle as well as neurons (Hasan, 1992; Griffith, 1994; Haghighi, 2003; Lu, 2003), but it is not known whether muscle CaMKII, which participates in retrograde signaling (Haghighi, 2003), is activated by Ca2+ flux through RyRs. Further study may show that the intimate functional relationship between the RyR and CaMKII exists on both sides of the synapse (Shakiryanova, 2007).

It was also found that a short-lived elevation in Ca2+ induces long-lasting mobilization and PTP. Hence, residual Ca2+, which is important for synaptic plasticity, does not sustain these responses. The downstream involvement of CaMKII suggests that phosphorylation leading to a sustained increase in vesicle mobility and secretion persists after residual Ca2+ has dissipated. This could reflect either slow substrate dephosphorylation because of limited phosphatase activity or prolonged activation of CaMKII, possibly by autophosphorylation on T287 of the Drosophila CaMKII. The presence of some autophosphorylated CaMKII at the resting NMJ might also explain the small reduction in DCV mobility induced by CaMKII inhibitors applied at rest. Unfortunately, because it is very difficult to specifically measure presynaptic autophosphorylation of CaMKII with phosphospecific antibodies in the context of far greater amounts of CaMKII in the postsynaptic side of the Drosophila NMJ, the prevalence of presynaptic autophosphorylated CaMKII is not known. Likewise, the participation of other Ca2+-signaling proteins cannot be excluded. Nevertheless, it is clear that CaMKII-induced phosphorylation is critical for sustained DCV mobilization and PTP after Ca2+ has returned to baseline levels (Shakiryanova, 2007).

The results also hint that the RyR-CaMKII pathway may not be the sole signaling mechanism for regulating DCV mobility in synaptic boutons. First, inhibiting Ca2+ release revealed a sustained small decrease in DCV mobility, which was not detected in the absence of Ca2+ influx (Shakiryanova, 2005). Previously, it was showed that DCV mobilization in type Ib boutons begins to reverse after ~5 min (Shakiryanova, 2005). Given that CaMKII is required for sustained mobilization, it is hypothesized that dephosphorylation resets DCV dynamics. Future experiments could explore whether an activity-sensitive phosphatase such as calcineurin reduces DCV mobility. Such a mechanism could be responsible for reducing steady-state phosphorylation in the absence of Ca2+ release and reversing the effects of additional CaMKII-mediated phosphorylation when RyRs are active. It was also observed that there was a small transient increase in DCV motion in many of the experiments in which RyRs and CaMKII were inhibited. The simplest explanation for this result is that this is a consequence of incomplete inhibition of mobilization combined with the above described demobilization mechanism. However, it was not excluded that this represents the direct, but inefficient, activation of CaMKII by Ca2+ influx. It would be interesting to test whether DCV mobility is transient and limited to near the cell surface when RyRs are inhibited, but this is beyond the resolution of current methods that can be used with the intact living NMJ (Shakiryanova, 2007).

The signaling responsible for increasing DCV mobility and PTP of neuropeptide secretion in Drosophila motor neuron terminals may be widely relevant. For example, this pathway could be responsible for mobilization in neuroendocrine cells (Allersma, 2006). Likewise, CaMKII-mediated DCV mobilization may contribute to the increase in activity-dependent neuropeptide release by dendritic spines (Lochner, 2006) induced by release of ER Ca2+ (Ludwig, 2002). The signaling described in this study could also be important in synapses capable of SSV mobilization (e.g., the NMJ), which shares many features with DCV mobilization. In fact, presynaptic RyR and CaMKII contribute to synaptic potentiation in Aplysia synapses and the neurotrophin-induced increase in spontaneous neurotransmitter release in developing NMJs. SSV mobility studies should be able to test whether mobilization occurs and is necessary in these cases of synaptic plasticity (Shakiryanova, 2007).

Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut

Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, this study identified two regulators of cytosolic Ca2+ levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca2+ signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. These findings provide a link between redox regulation and Ca2+ signaling and reveal a novel mechanism by which ISCs detect stress signals (Xu, 2017).

This study found that the two cation channels TRPA1 and RyR are critical for cytosolic Ca2+ signaling and ISC proliferation. Under homeostatic conditions, the basal activities of TRPA1 and RyR are required for maintaining cytosolic Ca2+ in ISCs to ensure their self-renewal activities and normal tissue turnover. Agonists, including but not limited to low levels of ROS, could be responsible for the basal activities of TRPA1 and RyR. Under tissue damage conditions, increased ROS stimulates the channel activities of TRPA1 to mediate increases in cytosolic Ca2+ in ISCs. As for RyR, besides its potential to directly sense ROS, it is known to act synergistically with TRPA1 in a positive feedback mechanism to release more Ca2+ from the ER into the cytosol upon sensing the initial Ca2+ influx through TRPA1 (Xu, 2017).

Previously, Deng (2015) identified L-glutamate as a signal that can activate metabotropic glutamate receptor (mGluR) in ISCs, which in turn modulates the cytosolic Ca2+ oscillation pattern via phospholipase C (PLC) and inositol-1,4,5-trisphosphate (InsP3). Interestingly, L-glutamate and mGluR RNAi mainly affected the frequency of Ca2+ oscillation in ISCs, while their influence on cytosolic Ca2+ concentration was very weak. Strikingly, the number of mitotic cells induced by L-glutamate (i.e. an increase from a basal level of ~5 per midgut to ~10 per midgut) is far less than what has been observed in tissue damage conditions (depending on the severity of damage, the number varies from ~20 to more than 100 per midgut following damage). Consistent with this, in a screen for regulators of ISC proliferation in response to tissue damage, this study tested three RNAi lines targeting mGluR (BL25938, BL32872, and BL41668, which was used by Deng, 2015), and none blocked the damage response in ISCs, suggesting that L-glutamate and mGluR do not play a major role in damage repair of the gut epithelium (Xu, 2017).

This study found that ROS can trigger Ca2+ increases through the redox- sensitive cation channels TRPA1 and RyR under damage conditions. In particular, it was demonstrated using voltage-clamp experiments that the TRPA1-D isoform, which is expressed in the midgut, is sensitive to the oxidant agent paraquat. In addition, the results of previous studies have demonstrated the direct response of RyR to oxidants via single channel recording and showed that RyR could amplify TRPA1-mediated Ca2+ signaling through the Ca2+-induced Ca2+ release (CICR) mechanism. Interestingly, expression of oxidant- insensitive TRPA1-C isoform in the ISCs also exhibits a tendency to induce ISC proliferation, indicating that ROS may not be the only stimuli for TRPA1 and RyR under physiological conditions. Possible other activators in the midgut may be irritant chemicals, noxious thermal/mechanical stimuli, or G-protein-coupled receptors (Xu, 2017).

Altogether, the concentration of cytosolic Ca2+ in ISCs appears to be regulated by a number of mechanisms/inputs including mGluR and the ion channels TRPA1 and RyR. Although mGluR might make a moderate contribution to cytosolic Ca2+ in ISCs, TRPA1 and RyR have a much stronger influence on ISC Ca2+ levels. Thus, it appears that the extent to which different inputs affect cytosolic Ca2+ concentration correlates with the extent of ISC proliferation (Xu, 2017).

Although, as a universal intracellular signal, cytosolic Ca2+ controls a plethora of cellular processes, we were able to demonstrate that cytosolic Ca2+ levels regulate Ras/MAPK activity in ISCs. Specifically, we found that trpA1 RNAi or RyR RNAi block Ras/MAPK activation in stem cells, and that forced cytosolic Ca2+ influx by SERCA RNAi induces Ras/MAPK activity. Moreover, Ras/MAPK activation is an early event following increases in cytosolic Ca2+, since increased dpErk signal was observed in stem cells expressing SERCA RNAi before they undergo massive expansion, and when Yki RNAi was co-expressed to block proliferation. It should be noted that a more variable pattern of pErk activation was observed with prolonged increases of cytosolic Ca2+, suggesting complicated regulations via negative feedback, cross-activation, and cell communication at late stages of Ca2+ signaling. This might explain why Deng failed to detect pErk activation after 4 days induction of Ca2+ signaling (Deng, 2015). Previously, Ras/MAPK activity was reported to increase in ISCs, regulating proliferation rather than differentiation, in regenerating midguts, which is consistent with the findings about TRPA1 and RyR (Xu, 2017).

The Calcineurin A1/CREB-regulated transcription coactivator/CrebB pathway previously proposed to act downstream of mGluR-calcium signaling (Deng, 2015) is not likely to play a major role in high Ca2+-induced ISC proliferation, as multiple RNAi lines targeting CanA1 or CrebB were tested and none of them suppressed SERCA RNAi-induced ISC proliferation. In support of this model, it was also found that the active forms of CanA1/ CRTC/ CrebB cannot stimulate mitosis in ISCs when their cytosolic Ca2+ levels are restricted by trpA1 RNAi, whereas mitosis induced by the active forms of Ras or Raf is not suppressed by trpA1 RNAi (Xu, 2017).

Prior to this study, it has been shown that paracrine ligands such as Vn from the visceral muscle, and autocrine ligands such as Spi and Pvf ligands from the stem cells, can stimulate ISC proliferation via RTK-Ras/MAPK signaling. It study found that multiple RTK ligands in the midgut are down-regulated by trpA1 RNAi expression in the ISCs, including spi and pvf1 that can be induced by SERCA RNAi. Further, it was demonstrated that high Ca2+ fails to induce ISC proliferation in the absence of EGFR. As spi is induced by EGFR-Ras/MAPK signaling in Drosophila cells, and DNA binding mapping (DamID) analyses indicate that spi might be a direct target of transcriptional factors downstream of EGFR-Ras/MAPK in the ISCs, the autocrine ligand Spi might therefore act as a positive feedback mechanism for EGFR-Ras/MAPK signaling in ISCs (Xu, 2017).

In summary, this study identifies a mechanism by which ISCs sense microenvironment stress signals. The cation channels TRPA1 and RyR detect oxidative stress associated with tissue damage and mediate increases in cytosolic Ca2+ in ISCs to amplify and activate EGFR-Ras/MAPK signaling. In vertebrates, a number of cation channels, including TRPA1 and RyR, have been associated with tumor malignancy. The current findings, unraveling the relationship between redox-sensing, cytosolic Ca2+, and pro-mitosis Ras/MAPK activity in ISCs, could potentially help understand the roles of cation channels in stem cells and cancers, and inspire novel pharmacological interventions to improve stem cell activity for regeneration purposes and suppress tumorigenic growth of stem cells (Xu, 2017).

Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling

Synapses and circuits rely on neuroplasticity to adjust output and meet physiological needs. Forms of homeostatic synaptic plasticity impart stability at synapses by countering destabilizing perturbations. The Drosophila melanogaster larval neuromuscular junction (NMJ) is a model synapse with robust expression of homeostatic plasticity. At the NMJ, a homeostatic system detects impaired postsynaptic sensitivity to neurotransmitter and activates a retrograde signal that restores synaptic function by adjusting neurotransmitter release. This process has been separated into temporally distinct phases, induction and maintenance. One prevailing hypothesis is that a shared mechanism governs both phases. This study shows the two phases are separable. Combining genetics, pharmacology, and electrophysiology, a signaling system consisting of PLCbeta, inositol triphosphate (IP3), IP3 receptors, and Ryanodine receptors was shown to be required only for the maintenance of homeostatic plasticity. It was also found that the NMJ is capable of inducing homeostatic signaling even when its sustained maintenance process is absent (James, 2019).

The Drosophila melanogaster NMJ is an ideal model synapse for studying the basic question of how synapses work to counter destabilizing perturbations. At this NMJ, reduced sensitivity to single vesicles of glutamate initiates a retrograde, muscle-to-nerve signaling cascade that induces increased neurotransmitter vesicle release, or quantal content (QC). As a result, the NMJ maintains a normal postsynaptic response level. Mechanistically, this increase in QC depends upon the successful execution of discrete presynaptic events, such as increases in neuronal Ca2+ influx and an increase in the size of the readily releasable pool (RRP) of synaptic vesicles. The field has termed this compensatory signaling process as presynaptic homeostatic potentiation (PHP) (Delvendahl, 2019). Two factors that govern the expression of PHP are the nature of the NMJ synaptic challenge and the amount of time elapsed after presentation of the challenge. Acute pharmacological inhibition of postsynaptic glutamate receptors initiates a rapid induction of PHP that restores synaptic output in minutes. By contrast, genetic lesions and other long-term reductions of NMJ sensitivity to neurotransmitter induce PHP in a way that is sustained throughout life (James, 2019).

Previously work has identified the Plc21C gene as a factor needed for PHP (Brusich, 2015). Plc21C encodes a Drosophila Phospholipase Cβ (PLCβ) homolog known to be neuronally expressed, but recent ribosomal profiling data also indicates possible muscle expression of Plc21C (Chen, 2017). In canonical signaling pathways, once PLCβ is activated by Gαq, it cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). DAG can affect synaptic function by activating Protein Kinase C (PKC), while IP3 binds its receptor (IP3R) to trigger release of calcium from intracellular stores. It is not understood which aspects of this signaling machinery are mobilized during PHP. Potential downstream consequences of PLCβ activity at the NMJ include phosphorylation of neuronal proteins, modulation of ion channel activity, and changes in localization of neurotransmission machinery (James, 2019).

This study scrutinized PLCβ-directed signaling further. Tests were performed to see whether PLCβ-directed signaling was required solely for the maintenance of PHP or if it could also be required for induction. In addition to PLCβ, the IP3 Receptor (Drosophila Itpr, herein IP3R) and Ryanodine receptor (Drosophila RyR) were identified as being part of the same signaling process. Neither PLCβ, nor IP3R, nor RyR are required for the rapid induction of PHP. Additionally, it was found that the rapid induction of PHP is still possible in synapses already sustaining PHP. Surprisingly, it was found that NMJs are capable of rapidly inducing PHP -- even when the sustained expression of PHP is already blocked by impairments in PLCβ, IP3R, or RyR signaling. Taken together, these data show that the induction and maintenance of PHP are separable. Even though there is compelling evidence that parts of the induction and maintenance signaling mechanisms overlap (Goel, 2017), it is also true that acute PHP is possible in scenarios where long-term PHP is not (James, 2019).

This study divides the acute induction and chronic maintenance stages of presynaptic homeostatic potentiation. The data support two core findings. The first is that the short-term induction and long-term maintenance of PHP are separable by genetic and pharmacological manipulations. The second is that an IP3-mediated signaling system is specifically required for the maintenance of PHP (see Model depicting PLCβ/IP3R/RyR signaling underling the maintenance of PHP in both muscle and neuron.) (James, 2019).

For several years, one assumption has been that both the acute and chronic forms of PHP are executed in a similar way, and possibly by shared mechanisms. The issue has been clouded by the fact that both PhTox and a GluRIIA deletion mutant -- the primary reagents utilized to induce PHP -- have the same molecular target, that is GluRIIA-containing glutamate receptors. The process of combining these acute and chronic forms of plasticity within a single genotypic background was cumbersome due to a lack of reagents available to conduct temporally separate targeting experiments (James, 2019).

Several groups ascertained insights into temporal requirements by targeting potential homeostatic signaling genes. The main finding has been that the majority of molecules identified are essential to both the acute and chronic forms of PHP. Neurons tightly control neurotransmitter release probability, and the core presynaptic machinery directly responsible for increasing quantal content is shared. These shared components include the CaV2-type voltage-gated calcium channel or factors gating influx through the channel. They also include factors that regulate the size of the readily releasable pool (RRP) of presynaptic vesicles or factors that control the baseline excitability or plasticity of the presynaptic motor neuron, and neurotransmitter fusion events themselves. As a result, both the acute and chronic forms of PHP signaling cause increases in readily releasable pool (RRP) size and CaV2-mediated calcium influx; and in turn, these presynaptic mechanisms underlie the increases in QC which constitute PHP (James, 2019).

This study shows that although the acute and chronic processes might overlap, they are functionally separable. The fact that they are separable is not necessarily surprising. This finding mirrors data for discrete molecules required for long-term PHP maintenance, such as Target of Rapamycin (Tor), the Rho-type guanine exchange factor Ephexin, the transcription factor Gooseberry, C-terminal Src Kinase, innate immune signals molecules IMD, IKKβ, Relish and the kinesin adaptor Arl8. Importantly, this list contains molecules implicated both in neuron and muscle. This study has added PLCβ (Brusich, 2015) and its effectors IP3R and RyR to this list (James, 2019).

Recent studies have augmented the idea of overlapping signaling pathways and added a degree of specificity. Both acute and chronic forms of PHP begin as instructive retrograde signals after perturbations are detected in the muscle. These forms of PHP involve a decrease in phosphorylation of muscle CaMKII levels, and converge upon the same signaling components in the presynaptic neuron. These studies suggest that Tor signaling converges on the same molecular targets as acute forms of PHP (Goel, 2017). However, the precise roles for Tor and CaMKII in either form of PHP are as yet unknown (James, 2019).

These data appear to contradict the idea of PHP pathway convergence (Goel, 2017). Yet, the current findings are not incompatible with this idea. Multiple lines of evidence indicate discrete signaling requirements for acute forms of PHP on both sides of the synapse. A convergence point is undefined. Accounting for the separation of acute and chronic forms of PHP -- as well as their discrete signaling requirements -- long-term maintenance of PHP might integrate multiple signals between the muscle and neuron over time. For future studies, it will be important to clearly define roles of signaling systems underlying PHP and how distinct signaling systems might be linked (James, 2019).

This work presents unexpected findings. The first is that even in the face of a chronic impairment or block of homeostatic potentiation, the NMJ is nevertheless capable of a full rapid induction of PHP. Given that most molecules required for PHP identified to date are needed for both phases, significant functional separation between them was not expected. A priori it was expected that a failure of the chronic maintenance of PHP would make core machinery unavailable for its acute induction. The second unexpected finding is how quickly the chronic maintenance of PHP can be nullified by pharmacology (10 min), resulting in a return to baseline neurotransmitter release probability after only minutes of drug exposure. This study showed that homeostatic potentiation in GluRIIA mutant larvae or GluRIIC knock-down larvae was abrogated by four different reagents previously known to block IP3R or RyR. Those findings are reminiscent of prior work showing that acute blockade of DAG/ENaC channels with the drug benzamil abolishes PHP in both a GluRIIA mutant background, as well as in the presence of PhTox (Younger, 2013). A difference between benzamil application and the pharmacological agents used in the current study is that the drugs employed in this study only abolished PHP in a chronically challenged background (James, 2019)?

It is unclear how signaling systems that drive homeostatic plasticity transition from a state of induction to a state of maintenance. It is also not understood how interdependent short-term and long-term HSP implementation mechanisms are. A more complete understanding of the timing and perdurance of these properties could have important implications for neurological conditions where synapse stability is episodically lost (James, 2019).

The current findings parallel recent data examining active zone protein intensities in the contexts of induction of PHP and maintenance of PHP at the NMJ. There are multiple results informative to this study. First, the expression of any form of PHP (acute or chronic) appears to correlate with an increased intensity of active zone protein levels, such as CaV2/Cacophony, UNC-13, and the Drosophila CAST/ELKS homolog Bruchpilot. Unexpectedly, however, two of these studies also reported that the rapid induction of PHP does not require this protein increase in order to be functionally executed. These are conundrums for future work. How does rapid active zone remodeling happen in minutes on a mechanistic level? In the absence of such remodeling, how is PHP able to be induced rapidly? Moreover, is the observed short-term active zone remodeling the kernel for the longer-term changes to the active zone and release probability - or is some other compensatory system triggered over long periods of developmental time (James, 2019)?

The current findings add a new dimension to those puzzles with the data that IP3 signaling is continuously required to maintain PHP. If active zone remodeling truly is instructive for PHP maintenance, then it will be interesting to test what roles IP3 signaling and intracellular calcium release play in that process. The screen did include a UAS-RNAi line against unc-13 and an upstream GPCR-encoding gene methuselah. Moreover, a previously study of PHP used a UAS-cac[RNAi] line (Brusich, 2015). Chronic PHP maintenance was intact for all of those manipulations. Those findings are not necessarily contradictory to the recent work from other groups. For instance, knockdown of an active zone protein by RNAi is not a null condition. As such, RNAi-mediated knockdown should leave residual wild-type protein around. In theory, that residual protein could be scaled with homeostatic need (James, 2019).

The data strongly suggest that intracellular calcium channel activation and store release fine tune neurotransmitter release that is implemented by PHP. The exact mechanism by which IP3R and RyR function to maintain PHP at the NMJ is unclear. It appears to be a shared process with IP3. If these store-release channels are acting downstream of IP3 activity, then the data suggest that this would be a coordinated activity involving both the muscle and the neuron, with loss of IP3 signaling in the neuron being more detrimental to evoked release (James, 2019).

It remains unclear what signals are acting upstream. PLCβ is canonically activated by Gαq signaling. From prior work, evidence was garnered that a Drosophila Gq protein plays a role in the long-term maintenance of HSP (Brusich, 2015). Logically, there may exist a G-protein-coupled receptor (GPCR) that functions upstream of PLCβ/IP3 signaling. A screen did not positively identify such a GPCR. Several genes encoding GPCRs were examined, including TkR86C, mAChR-A, GABA-B-R1, PK2-R2, methuselah, AdoR, and mGluR. Genes encoding Gβ subunits or putative scaffolding molecules were also examined, including CG7611 (a WD40-repeat-encoding gene), Gβ13F, and Gβ76C, again with no positive screen hits (James, 2019).

The data are consistent with dual pre- and post-synaptic functions of IP3. This could mean dual pre- and post-synaptic roles for calcium store release, through an undetermined combination of RyR and IP3R activities, again either pre- or post-synaptic. Both RyR and IP3R have been shown to be critical for specific aspects of neuroplasticity and neurotransmission. Activities of both RyR and IP3R can activate molecules that drive plasticity, such as Calcineurin and CaMKII. At rodent hippocampal synapses, electrophysiological measures like paired-pulse facilitation and frequency of spontaneous neurotransmitter release are modulated by RyR and/or IP3R function, as is facilitation of evoked neurotransmitter at the rat neocortex. In addition to vesicle fusion apparatus, activity of presynaptic voltage-gated calcium channels is modulated by intracellular calcium. Work from this lab at the NMJ has shown that impairing factors needed for store-operated calcium release can mollify hyperexcitability phenotypes caused by gain-of-function CaV2 amino-acid substitutions (Brusich, 2018; James, 2019 and references therein).

Within the pre-synaptic neuron, IP3R and RyR could activate any number of calcium-dependent molecules to propagate homeostatic signaling. Some candidates were tested in a screen, but none of those tests blocked PHP. One possibility is that the reagents utilized did not sufficiently diminish the function of target molecules enough to impact PHP in this directed screen. Detection of downstream effectors specific to muscle or neuron might also be ham>pered by the fact that attenuation of IP3 signaling in a single tissue is insufficient to abrogate PHP. Another possibility is that presynaptic store calcium efflux via IP3R and RyR may directly potentiate neurotransmitter release, either by potentiating basal calcium levels or synchronously with CaV2-type voltage-gated calcium channels (James, 2019).

Both pre- and post-synaptic voltage-gated calcium channels are critical for the expression of several forms of homeostatic synaptic plasticity. Much evidence supports the hypothesis that store-operated channels and voltage gated calcium channels interact to facilitate PHP. In various neuronal populations, both RyR and IP3R interact with L-type calcium channels physically and functionally to reciprocally impact the opening of the other channel. In presynaptic boutons, RyR calcium release follows action potential firing. Calcium imaging experiments show that both the acute expression and sustained maintenance of PHP requires an increase in presynaptic calcium following an action potential. Because IP3Rs are activated by both free calcium and IP3, elevated IP3 levels in the case of chronically expressed PHP could allow IP3Rs and RyRs to open in a way that is time-locked with CaV2-mediated calcium influx or in a way to facilitate the results of later CaV2-mediated influx (James, 2019).

Carbon monoxide, a retrograde messenger generated in postsynaptic mushroom body neurons, evokes noncanonical dopamine release

Dopaminergic neurons innervate extensive areas of the brain and release dopamine (DA) onto a wide range of target neurons. However, DA release is also precisely regulated. In Drosophila melanogaster brain explant preparations, DA is released specifically onto α3/α'3 compartments of mushroom body (MB) neurons that have been coincidentally activated by cholinergic and glutamatergic inputs. The mechanism for this precise release has been unclear. This study found that coincidentally activated MB neurons generate carbon monoxide (CO), which functions as a retrograde signal evoking local DA release from presynaptic terminals. CO production depends on activity of heme oxygenase in postsynaptic MB neurons, and CO-evoked DA release requires Ca(2+) efflux through ryanodine receptors in DA terminals. CO is only produced in MB areas receiving coincident activation, and removal of CO using scavengers blocks DA release. It is proposed that DA neurons use two distinct modes of transmission to produce global and local DA signaling (Ueno, 2020).

Dopamine (DA) is required for various brain functions, including the regulation of global brain states, such as arousal and moods. To perform these functions, individual DA neurons innervate extensive areas of the brain, and release DA onto a wide range of target neurons through a processes known as volume transmission. However, this extensive innervation is not suitable for precise, localized release of DA, and it has been unclear how widely innervating dopaminergic neurons can also direct DA release onto specific target neurons (Ueno, 2020).

In Drosophila, olfactory associative memories are formed and stored in the mushroom bodies (MBs) where Kenyon cells, MB intrinsic neurons which are activated by different odors, form synaptic connections with various MB output neurons, which regulate approach and avoidance behaviors. Dopaminergic neurons (DA neurons) modulate plasticity of synapses between Kenyon cells and MB output neurons. However, while there are ~2000-2500 Kenyon cells that form thousands of synapses with MB output neurons, plasticity at these synapses is regulated by relatively few DA neurons. This indicates that canonical action potential-dependent release cannot fully explain DA release and plasticity. It was recently determined that in Drosophila, synaptic vesicular (SV) exocytosis from DA terminals is restricted to MB neurons that have been activated by coincident inputs from odor-activated cholinergic pathways, and glutamatergic pathways, which convey somatosensory (pain) information. Odor information is transmitted to the MBs by projection neurons from the antennal lobe (AL), while somatosensory information is transmitted to the brain via ascending fibers of the ventral nerve cord (AFV). AL stimulation evokes Ca2+ responses in the MB by activating nAChRs, and AFV stimulation evokes Ca2+ responses in the MBs by activating N-Methyl-D-aspartate receptors (NMDARs) in the MBs. Significantly, when the AL and AFV are stimulated simultaneously (AL 1 AFV) or the AL and NMDARs are stimulated simultaneously (AL 1 NMDA), plasticity occurs such that subsequent AL stimulations causes increased Ca2+ responses in the a3/a93 compartments. This plasticity is known as long-term enhancement (LTE) of MB responses and requires activation of D1 type DA receptors (D1Rs) in the MBs. Furthermore, while activation of D1Rs alone is sufficient to produce LTE, neither AL nor AFV stimulation alone is able to cause SV exocytosis from presynaptic DA terminals projecting onto the a3/a93 compartments of the vertical MB lobes. Instead, exocytosis from DA terminals occurs only when postsynaptic Kenyon cells are activated by coincident AL 1 AFV or AL 1 NMDA stimulation. Strikingly, while MBs are bilateral structures and DA neurons project terminals onto both sides of MBs, SV exocytosis occurs specifically in MB areas that have been coincidently activated. Based on these results, it is proposed that coincident inputs specify the location where DA is released, whereas DA induces plastic changes needed to encode associations. However, it has been unclear how activated Kenyon cells send a retrograde 'demand' signal to presynaptic DA terminals to induce SV release (Ueno, 2020).

This study used a Drosophila dissected brain system to examine synaptic plasticity and DA release, and found that coincidentally activated postsynaptic Kenyon cells generate the retrograde messenger, carbon monoxide (CO). CO is generated by heme oxygenase (HO) in postsynaptic MB neurons, and induces DA release from presynaptic terminals by evoking Ca2+ release from internal stores via ryanodine receptors (RyRs). Thus, while individual DA neurons extensively innervate the MBs, on-demand SV exocytosis allows DA neurons to induce plasticity in specific target neurons (Ueno, 2020).

CO functions as a retrograde on-demand messenger for SV exocytosis in presynaptic DA terminals A central tenet of neurobiology is that action potentials, propagating from the cell bodies, induce Ca2+ influx in presynaptic terminals to evoke SV exocytosis. However, recent mammalian studies have shown that only a certain fraction of a large number of presynaptic DA release sites is involved in canonical SV exocytosis. In this study, a novel mechanism of SV exocytosis was identified in which activity in postsynaptic neurons evokes presynaptic release to induce plastic changes. This mechanism allows the timing and location of DA release to be strictly defined by activity of postsynaptic neurons (Ueno, 2020).

On-demand SV exocytosis uses CO as a retrograde signal from postsynaptic MB neurons to presynaptic DA terminals. CO fulfills the criteria that have been proposed for a retrograde messenger: (1) it was demonstrated that HO, which catalyzes CO production, is highly expressed in postsynaptic MB neurons, indicating that MB neurons have the capacity to synthesize the messenger; (2) it was shown that pharmacological and genetic suppression of HO activity in the MBs inhibits CO production, presynaptic DA release, and LTE, and (3) using a CO fluorescent probe, COP-1, it was demonstrated that CO is generated in the MBs following coincident stimulation of the MBs, and CO generation is restricted to lobes of MB neurons that receive coincident stimulation. It was further shown that direct application of CO, or a CO donor, induces DA release from presynaptic terminals, whereas addition of a CO scavenger, HemoCD, suppresses release. Fourth, it was demonstrated that CO activates RyR in presynaptic terminals to induce SV exocytosis. Strikingly, CO-dependent SV exocytosis does not depend on influx of extracellular Ca2+ but instead requires efflux of Ca2+ from internal stores via RyR. Finally, it was shown that pharmacological inhibition and genetic suppression of RyR in DA neurons impair DA release after coincident stimulation and CO application (Ueno, 2020).

Other retrograde signals, such as NO and endocannabinoids, enhance or suppress canonical SV exocytosis, but this study finds that CO-dependent DA release occurs even in conditions that block neuronal activity and Ca2+ influx in presynaptic DA terminals. This suggests that CO does not function to modulate canonical SV exocytosis, but may instead evoke exocytosis through a novel mechanism. Several previous studies have indicated that CO and RyR-dependent DA release also occurs in mammals. A microdialysis study has shown that CO increases the extracellular DA concentration in the rat striatum and hippocampus, either through increased DA release or inhibition of DA reuptake. Also, pharmacological stimulation of RyRs has been reported to induce DA release in the mice striatum. This release is attenuated in RyR3-deficient mice, while KCl-induced DA release, which requires influx of extracellular Ca2+, is unaffected, suggesting that RyR-dependent release is distinct from canonical DA release. However, it has been unknown whether and how CO is generated endogenously. Physiologic conditions that activate RyR to evoke DA release have also been unclear (Ueno, 2020).

While most neurotransmitters are stored in synaptic vesicles and released on neuronal depolarization, the release of gaseous retrograde messengers, such as NO and CO, is likely coupled to activation of their biosynthetic enzymes, NOS and HO. In mammals, an HO isoform, HO-2, is selectively enriched in neurons, and HO-2-derived CO is reported to function in plasticity. HO-2 is activated by Ca2+/calmodulin (CaM) binding, and by casein kinase II (CKII) phosphorylation. Previously, it was shown that coincident AL 1 NMDA stimulation induces a robust Ca2+ increase in the MBs that is greater than the increase from either stimulation alone (Ueno, 2017). It is proposed that this increase may activate Drosophila HO in the MB to generate CO during associative stimulation. While Drosophila has a single isoform of RyR, mammals have three isoforms, RyR1-RyR3. Skeletal muscle and cardiac muscle primarily express RyR1 and RyR2, and the brain, including the striatum, hippocampus, and cortex, expresses all three isoforms. RyRs are known to be activated by Ca2+ to mediate Ca2+ induced Ca2+ release. However, CO-evoked DA release occurs even in the presence of Ca2+-free extracellular solutions containing TTX and EGTA, suggesting that CO activates RyR through a different mechanism. In addition to Ca2+, RyR can be activated by calmodulin, ATP, PKA, PKG, cADP-ribose, and NO. NO can directly stimulate RyR1 nonenzymatically by S-nitrosylating a histidine residue to induce Ca2+ efflux. CO has been reported to activate Ca2+-activated potassium channels (KCa) through a nonenzymatic reaction in rat artery smooth muscle, raising the possibility that it may activate RyR through a similar mechanism. Alternatively, both NO and CO can bind to the heme moiety of soluble guanlylate cyclase leading to its activation. Activated soluble guanlylate cyclase produces cGMP, and cGMP-dependent protein kinase (PKG) rapidly phosphorylates and activates RyRs. Interestingly, NO increases DA in the mammalian striatum in a neural activity-independent manner. Since activation of RyRs also increases extracellular DA in the striatum, hippocampus, and cortex, NO may play a pivotal role in RyRs activation and DA release in mammals. However, NOS expression has not been detected in the MBs, suggesting that, in Drosophila, CO rather than NO may function in this process (Ueno, 2020).

Previous studies have shown that electrical activity from the AL and AFV is transmitted to the MBs by cholinergic and glutamatergic neurons acting on nAChRs and NMDARs, respectively. Although the cholinergic inputs from the AL are known to be delivered by projection neurons, the glutamate inputs are still unclear. Previous work identified glutamatergic neurons that innervate a3/a93 compartments of the MBs and show SV release on electrical stimulation of the AFV (Ueno, 2017). It is proposed that these neurons may transmit information regarding AFV stimuli to the MBs. Alternatively, while NMDARs are localized throughout MB lobes, vesicular glutamate transporter-positive terminals are found only sparsely on the MBs. This suggests that neurons expressing a currently uncharacterized vesicular glutamate transporter may convey information from the AFV to MBs (Ueno, 2020).

DA plays a critical role in associative learning and synaptic plasticity. In flies, neutral odors induce MB responses by activating sparse subsets of MB neurons. After being paired with electrical shocks during aversive olfactory conditioning, odors induce larger MB responses in certain areas of the MBs. This plastic change was modeled in ex vivo brains as LTE, and it was shown that DA application alone is sufficient to induce this larger response (Ueno, 2017). However, in the Drosophila brain, only a small number of DA neurons (~12 for aversive and ~100 for appetitive) regulate plasticity in ~2000 MB Kenyon cells. Thus, to form odor-specific associations, there should be a mechanism regulating release at individual synapses. CO-dependent on-demand DA release provides this type of control. If on-demand release is involved in plasticity and associative learning, knockdown of genes associated with release should affect learning. Indeed, this study shows that knocking down either dHO in the MBs or RyR in DA neurons impairs olfactory conditioning. While these knockdowns did not completely abolish olfactory conditioning, this may be due to inefficiency of the knockdown lines. Alternatively, on-demand release may not be the only mechanism responsible for memory formation, but may instead be required for a specific phase of olfactory memory (Ueno, 2020).

In ex vivo studies, this study found that DA release requires coincident activation of postsynaptic MB neurons by cholinergic and glutamatergic stimuli. However, other in vivo imaging studies have shown that DA neurons can be activated and release DA on odor stimulation or shock application alone. Notably, projection of DA terminals is compartmentalized on the MB lobes and shows distinct responses and DA release during sensory processing. In these studies, dopaminergic neurons innervating the the a3/a93 compartments at the tips of the MB vertical lobes were examined, whereas other studies focused on compartments located on the MB horizontal lobes. This suggests that plasticity in different MB compartments may be regulated by different mechanisms. Unfortunately, due the location of the microelectrode for AL stimulation which caused interference in fluorescent imaging of the horizontal lobes, it was not possible to obtain reliable imaging data from these lobes in this study. Another difference between ex vivo and in vivo studies is that in vivo imaging studies use living, tethered, dissected flies that are likely in different states of arousal/distress, are exposed to many different stimuli, and can form unintended associations. In contrast, brains in ex vivo preparations are in a more controlled environment and are likely exposed to fewer unintended sensory stimuli. This may also explain apparent discrepancies between ex vivo and previous in vivo results (Ueno, 2020).

In mammals, the role of CO in synaptic plasticity is unclear. Application of CO paired with low-frequency stimulation induces LTP, while inhibiting HO blocks LTP in the CA1 region of the hippocampus. However, HO-2-deficient mice have been reported to have normal hippocampal CA1 LTP. In contrast to CO, a role for NO in synaptic plasticity and learning has been previously reported. Thus, at this point, it is an open question whether CO or NO evokes DA release in mammals. Downstream from CO or NO, RyRs have been shown to be required for hippocampal and cerebellar synaptic plasticity (Ueno, 2020).

The current results suggest that DA neurons release DA via two distinct mechanisms: canonical exocytosis and on-demand release. Canonical exocytosis is evoked by electrical activity of presynaptic DA neurons, requires Ca2+ influx, and may be involved in volume transmission. This mode of release can activate widespread targets over time, and is suited for regulating global brain functions. In contrast, on-demand release is evoked by activity of postsynaptic neurons, requires Ca2+ efflux via RyR, and can regulate function of specific targets at precise times. DA neurons may differentially use these two modes of SV exocytosis in a context-dependent manner. Understanding how DA neurons differentially use these modes of transmission will provide new insights into how a relatively small number of DA neurons can control numerous different brain functions (Ueno, 2020).

Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis

Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In C. elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. This study reports that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. This study places Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca2+ sensor dSTIM (Stromal interaction molecule), and the Ca2+-release-activated Ca2+ channel dOrai (olf186-F) in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, these results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway (Cuttell, 2008).

Phagocytosis is a crucial process during development and in innate immunity of all multicellular organisms. It allows for rapid engulfment of dying cells and pathogens by specialized phagocytes, such as macrophages and neutrophils in mammals. Phagocytosis is also an essential function of dendritic cells that present processed antigens to lymphocytes, thus linking innate and adaptive immunity. In C. elegans, the death genes ced-2, 5, 10, and 12 activate the small GTPase CED-10 that triggers actin cytoskeleton rearrangement during phagocytosis; the parallel CED1/6/7 pathway also converges on CED-10, but its precise role in phagocytosis remains elusive (Mangahas, 2005; Cuttell, 2008 and references therein).

During Drosophila embryogenesis, two macrophage receptors, Croquemort (CRQ), a CD36 homolog, and Draper (DRPR), a CED-1 homolog (Manaka, 2004), play a role in apoptotic cell clearance, much like their counterparts in mammals or C. elegans. The Drosophila homolog of CED-6, Dmel/Ced-6 (hereafter called drCed-6), and DRPR are also required in glial cells for axon pruning and the engulfment of degenerating neurons (Awasaki, 2006; MacDonald, 2006; Cuttell, 2008 and references therein).

In a deficiency screen, a mutant was characterized in which embryonic macrophages poorly engulfed apoptotic cells. In an RNAi screen using S2 cells, undertaker/retinophilin (uta) was identified as being responsible for this phenotype. uta encodes a membrane occupational and recognition nexus (MORN) repeat-containing protein with homology to mammalian Junctophilins (JPs). JPs form junctional complexes between the plasma membrane (PM) and the endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+ storage compartment (Takeshima, 2000). These complexes allow for crosstalk between Ca2+ channels at the PM and the ER/SR Ca2+ channels, or Ryanodine receptors (RyRs), thus regulating Ca2+ homeostasis and functions of excitable cells (Takeshima, 2000). Although a role for Ca2+ in phagocytosis of various particles by mammalian phagocytes has been previously described, the molecular mechanisms underlying Ca2+ fluxes associated with these events are not known (Cuttell, 2008).

This study reports that, as for UTA, the Drosophila Ryanodine receptor, Rya-r44F (Xu, 2000), plays a role in phagocytosis of apoptotic cells in vivo. A requirement in phagocytosis was found for store-operated Ca2+ entry (SOCE) via dSTIM, a Ca2+ sensor of the ER/SR lumen (Roos, 2005), and CRACM1/dOrai, a Ca2+-release-activated Ca2+ channel (CRAC) (Feske, 2006; Vig, 2006). uta and rya-r44F genetically interact with drced-6 and drpr, and uta, drced6, and drpr are required for SOCE in S2 cells. Thus, these genes act in the same pathway that plays a general role in phagocytosis, as uta, dstim, dorai, drced-6, and drpr are also required for efficient phagocytosis of bacteria. These results provide a link between SOCE and phagocytosis, imply that UTA plays a similar role in macrophages to that of JPs in excitable cells, and shed light on a role for the CED1/6/7 pathway in Ca2+ homeostasis during phagocytosis (Cuttell, 2008).

Binding of various particles induces a rise in [Ca2+]i in mammalian phagocytes. In dendritic cells, [Ca2+]i increases upon apoptotic cell binding via integrin, and inhibition studies have suggested that both Ca2+ release from the ER/SR storage pool and extracellular Ca2+ entry into the cytosol are required for this process. Neutrophils also rely on such changes to promote particle engulfment. Yet, the molecular mechanisms underlying this rise in [Ca2+]i and what role it plays in phagocytes are poorly understood (Cuttell, 2008).

This study found that uta, a Drosophila gene encoding a JP-related protein, is required for phagocytosis of apoptotic cells. Genetic evidence is provided of a role for a Ryanodine receptor, Rya-r44F, and uta and rya-44F were genetically linked. SOCE via dstim and dorai promotes efficient apoptotic cell clearance. uta and rya-44F were genetically linked to drpr and drced-6, and a role was found for uta, drpr, and drced-6 in SOCE, thus demonstrating a functional link between the DRPR/drCed-6 pathway and SOCE during phagocytosis (Cuttell, 2008).

A model is proposed whereby apoptotic cell binding via DRPR (and possibly other receptors, such as CRQ) leads to ER Ca2+ release via Rya-r44F. DRPR, which bears an immunoreceptor tyrosine-based activation motif (ITAM) that is phosphorylated via Src and Syk family kinase-mediated signaling, appears to behave like an immunoreceptor (Ziegenfuss, 2008). In B and T lymphocytes, engagement of Fc immunoreceptors (the signaling of which also relies on phosphorylation on ITAMs) leads to a rise in [Ca2+]i. Thus, DRPR might play a similar role to that of Fc receptors in the signaling, leading to a rise in [Ca2+]i in macrophages (Cuttell, 2008).

UTA is localized in the ER and at the PM. Thus, it is proposed that, like JPs, UTA forms junctional complexes that link the PM events to the ER and trigger Ca2+ release from ER stores. The current studies, however, did not address whether the formation of UTA junctional complexes is required to trigger ER Ca2+ release via Rya-r44F, nor what triggers ER Ca2+ release. The resting membrane potential of mammalian phagocytes is depolarized upon contact with apoptotic cells. As in mammalian muscle cells, such changes in fly phagocytes might initiate ER Ca2+ release (Cuttell, 2008).

In S2 cells, Ca2+ imaging results with drpr and drced-6 RNAi and that of others with drced-6 RNAi (Vig, 2006) suggest that drpr and drced-6 are required for dOrai-mediated Ca2+ entry upon TG treatment (which bypasses the need for particle binding to the receptor). It is proposed that ER Ca2+ release feeds back onto DRPR and drCed-6 to activate their downstream signaling cascade. Although further studies will be required to test the validity of this proposal, several reports already support it: DRPR-mediated phagocytosis depends on Src and Syk family kinase signaling (Ziegenfuss, 2008), and the activity of such kinases can be Ca2+ dependent in mammalian cells (Cuttell, 2008).

It is then proposed that signaling downstream of DRPR and drCed-6 promotes and/or maintains the formation of UTA junctional complexes, thereby linking ER Ca2+ release to SOCE. dSTIM is indeed likely to act as an ER Ca2+ sensor that oligomerizes (Luik, 2008) and redistributes to ER-PM junctions upon ER Ca2+ depletion, as for its mammalian counterparts (Feske, 2007). It is proposed that UTA junctional complexes are needed to maintain a close proximity between the ER Ca2+ stores and the PM and to juxtapose dSTIM oligomers and dOrai, thereby promoting conformational changes and opening of dOrai. DRPR- and drCed-6-dependent signaling and/or UTA may also be required for dSTIM oligomerization. The resulting increase in [Ca2+]i then promotes engulfment of the particle (Cuttell, 2008).

Ca2+ may promote phagocytosis via several ways. It can enhance scavenger receptor (SR) activity: adhesion of mouse macrophages to a fibronectin-coated surface via integrin binding results in an increase in the number of SRs at their PM, which enhances their binding activity. This enrichment in SRs is dependent on extracellular Ca2+ influx, arguing in favor of a role for Ca2+ in SR trafficking and/or recycling. Several SRs or related receptors play a role in phagocytosis of apoptotic corpses, including the mammalian CD36 and its Drosophila homolog CRQ. Although no change was seen in CRQ expression in uta mutant macrophages, CRQ and UTA colocalize and genetically interact. One possible model is that CRQ is recruited to the phagocytic cup upon apoptotic cell binding after SOCE that depends on UTA, DRPR, and drCed-6, and that this might strengthen the binding and uptake of the corpse (Cuttell, 2008).

In C. elegans, CED-1 (DRPR homolog) is related to the endothelial scavenger receptor SREC. Its recruitment to the phagocytic cup depends on functional CED-7, and may occur by exocytosis. Components of the exocyst were implicated in phagocytosis. Moreover, Orai1 is required for degranulation of mast cells, which occurs by exocytosis. Thus, like Orai1, dOrai may be required for exocytosis and, whereas DRPR appears to always be present at the PM, CRQ may be recruited from its intracellular vesicular pool to the phagocytic cup by exocytosis, as previously proposed for CED-1, to promote apoptotic cell uptake (Cuttell, 2008).

A rise in Ca2+ was observed in mammalian neutrophils upon particle binding, and Ca2+ participates in phagocytosis by promoting F-actin breakdown and phagosome maturation. Mycobacterium tuberculosis is able to invade human macrophages without triggering an increase in [Ca2+]i: in the absence of Ca2+ signaling, phagosomes containing M. tuberculosis fail to mature, perhaps explaining the survival of this bacterium in the cell. A role for Ca2+ in particle binding and phagosome maturation in macrophages, however, was once discounted. uta, dstim, dorai, drCed-6, and drpr are required to trigger SOCE. Yet, although they are poorly phagocytic, macrophages in drced-6 hypomorphs and drpr null mutants engulf bacteria into fully matured phagosomes, arguing against Ca2+ being involved in phagosome maturation. This maturation, however, might still occur with lower efficiency when SOCE fails, as RNAi-treated S2 cells for all genes in this pathway poorly engulfed bacteria (Cuttell, 2008).

The findings that UTA links DRPR-mediated phagocytosis and Ca2+ homeostasis provide the opportunity to pursue the dissection of the DRPR pathway in Drosophila. DRPR is homologous to CED-1, which belongs to the CED1/6/7 pathway where CED-7 is an ABC transporter. Interestingly, an ABC transporter can modulate Ca2+ channel activity in plants, further supporting a link between the CED1/6/7-like pathways and Ca2+ homeostasis, which appears to have been conserved throughout evolution. Furthermore, a mutation in human Orai1 was found in some patients with severe combined immune deficiency (Feske, 2006). Thus, pursuing such studies might be relevant to mammalian systems and to human health (Cuttell, 2008).

Presynaptic ryanodine receptor-CamKII signaling is required for activity-dependent capture of transiting vesicles

Activity elicits capture of dense-core vesicles (DCVs) that transit through resting Drosophila synaptic boutons to produce a rebound in presynaptic neuropeptide content following release. The onset of capture overlaps with an increase in the mobility of DCVs already present in synaptic boutons. Vesicle mobilization requires Ca2+-induced Ca2+ release by presynaptic endoplasmic reticulum (ER) ryanodine receptors (RyRs) that in turn stimulates Ca2+/calmodulin-dependent kinase II (CamKII). This study shows that the same signaling is required for activity-dependent capture of transiting DCVs. Specifically, the CamKII inhibitor KN-93, but not its inactive analog KN-92, eliminated the rebound replacement of neuropeptidergic DCVs in synaptic boutons. Furthermore, pharmacologically or genetically inhibiting neuronal sarco-endoplasmic reticulum calcium ATPase (SERCA) to deplete presynaptic ER Ca2+ stores or directly inhibiting RyRs prevented the capture response. These results show that the presynaptic RyR-CamKII pathway, which triggers mobilization of resident synaptic DCVs to facilitate exocytosis, also mediates activity-dependent capture of transiting DCVs to replenish neuropeptide stores (Wong, 2009).

The function of nerve terminals depends on vesicular delivery of proteins synthesized in the soma to synaptic boutons. Transport vesicles are known to contain channels, active zone constituents and neuropeptides. In contrast to synaptic membrane proteins and classical transmitters that are recycled following exocytosis, neuropeptide release is irreversible. Thus, peptidergic transmission depends on replacement of neuropeptide-containing dense core vesicles (DCVs). This is potentially a very slow process because delivery of vesicles synthesized in the soma to nerve terminals by fast axonal transport can take days. However, a cell biological strategy has been discovered that bypasses such delays. Activity-dependent capture of transiting vesicles utilizes a pool of DCVs that rapidly pass through the resting nerve terminal, but that are captured in response to a burst of activity (Shakiryanova, 2006). The onset of this capture, which is evident as decreased DCV efflux and increased neuropeptide content in synaptic boutons, occurs over a period of minutes instead of the hours that would be required for conventional steady state DCV replacement. Essentially, the nerve terminal can tap into the transiting DCV pool to rapidly replenish neuropeptide stores without any direct involvement of the soma. Hence, activity-dependent capture of transiting DCVs eliminates the delay in delivering nascent DCVs, apportions resources based on activity and places control of synaptic neuropeptide storage at sites of release instead of the site of synthesis (i.e., the soma) (Shakiryanova, 2006). A similar recruitment process also occurs with neurotrypsin-containing vesicles, which were concluded to rapidly undergo exocytosis following stimulated capture (Frischknecht, 2008). Likewise, vesicle capture appears to be involved in release of presynaptic Wnt/Wingless protein (Ataman, 2008). Therefore, activity-dependent capture of transiting vesicles supports synaptic neuropeptide, enzyme and developmental peptide release (Wong, 2009).

The signaling required for activity-dependent capture of transiting DCVs is unknown. The long duration of this response in Drosophila motor neurons (i.e., for ~0.5 hour) coupled with the requirement for electrical activity suggests a potential involvement of Ca2+-induced phosphorylation. In fact, recent experiments have shown that such signaling increases the mobility of resident DCVs in synaptic boutons. Mobilization, which is triggered by Ca2+ influx and persists for ~10 minutes (Shakiryanova, 2005), requires Ca2+-induced Ca2+ release from presynaptic endoplasmic reticulum (ER) via ryanodine receptors (RyRs) (Shakiryanova, 2007). Subsequently, Ca2+/calmodulin-dependent protein kinase II (CamKII) is activated as a necessary step for DCV mobilization (Shakiryanova, 2007). The overlapping onset of the capture and mobilization responses in the first minutes following a brief tetanus stimulated an investigation of whether the RyR-CamKII pathway participates in activity-dependent capture of transiting vesicles (Wong, 2009).

In this study a GFP (Green Fluorescent Protein)-tagged neuropeptide was imaged at the intact Drosophila neuromuscular junction. The rebound in synaptic neuropeptide stores following activity-evoked release, which is caused by capture of transiting vesicles (Shakiryanova, 2006), requires RyR-mediated Ca2+ efflux from presynaptic ER and activation of CamKII. Therefore, RyR-CamKII signaling initiates both mobilization of resident DCVs within synaptic boutons and capture of DCVs from the rapidly transiting pool (Wong, 2009).

In vivo imaging has shown that a brief bout of activity elicits prolonged DCV mobilization and capture. These processes are independent because capture requires axonal transport while mobilization does not (Shakiryanova, 2005; Shakiryanova, 2006). Nevertheless, the onsets of mobilization of resident DCVs and capture of transiting DCVs overlap (i.e., both develop over minutes following seconds of activity). This observation stimulated a test of the hypothesis that these two mechanisms are initiated by the same signaling. Previous studies had established that Ca2+ influx triggers DCV mobilization by activating RyR-mediated Ca2+ release from presynaptic ER that in turn stimulates CamKII (Shakiryanova, 2007). The pharmacological and genetic experiments presented in this study establish that RyR-CamKII signaling is also required for activity-dependent capture of transiting DCVs (Wong, 2009).

This finding raises the issue of how a single signaling pathway produces responses with different durations: after seconds of activity, DCV mobilization lasts ~10 minutes, while the capture response lasts ~40 minutes (Shakiryanova, 2005; Shakiryanova, 2006). One possible consideration is that these kinetic differences could originate in the processes responsible for reversal of mobilization and capture. Specifically, RyR-CamKII signaling could initiate the two processes in parallel, but dephosphorylation of distinct CamKII substrates might occur at different rates, possibly because of the involvement of different phosphatases. This potential explanation suggests that identifying the CamKII substrates that mediate mobilization and capture will be important for understanding the diversity in long-lasting responses initiated by activity-triggered presynaptic RyR-CamKII signaling. Recently, CamKII-dependent phosphorylation of kinesin superfamily protein 17 (KIF17) was found to be essential for unloading NMDA receptor-carrying cargoes from microtubules near the postsynaptic density (Guillaud, 2008). Therefore, CamKII might induce capture by triggering dissociation of transiting DCVs from their molecular motor dynactin complex, which would contain both a kinesin-3 family member UNC-104/Kif1 and a dynein retrograde motor, while mobilization might depend on another CamKII substrate. Alternatively, some process downstream of dephosphorylation might be rate determining for reversal of capture. For example, once captured vesicles are committed to return to the transiting pool, they might need to recruit an unoccupied motor complex to support rapid transiting. If such complexes are rare, then recovery from capture would be very slow. In contrast, recovery from mobilization, which does not require exiting from the bouton, would not be limited in the same way. Regardless of the specific basis for the diverse time courses of mobilization and capture, the use of the same signaling pathway to induce both of these effects is an elegant means to ensure that facilitation of release is coupled to replacement of depleted synaptic neuropeptide stores (Wong, 2009).

Prolonged presynaptic posttetanic cyclic GMP signaling in Drosophila motoneurons

Ca2+ can stimulate cyclic nucleotide synthesis, but it is not known whether this signaling occurs in nerve terminals in response to activity. In this study, in vivo imaging of Drosophila motoneuron terminals shows that activity rapidly induces a long-lasting signal from a transgenically expressed optical indicator based on the epac1 (exchange protein directly activated by cAMP 1) cAMP-binding domain. The epac1-cAMP sensor (camps) response in synaptic boutons depends on extracellular Ca2+ and ryanodine receptor-mediated Ca2+-induced Ca2+ release from the endoplasmic reticulum. However, mutations that inhibit rutabaga Ca2+-stimulated adenylyl cyclase and dunce cAMP-specific phosphodiesterase (PDE) have no effect. Instead, the activity-dependent presynaptic epac1-camps signal reflects elevation of cGMP in response to nitric oxide-activated guanylyl cyclase. Posttetanic presynaptic cGMP is long-lived because of limited PDE activity. Thus, nerve terminal biochemical signaling induced by brief bouts of activity temporally summates on a time scale orders of magnitude longer than fast transmission (Shakiryanova, 2008).

The mechanisms that control cyclic nucleotide levels in the nerve terminal are not known. Activity-induced presynaptic Ca2+ influx could be involved because some cyclases are directly activated by Ca2+. For example, the rutabaga gene, which affects synaptic plasticity and development in Drosophila, encodes a neuronal Ca2+-stimulated adenylyl cyclase. However, this isoform is also stimulated by the Gsα G protein. Thus, it is not clear whether the presynaptic effects of the rutabaga adenylyl cyclase reflect a permissive background effect or activation by Gsα or Ca2+. Determining the mechanisms responsible for activating presynaptic cyclases has been difficult because it has not been possible to directly measure cyclic nucleotides in living nerve terminals (Shakiryanova, 2008).

Recently, a ratiometric FRET-based cAMP sensor (camps) that uses the cAMP binding domain of epac (exchange protein directly activated by cAMP) was generated to report activation of adenylyl cylase by forskolin and receptors. Here, in vivo imaging shows that activity rapidly induces a Ca2+-dependent epac1-camps response in Drosophila motoneuron synaptic boutons. Surprisingly, this response is long-lasting and is unaffected by mutants that disrupt rutabaga, which lowers neuronal cAMP, or the dunce cAMP-specific phosphodiesterase (PDE), which elevates neuronal cAMP. Pharmacological and genetic experiments are presented that identify the presynaptic biochemical signaling detected by epac1-camps and explain how it temporally summates on a time scale of minutes (Shakiryanova, 2008).

The presynaptic epac1-camps response depends on Ca2+ influx and Ca2+-induced Ca2+ release. First, removal of extracellular Ca2+ abolished the FRET change induced by a tetanus. Second, ryanodine (Ry) block of Ca2+-induced Ca2+ release by endoplasmic reticulum (ER) Ry receptors (RyRs), which eliminates activity-dependent vesicle mobilization and capture of transiting of transiting vesicles in Drosophila nerve terminals, reduced the epac1-camps response. To verify that RyRs were responsible for this partial inhibition, ER Ca2+ was depleted with thapsigargin (Tg). Under these conditions Ry had no effect, verifyng that RyR-mediated Ca2+-induced Ca2+ release was involved. Furthermore, Tg and Ry effects were statistically indistinguishable, implying that RyRs fully account for the participation of ER Ca2+ stores (i.e., there was no evidence of autoreceptor-induced inositol trisphosphate-mediated ER Ca2+ release). Together, these results show that Ca2+-induced Ca2+ release from the ER amplifies cyclic nucleotide signaling induced by Ca2+ influx (Shakiryanova, 2008).

In vivo imaging of epac1-camps in nerve terminals has yielded a number of striking results. First, activity-dependent cyclic nucleotide synthesis was demonstrated in synaptic boutons. This finding establishes a mechanism for coupling nerve terminal activity to cGMP signaling, which induces synaptic vesicle exocytosis at Drosophila motoneuron boutons and supports plasticity at other synapses. Second, previous studies of NOS-cGMP at synapses have focused on transynaptic effects (e.g., NO as a retrograde signal). However, because glutamatergic transmission was inhibited and postsynaptic muscle in this preparation does not produce cGMP in response to NO, NO generation and activation of guanylyl cyclase both occur presynaptically. This arrangement minimizes dilution of NO to produce robust activity-evoked cGMP responses in motoneuron terminals. Third, cGMP synthesized in response to brief bouts of activity is long-lived in synaptic boutons, because non-dunce PDE activity is limited in the nerve terminal. Hence, temporal summation of cGMP responses occurs on a time scale orders of magnitude longer than neurotransmission. Prolonged electrical activity-induced signaling may be important for a variety of NOS-dependent processes in insect nervous systems (Shakiryanova, 2008).

Calcium-stores mediate adaptation in axon terminals of olfactory receptor neurons in Drosophila

In vertebrates and invertebrates, sensory neurons adapt to variable ambient conditions, such as the duration or repetition of a stimulus, a physiological mechanism considered as a simple form of non-associative learning and neuronal plasticity. Although various signaling pathways, as cAMP, cGMP, and the inositol 1,4,5-triphosphate receptor (InsP3R) play a role in adaptation, their precise mechanisms of action at the cellular level remain incompletely understood. In Drosophila odor-induced Ca2+-response in axon terminals of olfactory receptor neurons (ORNs) has been shown to be related to odor duration. In particular, a relatively long odor stimulus (such as 5 s) triggers the induction of a second component involving intracellular Ca2+-stores. A recently developed in-vivo bioluminescence imaging approach was used to quantify the odor-induced Ca2+-activity in the axon terminals of ORNs. Using either a genetic approach to target specific RNAs, or a pharmacological approach, this study showed that the second component, relying on the intracellular Ca2+-stores, is responsible for the adaptation to repetitive stimuli. In the antennal lobes (a region analogous to the vertebrate olfactory bulb) ORNs make synaptic contacts with second-order neurons, the projection neurons (PNs). These synapses are modulated by GABA, through either GABAergic local interneurons (LNs) and/or some GABAergic PNs. Application of GABAergic receptor antagonists, both GABAA or GABAB, abolishes the adaptation, while RNAi targeting the GABABR (a metabotropic receptor) within the ORNs, blocks the Ca2+-store dependent component, and consequently disrupts the adaptation. These results indicate that GABA exerts a feedback control. Finally, at the behavioral level, using an olfactory test, genetically impairing the GABABR or its signaling pathway specifically in the ORNs disrupts olfactory adapted behavior. Taken together, these results indicate that a relatively long lasting form of adaptation occurs within the axon terminals of the ORNs in the antennal lobes, which depends on intracellular Ca2+-stores, attributable to a positive feedback through the GABAergic synapses (Murmu, 2011).

This study provides evidence that the bioluminescent (GFP-aequorin) Ca2+-sensor is sensitive enough to monitor the Ca2+-response following various protocols (duration and repetition-frequency) of odor application. 1 s of odor induces a response which does not significantly decrease if repeated every 5 min, whereas a longer stimulus, such as 5 s, is sufficient to induce a decrease in response following repeated odor stimulations (adaptation). Similarly, using a 5 s odor stimulation and increasing the frequency of repetition to 1-min intervals also induces, in an odor specific manner, a faster adaptation. It was also demonstrated that prolonged odor application (up to 2 min) generates a sustained Ca2+-response within the ORN axon terminals, indicating that the ORNs are capable of responding as long as the odor is presented, of even longer. This work also indicates that the GFP-aequorin probe is not a limiting factor for the detection of the Ca2+-activity. These physiological results (reduction of the Ca2+-activity according to prolonged/sustained odor duration and/or odor repetition) are consistent with previous studies which report that adaptation depends both on the duration of a stimulus and on the frequency of its repetition (Murmu, 2011).

Different physiological approaches, based either on fluorescence brain imaging or electrophysiological techniques have previously reported odor-induced activity in different interconnected neurons in the antennal lobes of different invertebrate model organisms, including honeybees with the goal of deciphering the neural odor code. However, except for one study performed in locusts, which indirectly described a form of adaptation, long-lasting forms of adaptation within ORNs such as that described in this study have not been reported. This is likely due to the experimental design of these previous studies, which either generally took into account the odor-induced signal solely after the response was stabilized (generally after about 5 successive odor applications), or used a shorter odor stimulation duration (< = 1 s), which as demonstrated in this study, is not sufficient to induce detectable and reliable adaptation. Additionally, others have relied on extracellular recordings of the sensillae of the antennae which reflects the activity occurring in the cell-bodies of the ORNs. In this study, monitoring the axon terminals of the ORNs, 5 s odor stimulations, repeated at 5-min intervals, induced a relatively long-lasting adaptation that resembles in term of kinetics, the long-lasting adaptation (LLA) reported in ORNs in salamanders. Indeed and interestingly, the recovery time (15 min for spearmint and octanol and 30 min for citronella) occurs over a similar time scale in salamander ORNs (which are different from the long-lasting olfactory adaptation described in C. elegans. However, in contrast to long-lasting adaptation, which was reported in isolated ORNs, the adaptation described in this study seems to rely on different mechanisms, since it is sensitive to a 'feedback control' provided by GABAergic synaptic transmission within the antennal lobes (Murmu, 2011).

In Drosophila, mutants lacking InsP3R are defective in olfactory adaptive behavior. In vertebrates, different forms of olfactory adaptation have also been reported in the ORNs. First, this study shows in Drosophila that an adaptation mechanism occurs in axon terminal of the ORNs in the antennal lobes. Second, using two independent approaches, pharmacological and genetic, it was shown that odor-induced specific adaptation relies principally on InsP3R and RyR. When these two different receptors are blocked or knocked-down, although some difference (variability) can be observed between different conditions, overall the odor-induced Ca2+-response no longer adapts or is severely affected. More specifically, it seems that the lack of adaptation is due to the non-induction of the 'second delayed and slow rising component' of the Ca2+-response, which is triggered in particular and specific conditions: when the duration of an odor stimulation is relatively long (1 s does not induce it, while 5 s induces an important second component. Alternatively, the second component of the response is also induced and visible particularly on the first and to a lesser extent, on the second odor applications, especially when the odor is successively repeated. This second component gradually vanishes with sequential repetition. That is, adaptation is not directly due to a decrease in the response, but rather indirectly to a defect in presynaptic Ca2+-increase, due to a lack of triggering release of intracellular Ca2+-stores, normally occurring in the first and successive responses following either a sufficiently strong (long stimulus) or repeated stimuli. These results suggest that one of the major intracellular mechanisms of adaptation depends on internal Ca2+-stores. In brief, the intracellular mechanism was blocked that allows the cell to adapt to long lasting or repetitive stimuli. Interestingly, in mammals, in hippocampal CA3 pyramidal neurons, intracellular Ca2+-stores, which are controlled by InsP3R and/or RyR at the presynaptic terminal, have been previously implicated in neurotransmitter release as well as in synaptic plasticity (Murmu, 2011).

In vertebrates, neuronal plasticity related to odor representation occurs at the synapse between the ORNs and the second-order neurons in the olfactory bulb glomeruli, a region analogous to the invertebrate antennal lobes. At this synapse, signal transmission is modulated presynaptically by several mechanisms, a major one being via the metabotropic GABAB receptors. This suppresses presynaptic Ca2+-influx and subsequently transmitter release from the receptor neurons terminal. At least two kinds of presynaptic inhibition (intra- and interglomerular) are mediated by GABAB receptors. Intraglomerular presynaptic inhibition seems to control input sensitivity, while interglomerular presynaptic inhibition seems to increase the contrast of sensory input (although the two studies addressing this question in-vivo show contradictory results). In Drosophila, a similar mechanism seems to occur, as interglomerular presynaptic inhibition, mediated by both ionotropic and metabotropic receptors on the same axon terminal of the ORNs, mediate gain control mechanism, serving to adjust the gain of PN in response to ORN stimulation (Olsen, 2008). Yet another study has suggested that GABAB but not GABAA receptors are involved in presynaptic inhibition (Root, 2008) yielding a contradiction. In this study, by monitoring the Ca2+-release from the axon terminals of ORNs, in experimental conditions that generate a long-lasting form of adaptation, it was shown that GABAergic synaptic transmission plays a role in adaptation. Both ionotropic GABAAR antagonists, bicuculline and picrotoxin, block partially or completely the Ca2+-response, while, CGP54626, a metabotropic GABABR antagonist, also blocks the adaptation, albeit not completely. It should be mentioned that application of picrotoxin per se induces a strong transient Ca2+-release within the axon terminals of the ORNs, even without odor application. This 'transient release effect' likely disturbs the resting state of the neurons, which probably accounts for the important reduction observed in the amplitude of the odor-induced response. Nevertheless, these results suggest that both types of GABA receptors (A and B) are involved in adaptation. Moreover, as proposed by the study of Olsen and Wilson (2008), it cannot be ruled-out that ORNs also express different subtypes of GABAAR (homo- and/or heteromultimers), since the results showed that picrotoxin and particularly bicuculline, two distinct inhibitors of GABAAR, block adaptation. Another possibility is that the effect of the two GABAAR antagonists results from the blockage of GABAAR on other neurons in the antennal lobes, as the LNs or certain PNs (which have not yet been demonstrated). Lastly and unfortunately, this pharmacological approach does not allow distinguishing by which precise neurons this GABAergic-dependent adaptation is mediated. With the goal of clarifying precisely in which neurons GABAergic transmission acts, the metabotropic GABABR (GABABR2-RNAi) or its signaling pathway (UAS-PTX) were blocked directly within ORNs. This yields defects in long-lasting adaptation for several conditions, seemingly in an odor specific manner. Therefore, although GABAergic effects have been described in ORNs of both Drosophila and mammals, to support 'feedback inhibition', this study reports that in different experimental conditions such as a long odor duration (5 s) and/or repetition of the stimulus, it also participates in the adaptation process. Indeed, the results suggest that GABA signaling support a positive (excitatory) feedback control instead of an inhibitory feedback, as formerly reported by other studies. Though these results seem to be contradictory, some explanations can be provided. First, as aforementioned, the experimental conditions are different: this study used a relatively high odor concentration with relatively long odor duration (5 s). In addition, recordings were taken immediately from the first odor application and the successive one, while in the experimental protocol of certain studies, the odor is generally presented several times (priming) before the beginning of recording. Consequently, it seems that these previous studies were performed on already adapted ORNs. This implies that the neuronal network in the antennal lobes was already stimulated, and therefore its dynamics was probably already modified, since as described in this study, an important effect occurs immediately after the first odor application. Moreover, GFP-aequorin allows monitoring, in continuity over a long time period, the intracellular level of calcium with high sensitivity to [Ca2+] (from ~ 10-7 to 10-3). In addition, although it is not possible to precisely assign which glomeruli are activated, this approach allows visualizing simultaneously the odor-induced Ca2+-activity from the entire antennal lobes (the overall depth). Therefore, the outcome of the overall response of the antennal lobes is being monitored, instead of the response from single or a few glomeruli. Finally, in vertebrates it has been reported that in certain experimental conditions, GABA could be excitatory, although this contradiction cannot yet be precisely explained. Furthermore, it seems that a given synapse can display inhibitory effects under one protocol and an excitatory effect with another. Notably, it has been reported that a short stimulation of GABA is inhibitory, while during a long stimulation, the GABA effect can switch from inhibitory to excitatory. Interestingly, this particular 'switching effect' could potentially explain the current 'contradictory' situation reported in this study: in the current experimental conditions, in which a relatively long odor stimulus (5 s) was used, GABA generates an excitatory effect, whereas in previous studies based on short (<1 s) odor stimuli, GABA seems to be inhibitor. This difference in the duration of stimuli could perhaps account for such inverted or 'switching effects' (Murmu, 2011).

To explore the behavioral and functional consequences of disturbing the GABAergic signaling pathway, flies were studied with a GABABR2 (RNAi) ORN-specific genetic knockdown, as well those with a component of its signaling pathway, the G-protein, blocked by the pertussis toxin. Both groups of flies present strong behavioral deficits, as adaptation-disrupted flies are not able to discern between odors and air after 5-min of exposure to odor. Interestingly, control flies reverse their choice preferring odor after a 5-min pre-exposure (adaptation) suggesting that in these experimental conditions, the meaning of the odor changes in the fly's adapted state. These results are consistent with previous studies suggesting that adaptation could serve to extend the operating range of sensory systems over different stimulus intensities. In other terms, adaptation modifies the sensitivity (threshold) to the odor, as previously reported in different organisms, such as C. elegans and vertebrates including humans. This phenomenon is similar to that in other sensory modalities, as in visual system, where light adaptation in photoreceptors sets the gain, allowing vision at both high and low light levels. As previously reported, odors could be repulsive (at high concentrations) or attractive (at low concentrations). In the current experimental conditions in control flies the odors are repulsive. However, after 5-min of preexposure, the flies adapt to this odor concentration, and when tested at the same concentration odors are then likely only weakly perceived and therefore might correspond to an attractive 'weak-odor concentration'. In a former study in similar experimental conditions, it was reported that the flies are attracted by each of these three odors for a weak odor concentration. Interestingly, reverse odor preference has already been reported in C. elegans, resulting from presynaptic changes involving a receptor-like guanylate cyclase (GCY-28) via the diacylglycerol/protein kinase C pathway. Finally, the fact that without pre-exposure all groups of flies preferred the control arm and were repelled by the odorants indicates that the odor acuity of these flies is intact. In other words, odor-adaptation and not odor-acuity is affected in each of these groups of flies. These results strengthen the idea that odor perception and adaptation are indeed two distinct and separable processes (Murmu, 2011).

This study has demonstrated that the adaptation process occurring specifically in the axon terminals of the ORNs depends on intracellular Ca2+-stores, through InsP3 and ryanodine receptors. Moreover, evidence is provided that this Ca2+-release requires synaptic transmission, since it does not occur when the cholinergic receptors are blocked (α-bungarotoxin experiment). It also requires a feedback control through GAB A, since blocking GABAB signaling within ORNs prevents or strongly affects adaptation, suggesting that a local neuronal network mediated by GABAergic neurons is involved (for a more complete overview, see the schematic model of synaptic interactions within the antennal lobes. In complement to the brain imaging data, knocking-down the metabotropic GABABR2, or its signaling pathway specifically in the ORNs, yields olfactory functional behavioral deficits. These results, combined with the results of blocking the InsP3R or RyR suggest that a crucial olfactory integration process that can be ascribed to a form of neuronal plasticity and/or short-term memory occurs directly in the ORNs immediately after the first odor application or during a prolonged odor application. Thus, this effect could resemble the long-lasting form of odor adaptation described previously at the cellular and systems levels in vertebrates, including humans. By extension, it is hypothesized that in humans, the well-known 'odor-specific transient functional anosmia' following a prolonged odor exposure, which results from an adaptation, may also rely on intracellular Ca2+-stores (Murmu, 2011).

A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila

Long-term exposure to environmental oxidative stressors, like the herbicide paraquat (PQ), has been linked to the development of Parkinson's disease (PD), the most frequent neurodegenerative movement disorder. PQ is thus frequently used in the fruit fly Drosophila melanogaster and other animal models to study PD and the degeneration of dopaminergic neurons (DNs) that characterizes this disease. This study shows that a D1-like dopamine (DA) receptor, DAMB (Dopamine 1-like receptor 2), actively contributes to the fast central nervous system (CNS) failure induced by PQ in the fly. Firstly, it was found that a long-term increase in neuronal DA synthesis reduced DAMB expression and protected against PQ neurotoxicity. Secondly, a striking age-related decrease in PQ resistance correlated to an augmentation of DAMB expression. This aging-induced increase in oxidative stress vulnerability not observed in a DAMB-deficient mutant. Thirdly, targeted inactivation of this receptor in glutamatergic neurons (GNs) markedly enhanced the survival of Drosophila exposed to either PQ or neurotoxic levels of DA, while, conversely, DAMB overexpression in these cells made the flies more vulnerable to both compounds. Fourthly, a mutation in the Drosophila ryanodine receptor (RyR), which inhibits activity-induced increase in cytosolic Ca2+, also strongly enhanced PQ resistance. Finally, this study found that DAMB overexpression in specific neuronal populations arrested development of the fly and that in vivo stimulation of either DNs or GNs increased PQ susceptibility. This suggests a model for DA receptor-mediated potentiation of PQ-induced neurotoxicity. Further studies of DAMB signaling in Drosophila could have implications for better understanding DA-related neurodegenerative disorders in humans (Cassar, 2014).

Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila

A mutation (Cam7) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. The biochemical properties of V91G D-CaM were investigated. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described the this paper establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM (Wang, 2004).

Drosophila Pkd2 is haploid-insufficient for mediating optimal smooth muscle contractility

Humans heterozygous for PKD1 or PKD2 develop autosomal dominant polycystic kidney disease, a common genetic disorder characterized by renal cyst formation and extrarenal complications such as hypertension and vascular aneurysms. Cyst formation requires the somatic inactivation of the wild type allele. However, it is unknown whether this recessive mechanism applies to life-threatening vascular aneurysms, which could involve weakening of the endothelial lining or surrounding vascular smooth muscle cells (SMCs). Drosophila Polycystic kidney disease gene-2 at 33E3 (Pkd2) encodes a PKD2 family of Ca(2+)-activated Ca(2+)-permeable cation channels. Loss-of-function Pkd2 mutations severely reduced the contractility of the visceral SMCs, which was restored by expressing wild type Pkd2 cDNA via a muscle-specific Gal4 driver. The effect of Pkd2 mutations alone on the skeletal muscle was minimal but was exacerbated by ryanodine-induced perturbation of intracellular Ca(2+) stores. Consistent with this, Pkd2 interacted strongly with a ryanodine receptor mutation, causing a synergistic reduction of larval body wall contraction rate that is normally regulated through Ca(2+) oscillation during excitation-contraction coupling in the skeletal muscle. These results suggest that PKD2 cooperates with the ryanodine receptor to promote optimal muscle contractility through intracellular Ca(2+) homeostasis. Further genetic analysis indicated that Pkd2 is strongly haploinsufficient for normal SMC contractility. Since Ca(2+) homeostasis is a conserved mechanism for optimal muscle performance, these results raise the possibility that inactivation of just one PKD2 copy is sufficient to compromise vascular SMC contractility and function in PKD2 heterozygous patients, thus explaining their increased susceptibility to hypertension and vascular aneurysms (Gao, 2004).

Biochemical characterization, distribution and phylogenetic analysis of Drosophila melanogaster ryanodine and IP3 receptors, and thapsigargin-sensitive Ca2+ ATPase

The biochemistry, distribution and phylogeny of Drosophila ryanodine (RyR) and inositol triphosphate (IP3R) receptors and the endoplasmic reticulum Ca2+-ATPase (SERCA) were characterized by using binding and enzymatic assays, confocal microscopy and amino acid sequence analysis. 3H-ryanodine binding in total membranes was enhanced by AMP-PCP, caffeine and xanthine, whereas Mg2+, Ruthenium Red and dantrolene were inhibitors. 3-ryanodine binding showed a bell-shaped curve with increasing free [Ca2+], without complete inhibition at millimolar levels of [Ca2+]. 3-IP3 binding was inhibited by heparin, 2-APB and xestospongin C. Microsomal Ca2+-ATPase activity was inhibited by thapsigargin. Confocal microscopy demonstrated abundant expression of ryanodine and inositol triphosphate receptors and abundant Ca2+-ATPase in Drosophila embryos and adults. Ryanodine receptor was expressed mainly in the digestive tract and parts of the nervous system. Maximum parsimony and Neighbour Joining were used to generate a phylogenetic classification of Drosophila ryanodine and insitol triphosphate receptors and Ca2+-ATPase based on 48 invertebrate and vertebrate complete sequences. The consensus trees indicated that Drosophila proteins grouped with proteins from other invertebrates, separately from vertebrate counterparts. Despite evolutionary distances, functional results demonstrate that Drosophila ryanodine and inositol triphosphate receptors and Ca2+-ATPase are reasonably similar to vertebrate counterparts. Protein expression data are consistent with the known functions of these proteins in the Drosophila digestive tract and nervous system. Overall, results show Drosophila as a valuable tool for intracellular Ca2+ dynamics studies in eukaryotes (Vázquez-Martínez, 2003).

The ryanodine receptor is essential for larval development in Drosophila

The study investigated role of the ryanodine receptor in Drosophila development by using pharmacological and genetic approaches. A P element insertion was identified in the Drosophila ryanodine receptor gene, Ryanodine receptor 44F (Ryr), and it was used to generate the hypomorphic allele Ryr16. An examination of hypodermal, visceral, and circulatory muscle showed that, in each case, muscle contraction was impaired in Ryr 16 larvae. Treatment with the drug ryanodine, a highly specific modulator of ryanodine receptor channel activity, also inhibited muscle function, and, at high levels, completely blocked hypodermal muscle contraction. These results suggest that the ryanodine receptor is required for proper muscle function and may be essential for excitation-contraction coupling in larval body wall muscles. Nonmuscle roles of Ryr were also investigated. Ryanodine-sensitive Ca2+ stores had previously been implicated in phototransduction; to address this, Ryr16 mutant clones were generated in the adult eye and whole-cell, patch-clamp recordings were performed on dissociated ommatidia. The results do not support a role for Ryr in normal light responses (Sullivan, 2000).

The Drosophila genome contains a single RyR gene at cytological position 44F. Genomic sequence was obtained flanking each P element in the 44F region and it was determined that P{lacW}l(2)k04913 was inserted in an intron of Ryr, 255 bp upstream of the first coding exon. l(2)k04913 was excised, and it was found that 80% of the independent excision lines were viable, indicating that the lethal lesion of l(2)k04913 corresponded to the P element insertion site and that there were no other lethal mutations on the chromosome. All but one of the lethal excisions failed to complement l(2)k04913 and Df(2R)Np3, a deficiency that uncovers Ryrk04913, confirming that these mutations were allelic to the P element. Using PCR, four lethal excision lines were identified that had deletions specifically in Ryr. The largest, Ryr 16, removed the first coding exon and extended into the first and second introns (Sullivan, 2000).

Ryr encodes a protein >5,000 amino acids in length that contains many methionine residues. The transmembrane, channel-forming domain is found at the extreme C terminus, and it has been shown for RyR1 that the C-terminal third of the protein is sufficient to make a functional channel (Sullivan, 2000).

Ryr is widely expressed. RNA in situs were performed on embryos using both genomic- and cDNA-derived probes. Ryr expression was first detected in the mesoderm around stage 9 and then increased starting at stage 13. The highest levels were seen in hypodermal muscles and in the visceral muscles surrounding the gut. The transcript was also detected at lower levels in other tissues, notably the central nervous system (Sullivan, 2000).

Based on the in situ results, the role of Ryr was examined in hypodermal or body wall muscles, which are analogous in structure and function to vertebrate skeletal muscle. Forward movement is initiated by contraction of the posterior body wall muscles, moving the tail up, forward, and down. The contraction propagates as a constriction anteriorly, narrowing and lengthening the larval body, and finally terminates on extension of the mouth hooks up and forward. Initiation of a new body wall contraction (BWC) typically does not occur until termination of the previous one; however, initiation and propagation appear to be independent processes. For example, in calmodulin null mutants, initiation of BWCs was greatly decreased, whereas propagation occurred at normal speeds once initiated (Sullivan, 2000).

BWC was quantified by counting the number of end-to-end contractions a single larva underwent per minute. In Ryr 16 larvae, the timing of BWC initiation appeared normal, but the contractions propagated more slowly than in wild-type and Ryr 16/CyO-GFP arm controls, such that the rate of BWC was reduced by 50%. Furthermore, contraction in Ryr 16 animals was attenuated and often had associated tremors; this muscle weakness is probably responsible for the altered larval appearance. As expected, Ryr 16/Df(2R)Np3 larvae had a more severe defect in BWC propagation, which was reduced by over 90%. The weakest allele, Ryr k04913, had a small but significant decrease in the rate of BWC propagation compared with wild-type and Ryr k04913/CyO-GFP arm controls. By the second instar, Ryrk04913 larvae had visible contraction defects in hypodermal muscle, which included tilting and dragging of the mouth hooks (Sullivan, 2000).

The drug ryanodine is a highly specific modulator of the RyR channel. In vitro studies have demonstrated that low doses of ryanodine (<10 microM) activate the channel, medium doses (>10 microM) lock the channel into a subconductance state, and high concentrations (~100 microM) completely inactivate it. A range of ryanodine concentrations was fed to newly hatched larvae in yeast paste, and the effect on BWC rates was determined. Low concentrations of ryanodine (<5 microM) had no significant effect on BWC rates when compared to larvae fed yeast paste doped with solvent alone. Higher ryanodine levels (5-100 microM) decreased the rate of BWC propagation, but not initiation, and at the highest doses (~100 microM), BWC was completely inhibited. Ryanodine similarly inhibited BWC in second and third instar larvae. Ryanodine concentrations ~10 microM also caused larvae to round up, altering their appearance in a manner similar to that of Ryr 16. These results demonstrate that Ryr 16 is phenocopied by inhibitory concentrations of ryanodine, which is expected if the drug and the mutation both target Ryr. The complete inhibition of BWC by ryanodine provides further evidence that Ryr16 is not a null allele and suggests that the ryanodine receptor is essential for ECC in hypodermal muscles. In contrast, C. elegans (Maryon, 1998) can undergo muscle contraction even in the absence of RyR activity (Sullivan, 2000).

Visceral muscle function was analyzed in wild-type and Ryr 16 larvae by assaying the ingestion and excretion of food. The dye bromophenol blue is nontoxic, readily ingested, and completely excreted by larvae, as well as easy to detect through the cuticle. In a typical ingestion assay, 96%-100% of wild-type, Itp-r 1, and Ryr 16/CyO-GFP arm larvae scored positive for ingestion within 30 min. A very mild, but reproducible, ingestion defect was seen in Ryrk04913 larvae. In sharp contrast, Ryr 16 larvae ingested food much more slowly, and the percentage that scored positive failed to reach 100 even after 2 days. Additionally, Ryr 16 larvae accumulated food in the pharynx, which was never seen in controls, and those that failed to ingest food were indistinguishable in growth and movement from those that did. Ryr16 mutants may have additional defects in nutrient absorption or metabolism; however, it may simply be that none of the Ryr 16 larvae ingest sufficient food for growth. These results demonstrate that Ryr 16 mutants have a severe defect in the ingestion and passage of food into the gut, suggesting that the head and visceral muscles are impaired (Sullivan, 2000).

Pulse-chase and defecation assays were used to measure visceral muscle function specifically. Newly hatched larvae were fed blue yeast for 4 h, then transferred to undyed yeast and scored for complete loss of the dye. In a typical excretion time course, 100% of Ryr 16/CyO-GFP arm, Itp-r 1, and wild-type larvae had completely excreted the dye within 3 h. In contrast, Ryr 16 larvae excreted the dye extremely slowly, such that roughly half still retained dye in the gut when assayed for up to 12 h. As a second test for visceral muscle function, the rate of defecation was measured. Wild-type and heterozygous larvae on average defecated once every 4 min, Ryr k04913 once every 7 min, and Ryr16 less than once per 60 min. Both assays demonstrate that Ryr 16 larvae have a severe defect in excretion, consistent with impaired visceral muscle function (Sullivan, 2000).

Drosophila visceral muscles, although considered striated muscles, are distinct from those of the hypodermus: they are much smaller and mononucleate; the contractile elements are reduced in density and more disorganized; and the sarcoplasmic reticulum (SR) is less developed. It has been proposed that the extent of SR in a given muscle type correlates with the requirement for intracellular Ca2+, and thus the RyR, in contraction. However, this hypothesis is inconsistent with the severe visceral defect seen in Ryr16 larvae. One possibility is that visceral muscle ECC requires Ca2+ release from internal stores, but not the rapid, widespread release enabled by extensively developed stores. Alternatively, intracellular Ca2+ stores may be essential for some other process equally vital for the passage of food through the gut, such as Ca2+ homeostasis or muscle relaxation (Sullivan, 2000).

The DV is the major pulsatile organ of the Drosophila circulatory system. The contractile region or heart is a tube-shaped chamber of cardial cells in the posterior segments that circulates the hemolymph by lateral constriction. The aorta extends anteriorly and connects to the ring gland and lymph nodes. Little is known about the structure or physiology of the circulatory muscle, although L-type channels have been implicated in DV contraction, as is the case in vertebrate cardiac cells. DV function and development have been compared with that of the vertebrate heart, although it has recently been argued that visceral muscle development is the more analogous (Sullivan, 2000).

By expressing GFP in muscles, the larval heartbeat was examined in vivo for all three larval stages; it remained relatively consistent. The heartbeat frequency ranged from slow to fast to fibrillating, with the heart occasionally pausing for up to several seconds; more rarely, localized contractions or twitches occurred. There was no obvious correlation between larval behavior or movement and heart rate. Ryr 16/CyO and wild-type heart rates and behavior were indistinguishable. In Ryr 16 larvae, the heart rate was reduced by ~75% relative to the heterozygotes. The decrease in heart rate was caused by loss of the fast and fibrillating contractions but not an increase in pausing (Sullivan, 2000).

The effect of ryanodine on the dorsal vessel was examined in wild-type first and second instar larvae. Feeding larvae ~25 microM ryanodine decreased heart rates, and ~100 microM ryanodine reduced it by ~85% relative to wild type. It was not possible to completely inhibit circulatory muscle contraction with ryanodine as it was in the case of hypodermal muscles. This may reflect experimental limitations, because the larvae must be washed to observe the GFP signal. As detected by increasing BWC rates, larvae rapidly recovered when washed out of ryanodine. Alternatively, the ryanodine receptor may not be essential for ECC in circulatory muscles (Sullivan, 2000).

In previous studies (Amon, 1997), it was observed that depletion of Ca2+ stores in dissected photoreceptor cells by ryanodine inhibited subsequent light responses. This effect could be rescued by the addition of Ca2+-calmodulin, which inhibits Ca2+ release by the ryanodine receptor. Based on these results, it was proposed that Ca2+ release through the RyR is required for phototransduction. However, these experiments could not distinguish a pleiotropic effect of the pharmacological depletion of internal stores from an actual role for the RyR in light transduction. To test this, Ryr16 mutant eye clones were generated in heterozygous adults by using the Flp/FRT system. Clones representing >90% of the eye were generated using eyFlp and a marker chromosome carrying the minute (M53) mutation, which slows cellular growth. The eyeless enhancer drives Flp expression specifically in the eye disc, and the resulting Ryr16 mutant cells outgrow both M53/Ryr 16 and M53/M53 cells in mosaic tissue. Ryr16 clones appeared morphologically normal; furthermore, whole-cell, voltage-clamp recordings on Ryr 16 ommatidia showed responses indistinguishable from wild type. Taken together, these results are inconsistent with the postulate that Ryr is required for phototransduction (Sullivan, 2000).

To examine the role of Ryr in embryonic development, females were generated with germ-line clones homozygous for Ryr 16 using the dominant female sterile technique. However, the progeny of these females, when crossed to Ryr 16 males, were phenotypically identical to either wild type or Ryr16. Consistent with this, RNA interference using double-stranded RNA transcribed from Ryr cDNA had no apparent effect on embryogenesis, although a majority of the injected embryos that hatched did have muscle contraction defects. The overall behavior of Ryr 16 larvae was indistinguishable from wild type: they detected and migrated to food sources; had an enhanced rate of food ingestion after starvation; had normal salt avoidance and response to mechanical stimulation; and showed no spontaneous avoidance behavior. BrdUrd labeling of Ryr16 larval brains showed that cell cycle progression is normal in these mutants. Thus, at present these results provide no evidence that Ryr has any nonmuscle roles in Drosophila development (Sullivan, 2000).


Search PubMed for articles about Drosophila Ryanodine receptor

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Cassar, M., Issa, A. R., Riemensperger, T., Petitgas, C., Rival, T., Coulom, H., Iche-Torres, M., Han, K. A. and Birman, S. (2014). A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25158689

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