Here the effects of cAMP on inositol 1,4,5-trisphosphate receptors (IP3Rs) are briefly reviewed. All three subtypes of IP3R are phosphorylated by cAMP-dependent protein kinase (PKA). This potentiates IP3-evoked Ca2+ release through IP3R1 and IP3R2, but probably has little effect on IP3R3. In addition, cAMP can directly sensitize all three IP3R subtypes to IP3. The high concentrations of cAMP required for this PKA-independent modulation of IP3Rs is delivered to them within signalling junctions that include type 6 adenylyl cyclase and IP3R2.Ca The three large, closely related subunits assemble into homo- and hetero-tetrameric structures, which form large-conductance Ca2+-permeable channels within intracellular membranes, primarily those of the endoplasmic reticulum 3 to all four IP3R subunits 3R 2+, which then triggers opening of the pore. Hence, the IP3R is itself a coincidence detector, responding only when provided with both cytosolic IP3 and Ca2+. High-resolution structures of the N-terminal region of an IP3R with and without IP3 bound 3R in a closed state 3R machinery. However, the mechanisms linking IP3 binding to channel gating are not yet fully resolved. While IP3 and Ca2+ are the essential regulators of IP3R gating, many additional signals modulate IP3R behaviour 3Rs to integrate information provided by different signalling pathways.A ubiquitous pathway from extracellular stimuli to cytosolic Ca23-evoked Ca2+ signalling. PKA, for example, stimulates Ca2+ uptake into the sarcoplasmic reticulum, and EPACs through the small G protein rap2B stimulate PLCε 3Rs. The three IP3R subtypes are closely related, but each has a distinctive distribution of PKA phosphorylation sites. The many effects of cAMP within Ca2+ signalling pathways were sources of some confusion in the pioneering studies of IP3R phosphorylation 3R1 and IP3R2 enhances their activity, while the functional significance of such phosphorylation for IP3R3 is less clear Cyclic AMP-dependent protein kinase , exchange proteins activated by cAMP (EPACs), cyclic nucleotide-activated cation channels (CNGs), and some cyclic nucleotide phosphodiesterases (PDEs) are the major targets of cAMP in mammals. At least some of these targets regulate IP1589 and S1755) within the central cytosolic domain of IP3R1 are phosphorylated by PKA, and their replacement by non-phosphorylatable alanine residues confirms that they are the only sites 3R1 by PKA or introduction of phosphomimetic residues (S1589E/S1755E) do not themselves open the channel, but they increase the open probability (Po) of channels activated by IP3. The increased Po results from shortening of the gaps between bursts of channel openings and an increase in the duration of the bursts, with no obvious effect on IP3 binding or the sensitivity to Ca2+ regulation 3R1 by PKA improves the coupling of IP3 and Ca2+ binding to channel gating by both stabilizing the bursting state of the IP3R and destabilizing a prolonged closed state. An alternative splice site , which encodes 40 residues and is removed from non-neuronal IP3R1, abuts the second phosphorylation site (S1755). For the neuronal S2+ form of IP3R1, S1755 entirely mediates the effects of PKA, while in the peripheral S2− form both residues (S1589 and S1755) must be phosphorylated for PKA to enhance IP3-evoked Ca2+ release 3R1 are facilitated by tethering of PKA to IP3R1 by AKAP9 (A-kinase-anchoring protein 9) 3R1 Two residues (S1755 in IP3R1S2+) are phosphorylated by either kinase. Yet in native tissues PKG and PKA often exert opposing effects on IP3-evoked Ca2+ release. The difference may, at least in part, be due to expression of IRAG (IP3R-associated cGMP kinase substrate), which blocks phosphorylation of IP3R1 by PKA, and IRAG phosphorylated by PKG inhibits IP3R 3R1 with its endogenous antagonist, IRBIT (IP3R-binding protein released by IP3), apparently decreasing the affinity for IRBIT so that IP3 more effectively competes for occupancy of their shared binding site on the IP3R 3 synergistically stimulate release of IRBIT from IP3Rs, and IRBIT then directly stimulates two of the ion transporters that sustain fluid transport The consensus sequences for PKA and cGMP-dependent protein kinase (PKG) are similar, such that some residues  and β-adrenoceptors (which stimulate formation of cAMP) in hepatocytes suggested that cAMP might enhance receptor-mediated Ca2+ release from intracellular stores 3Rs 3-evoked Ca2+ release 3R2, the major IP3R subtype in hepatocytes, is phosphorylated by PKA at a single residue (Ser937), although others suggest that IP3R2 is a rather poor substrate for PKA 937 is unique to IP3R2, but the functional consequences of the phosphorylation appear similar to those seen with IP3R1, namely enhanced bursts of IP3R gating 3 binding affinity 3Rs into functional Ca2+ stores 3R2 in intact cells.Long before the discovery IP3R3 have been least explored. In intact cells, IP3R3 is phosphorylated by PKA at three sites  that are unique to IP3R3, with S934 being the most extensively phosphorylated 3R3, PKA has no effect on IP3-evoked Ca2+ release triggered by cell-surface receptors 3R3 behaviour remain to be determined.The effects of PKA on IP32+ concentration ([Ca2+]c). However, PTH potentiates the increase in [Ca2+]c evoked by receptors that stimulate PLC, the endogenous muscarinic M3 receptors of HEK-293 cells, for example, which can be activated by carbachol  3, are important because cAMP can, through EPACs, stimulate PLCε 3 −evoked increases in [Ca2+]c are not due inhibition of Ca2+ extrusion from the cytosol by cAMP 3-evoked Ca2+ release in permeabilized cells 3-gated channel activity in nuclear patch-clamp recordings of IP3R 3R by IP3, seem consistent with the many reports suggesting that phosphorylation of IP3R1 and IP3R2 by PKA enhances responses to IP3 (see preceding section). However, several lines of evidence demonstrate that this is not a sufficient explanation:1.2+ signals evoked by carbachol 2+ signals by β2-adrenoceptors is insensitive to inhibition of PKA in HEK-293 cells 2+ signals by PTH was unaffected by inhibition of PKA When PTH-evoked protein phosphorylation is blocked by inhibition of either PKA activity (using H89) or the association of PKA with A-kinase-anchoring proteins , there is no effect on the ability of any concentration of PTH to potentiate the Ca2.3R and a barely detectable increase in the sensitivity of the Ca2+ stores to IP3, while cAMP and 8-Br-cAMP cause substantial increases in IP3 sensitivity In permeabilized HEK-293 cells, the catalytic subunit of PKA causes minimal phosphorylation of IP3.3Rs to IP3 are much higher than those required to activate PKA The concentrations of 8-Br-cAMP (in intact cells) and of cAMP (in permeabilized cells) needed to sensitize IP4.3R subtypes, high concentrations of cAMP potentiate IP3-evoked Ca2+ release through IP3R1, IP3R2 and IP3R3 3R1 and IP3R2 In DT40 cells expressing single IP5.3R2, cAMP potentiates the Ca2+ release evoked by IP3, and the effect of cAMP is unaffected by addition of either H89 (to inhibit PKA) or the catalytic subunit of PKA.In permeabilized DT40 cells expressing IP6.3R2 expressed in DT40 cells and stimulated with IP3, cAMP increases channel activity in the absence of ATP In nuclear patch-clamp recordings from IPIn HEK-293 cells stably expressing human type 1 receptors for parathyroid hormone (PTH), PTH stimulates formation of cAMP, but it does not alone evoke an increase in cytosolic Caarbachol A. This e by cAMP B [35], [3R activity via both PKA and by mechanisms that do not require activation of either PKA or EPACs. The observations are intriguing because they suggest an effect of cAMP that is not mediated by any of its conventional targets. Our results indicate that while cAMP alone cannot activate IP3Rs and nor does cAMP affect IP3 binding to IP3Rs 3 and Ca2+, stimulate channel opening. The mechanisms underlying these non-canonical actions of cAMP are not yet resolved. In light of evidence that the effects of cAMP are preserved in isolated nuclei and permeabilized cells 3R itself or a tightly associated protein mediates this allosteric regulation of IP3Rs by cAMP.These observations suggest that cAMP can regulate IP43-evoked Ca2+ signals 3Rs require much higher concentrations of cAMP than are needed for activation of PKA or EPACs, and probably much higher than the average concentrations achieved in stimulated cells 2+ signals, the relationship between their effects on cAMP and Ca2+ are entirely different for different stimuli. For example, for concentrations of PTH and isoproterenol (which activates β-adrenoceptors) that cause similar submaximal potentiation of Ca2+ signals, PTH evokes a more than 10-fold greater increase in intracellular cAMP concentration than does isopreterenol 3R activity cannot be uniformly distributed in the cytosol. Thirdly, and more troublesome, are the many manipulations of cAMP formation that fail to affect the carbachol-evoked Ca2+ signals. Hence substantial inhibition of cAMP formation by either low-affinity inhibitors of adenylyl cyclase  and it provides a mechanism that would allow IP3Rs to be exposed to high concentrations of cAMP. It also accommodates the results showing that even manipulations of cAMP concentration fail to effect the Ca2+ signals evoked by PTH .It is easy to envisage how the effects of a maximal concentration of PTH might be unaffected by even very substantial inhibition of AC if there are ‘spare receptors’, such that maximal activation of the receptors can provide more cAMP than needed to cause maximal activation of IPed IP3Rs C. The co3R2 in HEK-293 cells, consistent with targeted delivery of cAMP from AC to IP3R. Loss of IP3R2 (using siRNA) selectively attenuates the potentiation of carbachol-evoked Ca2+ signals by PTH. Global inhibition of AC activity by low-affinity inhibitors reduces PTH-evoked cAMP formation without affecting Ca2+ signals, whereas the converse occurs when expression of AC6 is reduced. Loss of AC6 has no perceptible effect on cAMP levels, but it attenuates the potentiation of carbachol-evoked Ca2+ signals by PTH. The rationale, we suggest, is that all signalling junctions feel the effect of the low-affinity inhibitors, which thereby reduces the cAMP delivered within junctions but not sufficiently to obliterate the safety margin, whereas removing AC6 from individual junctions (each perhaps containing only a single AC) will incapacitate that junction. Finally, in cells with diminished expression of αs, which couples receptors to AC, the safety margin is reduced such that further inhibition of AC (using the low-affinity AC inhibitors) does reduce the ability of PTH to potentiate carbachol-evoked Ca2+ signals The involvement of signalling junctions is supported by additional evidence 3R gating. Phosphorylation of IP3R1 and IP3R2 by PKA increases the effectiveness with which IP3 and Ca2+ evoke bursts of channel openings. In addition, binding of cAMP to a low-affinity site that seems to be closely associated with the IP3R can also increase the apparent efficacy of IP3 and Ca2+ in gating each IP3R subtype. The need for high concentrations of cAMP for this direct action demands local delivery of cAMP to IP3Rs, and that has so far been shown to occur for only IP3R2 3Rs for cAMP effectively insulates them from global changes in cytosolic cAMP concentration and allows them to respond only to cAMP delivered to them within signalling junctions (3Rs, the junction behaves as a robust digital switch that can rapidly respond to changes in extracellular stimulus intensity. The cAMP is delivered rapidly and at a high concentration driving rapid association of cAMP with IP3Rs, and as soon as the AC stimulus is removed the focal concentration of cAMP dissipates by diffusion, rapidly terminating the effects of cAMP on IP3Rs.In conclusion, there are at least two routes through which cAMP can directly modulate IPunctions C. Becaus


Here we investigated whether Ca2+ sparks regulate SMC global [Ca2+]i and diameter in the spiral modiolar artery (SMA) by activating BK channels.In many vascular smooth muscle cells (SMCs), ryanodine receptor-mediated Ca2+-sensitive flourescent dye fluo-4 and pressurized using a concentric double-pipette system. Ca2+ signals and vascular diameter changes were recorded using a laser-scanning confocal imaging system. Effects of various pharmacological agents on Ca2+ signals and vascular diameter were analyzed.SMAs were isolated from adult female gerbils, loaded with the Ca2+ sparks and waves were observed in pressurized SMAs. Inhibition of Ca2+ sparks with ryanodine increased global Ca2+ and constricted SMA at 40 cmH2O but inhibition of Ca2+ sparks with tetracaine or inhibition of BK channels with iberiotoxin at 40 cmH2O did not produce a similar effect. The ryanodine-induced vasoconstriction observed at 40 cmH2O was abolished at 60 cmH2O, consistent with a greater Ca2+-sensitivity of constriction at 40 cmH2O than at 60 cmH2O. When the Ca2+-sensitivity of the SMA was increased by prior application of 1 nM endothelin-1, ryanodine induced a robust vasoconstriction at 60 cmH2O.Ca2+ sparks, while present, do not regulate vascular diameter in the SMA by activating BK channels and that the regulation of vascular diameter in the SMA is determined by the Ca2+-sensitivity of constriction.The results suggest that Ca Stock solutions of ryanodine , tetracaine , paxilline  and papaverine  were prepared in DMSO and stored at -20 °C and freshly diluted to target concentration in solution when required taking care that the final DMSO concentration in solution did not exceed 0.1 %. Endothelin-1  and iberiotoxin  were always freshly prepared in PSS for immediate use.HEPES-buffered PSS contained (in mM): 150 NaCl, 5 HEPES, 3.6 KCl, 1 MgCl2+ sparks and waves were recently reported in the intact unpressurized gerbil SMA [2+ sparks and Ca2+ waves in SMCs of the pressurized gerbil SMA , corresponding to ~47 % of one cell, giving a spark density of 2.8 ± 0.3 spark sites/cell. The frequency of Ca2+ spark occurrence per site and other temporal parameters were measured in pressurized SMA using line-scans  SMA, application of 10 μM ryanodine inhibited Ca2+ sparks and appeared to similarly increase the average global cytosolic Ca2+ followed by a robust vasoconstriction , which are open at the physiological resting membrane potential in SMCs. Evidence for active VDCCs in the SMA comes from the observation of a decrease in global Ca2+ upon application of a reversible VDCC inhibitor, 2 μM nifedipine, in the presence of 100 μM tetracaine . It is well-established that at a concentration of 10 μM, ryanodine binds to open ryanodine receptors and modifies the channel to lock them in an irreversible sub-conductance state of 234 pS [2+ release and instead “leaks” SR Ca2+ into the cytosol. This is reflected in the transient increase in global Ca2+ immediately upon application of 10 μM ryanodine, followed by a slowly decaying plateau phase devoid of Ca2+ oscillations, indicating the relatively slow emptying of the SR through the partially open ryanodine receptors  for 1 min. It has been previously shown that endothelin-1 acts via ETA receptors to increase the Ca2+ sensitivity of the SMA in a rho-kinase dependent manner [2+ concentration and a persistent vasoconstriction, consistent with an increase in the Ca2+ sensitivity  Inhibition of Ca2+ sparks with ryanodine increases global Ca2+ and causes a robust vasoconstriction, however, ryanodine-induced effects on global Ca2+ and vascular diameter are not reproduced by other inhibitors of Ca2+ sparks or by inhibitors of BK channels as would be expected if Ca2+ sparks activated BK channels to regulate membrane potential, global Ca2+ and vascular diameter. 3) The ryanodine-induced vasoconstrictions depends on the Ca2+ sensitivity, which is higher at 40 cmH2O compared to that at 60 cmH2O and can be enhanced with endothelin-1.Salient findings of the present study are 1) Ca2+ sparks in the pressurized SMA occurred with a lower frequency than in unpressurized SMA [2+ spark frequency in response to a 20 cmH2O (~ 14 mmHg) increase in pressure, is also consistent with observations made in cerebral arteries [2+ sparks was far shorter (~17–19 ms) than that observed for Ca2+ sparks in cerebral arteries, but closer to that found in rat heart [2+ spark amplitudes and decay times are generally a reflection of the number as well as the isoform of ryanodine receptors (RyRs) present in a spark cluster. Typically, Ca2+ spark sites are composed of 4 – 6 RyRs, giving a punctate staining pattern in immunolocalization studies. In the SMA, the distribution pattern of RyRs in SMCs shows a uniform expression throughout the SR rather than a punctate expression expected of ryanodine receptors clustered in spark sites [2+ sparks in the SMA.Caized SMA , but witized SMA , 19, 28 ized SMA , 29. Theat heart , 30. Ca22+ spark/BK channel signaling complex provides an important vasodilatory mechanism in preventing or mitigating pressure- or agonist-induced vasoconstrictions in arteries. This negative feedback mechanism in regulating vascular tone is evident from observations that pharmacological inhibition of Ca2+ sparks and/or BK channels or SMC-specific genetic manipulation of BK channel or RyR expression leads to the loss of this hyperpolarizing signal leading to membrane depolarization, increased VDCC activation, increase in Ca2+ influx and global Ca2+ and enhanced vasoconstriction [2+ sparks have not always been linked to a hyperpolarizing or vasodilatory mechanism. Other studies have observed excitatory roles for Ca2+ sparks and RyR-mediated Ca2+ release in small diameter arterioles. Kur et al. [2+ sparks in retinal arterioles combined to form Ca2+ waves and enhanced the myogenic tone. Westcott et al. [2+ sparks and no coupling with BK channels, while SMCs of upstream feed arteries exhibited clustered staining of RyRs, robust Ca2+ sparks and spatial and functional coupling to BK channels, indicating heterogeneity of RyR function within the same vascular tree. In the SMA, the effects of ryanodine on global Ca2+ and vascular diameter at 40 cmH2O seemed to suggest, at first, a vasodilatory mechanism for Ca2+ sparks acting via BK channels. However, the failure of tetracaine, an RyR inhibitor, which inhibits Ca2+ sparks without depleting the SR, and iberiotoxin, a BK channel inhibitor, to produce similar effects on global Ca2+ and diameter as ryanodine  that are relevant for human auditory perception, compared to the much higher thresholds in mice and rats for the same frequency range . Thus, t


We find that Drosophila astrocytes exhibit robust Ca2+ oscillatory activity manifested by fast, recurrent microdomain Ca2+ fluctuations within processes that infiltrate the synaptic neuropil. Unlike the enhanced neuronal activity and behavioral seizures that were previously observed during manipulations that trigger Ca2+ influx into Drosophila cortex glia, we find that acute induction of astrocyte Ca2+ influx leads to a rapid onset of behavioral paralysis and a suppression of neuronal activity. We observe that Ca2+ influx triggers rapid endocytosis of the GABA transporter (GAT) from astrocyte plasma membranes, suggesting that increased synaptic GABA levels contribute to the neuronal silencing and paralysis. We identify Rab11 as a novel regulator of GAT trafficking that is required for this form of activity regulation. Suppression of Rab11 function strongly offsets the reduction of neuronal activity caused by acute astrocyte Ca2+ influx, likely by inhibiting GAT endocytosis. Our data provide new insights into astrocyte Ca2+ signaling and indicate that distinct glial subtypes in the Drosophila brain can mediate opposing effects on neuronal excitability.Maintenance of neural circuit activity requires appropriate regulation of excitatory and inhibitory synaptic transmission. Recently, glia have emerged as key partners in the modulation of neuronal excitability; however, the mechanisms by which glia regulate neuronal signaling are still being elucidated. Here, we describe an analysis of how Ca Drosophila astrocytes exhibit endogenous spontaneous Ca2+ oscillatory activity. Acute elevation of astrocyte Ca2+ suppresses neuronal activity and causes rapid paralysis. This is accompanied by a rapid reduction in the levels of membrane GABA transporter (GAT), a transporter for the inhibitory neurotransmitter GABA. We identify Rab11 as a novel regulator of GAT trafficking that is required for this form of Ca2+-dependent astrocyte regulation of neuronal activity. Our study provides new insight into the acute regulation of neuronal activity by glia.Complex brain functions require precise control of neuronal activity, which is often disrupted in neurologic disorders such as epilepsy. Here, we show that 2+ dynamics , eliminate microdomain Ca2+ oscillations in cortex glia and lead to higher intracellular Ca2+ levels. This increase in cortex glial Ca2+ leads to enhanced seizure susceptibility, as does acute induction of Ca2+ influx through ectopic expression of TRP channels in cortex glia . The following stocks were used: UAS-TrpA1 (BDSC 26263), Alrm-Gal4  by Bestgene.To construct the plasmid pBID-UASc-myrGCaMP6s, GCaMP6s cDNA (Addgene #40753) was PCR amplified by designing primers with restriction enzyme cutting sites added on each end. Plasmid pBID-UASc-myrGCaMP5G  was dige2 anesthetization. These flies were then allowed to recover at room temperature in a closed Petri dish for 1 h before being transferred to Petri dishes preheated to the indicated temperature.For testing the temperature sensitive behavioral phenotype of adult flies, 2-d-old flies were used. A total of 10-12 flies were transferred to each vial and placed in a water bath held at the indicated temperature. The number of flies with motor impairment was observed and manually recorded for up to 5 min, with an interval of 15 s in the first minute and 30 s in the following 4 min. Paralysis of adult flies was defined by complete loss of movement. For RNAi screening conducted at 30°C, Alrm > TrpA1 flies do not show 100% penetration of the paralysis phenotype. In this case, the number of flies that showed severe motor impairment and were unable to climb up the vial wall were recorded at each time point. For testing the behavior of headless flies, heads of adult flies were severed by fine scissors under CO3, 4 mM MgCl2, 5 mM trehalose, 115 mM sucrose, and 5 mM HEPES; pH 7.2; For immunofluorescence, 3rd instar stage larvae were dissected in HL3.1  and transferred to nitrocellulose membrane. Membranes were blocked with blocking buffer  and then incubated with appropriate antibodies for immunoblotting. Primary antibody incubation were performed with gentle shaking at 4°C overnight followed by four 5-min washes using TBS containing 0.01% Tween 20, secondary antibody incubation for 1 h at room temperature, and four more 5-min washes using TBS containing 0.01% Tween 20. Then the membrane was briefly rinsed with TBS, and the blot was visualized using an Odyssey infrared scanner (Li-Cor). Antibodies for Western blotting: rabbit anti-GAT, 1:5000 . Alexa Fluor 680-conjugated anti-rabbit and Alexa Fluor 800-conjugated anti-mouse, 1:10,000 (Invitrogen). All antibodies were diluted using blocking buffer.Adult flies were snap frozen in liquid nitrogen and five heads per sample were collected and homogenized in 50 µl of 1× Laemmli loading buffer , followed by centrifugation at 16,000 × 2+. Saline was heated using an in-line solution heater (Harvard Apparatus), controlled by a Dual Channel Bipolar Temperature Controller (CL-200A). Additionally, the recording dish was temperature controlled using a Peltier device regulated by a fan-based heat sink and a Dual Channel Temperature Controller .Extracellular recordings were obtained from dissected 3rd instar larvae using a fire-polished patch electrode. To record central pattern generator (CPG) activity, the larval brain was left intact and care was taken during dissection to avoid damaging segmental nerves. Recordings were amplified using an AxoClamp 2B amplifier and digitized via an Molecular Devices Digidata 1550 (Molecular Devices). Clampfit 6.1 software was used to record and process electrophysiological data. Larvae were constantly perfused via a gravity system with HL3.1 solution containing 1.5 mM CaUAS-myrGCaMP6s was expressed in astrocytes using the Alrm-Gal4 driver. L2 stage larvae were mounted on their ventral side in a small drop of water on a 25 × 60 mm coverslip. A single layer of scotch tape was applied on each side of the larva as a spacer to avoid damaging the animal when an 18 × 18 mm coverslip was placed on top. An upright PerkinElmer Ultraview Vox spinning disk confocal microscope equipped with a high-speed EM CCD camera, and a 43× 1.3 NA oil objective was used for image acquisition. Velocity software was used for data analysis. When imaging, the assembly was flipped so that the ventral side of the larvae faced the objective. Time series were acquired at a speed of ∼9 Hz using a single optical plane in the neuropil layer of the larval ventral nerve cord (VNC).p values are used to refer to the corresponding comparisons described in the table.Statistical analysis was performed using GraphPad Prism 6. Data are presented as mean ± SEM in all figures. Statistical analysis is summarized in 2+ indicator (GECI) in cortex glia, a type of glia that associate with neuronal cell bodies in the Drosophila CNS, has revealed that these cells display robust near-membrane microdomain oscillatory activities .Expression of a genetically encoded membrane targeted myristoylated Cativities . To detetex glia . We alsoMovie 1.Drosophila astrocyte near-membrane Ca2+ activity detected with myrGCaMP6s. Ca2+ transients were recorded from the VNC of undissected 2nd instar larvae. Video speed, 2× real time. Scale bar, 20 µm.10.1523/ENEURO.0340-16.2017.video.12+ transients had an average duration of 3.69 ± 0.24 s, a half decay time of 1.00 ± 0.06 s, and a mean ΔF/Favg of 34.30 ± 1.70% . These events were similar to Ca2+ microdomains previously described in cortex glia, which had an average duration of 1.35 s, and a mean ΔF/Favg of 35% , led to elevated intracellular Ca2+ in cortex glia and enhanced seizure susceptibility, suggesting a role for glial Ca2+ signaling in regulating neuroexcitability  of 3rd instar stage larvae. Both control and Alrm > TrpA1 animals demonstrated normal spaced bursting activity that underlies larval locomotion at room temperature C. HoweveDrosophila, astrocytes extend fine processes and associate with synapses in the neuropil of both the central brain and the ventral ganglion . Alternatively, the observed paralysis could be the consequence of direct interactions between astrocytes and local neurons in the ventral ganglion. To determine whether central brain function is necessary for conveying the astrocyte Ca2+ influx-induced paralysis, we severed the heads of adult flies and preformed behavioral tests on headless flies. Headless flies expressing TrpA1 in astrocytes rapidly became paralyzed when the temperature was shifted to 33°C  involved in vesicle transport, (2) enriched in astrocytes, or (3) involved in regulation of Ca2+ signaling. Using this collection, we identified 13 RNAi lines that either enhanced or suppressed the motor impairments of Alrm > TrpA1 flies . We verified the RNAi knockdown result using a dominant-negative mutant form of Rab11, Rab11N124I . Rab11 is a member of the Rab family of small-GTPases, and it is crucial for recycling endosome function and post-Golgi transport .Among the RNAi lines that suppressed the paralysis phenotype of Alrm > TrpA1 flies, Rab11-knockdown displayed the strongest effect . These data suggest that Rab11 is a novel regulator of GAT trafficking and likely ameliorates the acute astrocyte Ca2+ influx-induced paralysis of Alrm > TrpA1 flies by indirectly regulating GABA uptake.One of the major functions of astrocytes is to uptake neurotransmitters following synaptic activity . Drosophter GABA . It has ter GABA . If GAT membrane E. Overexlization E. We alsn = 8-10 single optical sections for each group, pf = 0.4974). However, after subjecting animals to 33°C, Alrm > TrpA1 larvae showed a severe reduction of surface GAT expression compared with controls . Large, GAT-containing membranous structures were frequently observed in the cytoplasm following acute Ca2+ influx in astrocytes, indicating Ca2+ elevation may drive GAT endocytosis , it will be interesting to explore the role of chronic increases in glial Ca2+ signals in the pathogenesis of neurologic diseases using the Drosophila model.Perturbations in glial Caisorders . For exafunction . AlthougDrosophila CNS, astrocytes invade the entire neuropil with their meshwork of fine processes, and each astrocyte establishes unique and stereotypical territory (Drosophila is still largely unexplored. Here, as a first step, we show that astrocytes in the ventral ganglion are sufficient to convey the suppression of neuronal activity caused by acute Ca2+ influx.In the erritory . Althougerritory , the funDrosophila astrocytes are developmentally regulated and sensitive to GABAergic neuronal output during synaptogenesis (B receptor signaling reduces GAT levels and suppresses seizure induction in hyperexcitable Drosophila seizure mutants, likely through dampening the rate of GABA uptake (Modulation of GABA uptake is an important glial mechanism to actively participate in the regulation of neuronal activity. It has been previously shown that global GAT levels in ogenesis . SuppresA uptake .2+ influx induces a rapid decrease in membrane GAT levels via endocytosis, which correlates with acute suppression of neural activity, representing a mechanism for glial cells to actively modulate neuronal function.Our study suggests the existence of a new mechanism for astrocytes to acutely regulate neuronal activity through fast modulation of GAT turnover. Compared with other known membrane proteins, GAT has one of the highest turnover rates . Plasma Drosophila astrocytes. The accumulation of GAT-containing vesicles in rab11-knowdown animals likely represents a strong suppression of post-Golgi transport of GAT to the plasma membrane. However, it is important to note that suppression of Rab11 function did not totally abolish GAT localization to astrocyte membranes (rab11-knowdown flies do not have strong locomotion defects like GAT-deficient animals (rab11-knockdown did not alter total GAT levels (Drosophila eye, suppression of Rab11 function decreases the rate of endocytosis and eliminates the formation of endocytosis-dependent multivesicular bodies (Drosophila Garland cells (2+ influx, leading to sustained GABA uptake and a suppression of the paralysis and loss of neural activity normally observed following excessive astrocyte Ca2+ influx (Our data reveal Rab11 as a novel regulator of GAT trafficking in embranes D. Consis animals . Indeed, animals . Since rT levels E,F, consr bodies . Ectopicnd cells . Consist+ influx C.2+ signaling in regulating neuronal activity. Acute modulation of the rate of neurotransmitter uptake through Ca2+-dependent glial signaling provides a robust mechanism for astrocytes to actively modulate the function of neural circuits.Taken together, our study reveals an important function of glial CaMovie 4.2+ influx in adult ventral ganglion astrocytes causes paralysis. Headless adult Alrm > TrpA1 flies show paralysis when transfer to a preheated Petri dish held at 33°C. The video started 30 s after the flies were transferred to the Petri dish. Video speed is real time.Ca10.1523/ENEURO.0340-16.2017.video.4


They are gated by IP3, produced upon external stimulation and activation of G protein-coupled receptors on the plasma membrane (PM). IP3-mediated Ca2+ release, and the resulting depletion of the ER store, triggers entry of extracellular Ca2+ by store-operated Ca2+ entry (SOCE). Mutations in IP3R attenuate SOCE. Compromised IP3R function and SOCE during pupal development of Drosophila leads to flight deficits and mimics suppression of neuronal activity during pupal or adult development. To understand the effect of compromised IP3R function on pupal neuronal calcium signaling, we examined the effects of mutations in the IP3R gene (itpr) on Ca2+ signals in cultured neurons derived from Drosophila pupae. We observed increased spontaneous Ca2+ influx across the PM of isolated pupal neurons with mutant IP3R and also a loss of SOCE. Both spontaneous Ca2+ influx and reduced SOCE were reversed by over-expression of dOrai and dSTIM, which encode the SOCE Ca2+ channel and the ER Ca2+-sensor that regulates it, respectively. Expression of voltage-gated Ca2+ channels  was significantly reduced in itpr mutant neurons. However, expression of trp mRNAs and transient receptor potential (TRP) protein were increased, suggesting that TRP channels might contribute to the increased spontaneous Ca2+ influx in neurons with mutant IP3R. Thus, IP3R/SOCE modulates spontaneous Ca2+ influx and expression of PM Ca2+ channels in Drosophila pupal neurons. Spontaneous Ca2+ influx compensates for the loss of SOCE in Drosophilaitpr mutant neurons.Inositol 1,4,5-trisphosphate receptors (IP IP3Rs are gated by the second messenger IP3, which is produced upon external stimulation and activation of G protein-coupled receptors (GPCRs) on the plasma membrane (PM). IP3-mediated Ca2+ release, and the resulting depletion of the ER store, triggers entry of extracellular Ca2+ by store-operated Ca2+ entry (SOCE). Drosophila mutants for the IP3R are flightless  and of genes encoding other calcium signaling molecules, such as GPCRs and the SOCE molecules, dSTIM and dOrai also result in flight deficits  and can be decoded by the frequency and amplitude of spontaneous Ca2+ transients  in Drosophila pupal neurons are also mediated through VGCCs  screen; detailed molecular information on these alleles has been published  was obtained from Bloomington Stock Center, Indiana University, Bloomington, IN, USA. Canton S (CS), in the background of which all mutant and transgenics were back-crossed, was used as the wild-type control. RNAi lines for itpr (1063) was obtained from National Institute of Genetics, Japan, and for dSTIM (47073), cac (104186), Ca-β (102188), Ca-α1D (51491) and Ca-α1T  were obtained from Vienna Drosophila Resource Centre, Austria. Fly strains used in this study were generated by standard genetic methods using individual mutant and transgenic fly lines described above.Single point mutants in the 2PO4/0.17 mM NaH2PO4/43.8 mM sucrose/33.3 mM glucose/9.9 mM HEPES, pH 7.3 with NaOH). The brain tissue was dissociated with gentle pipetting. The lysate containing a mixture of tissue clumps and single cells was spun down, re-suspended and plated onto poly lysine-coated glass coverslip mounted to the bottom of a petri dish. Cells were resuspended and cultured in DMEM/F12-1065 , containing Glutamax-I, 2.438 sodium bicarbonate and sodium pyruvate, supplemented with 50 U/ml penicillin , 50 μg/ml streptomycin , and 10 μg/ml amphotericin B , 1 mg/ml sodium bicarbonate, 20 mM HEPES, 100 μM putrescine, 20 ng/ml progesterone, 50 μg/ml insulin, 1 μg/ml 20-hydroxyecdysone. The cells were incubated at 25°C in a humidified incubator with 5% CO2 for 14–16 h. All chemicals for cell culture were obtained from Sigma-Aldrich  unless otherwise stated.The protocol for pupal neuronal culture was adapted from Sicaeros and O’Dowd . The bra2/35 mM sucrose, pH 7.2 with NaOH) with (1 mM CaCl2) or without (2 mM EGTA) Ca2+. Data were acquired using 488 nm excitation and 520 nm emission filter sets at 15 s intervals. An Olympus IX81-ZDC2 inverted wide field microscope with epifluorescence and Z-drift compensation and a 60×/1.35 NA (oil) objective lens, was used for calcium imaging. Excitation of fluorescent Ca2+ indicator dyes was performed using specific wavelength illuminations from a halogen arc lamp with TILL Polychrome 5000 monochromator  for variable bandwidth and intensity. Emitted light was detected through band pass filter sets . Image acquisition was performed using the Andor iXON 897E EMCCD camera and Andor iQ 2.4.2 imaging software. The time lapse acquisition mode of the software was used to follow fluorescence changes over time.For measurement of spontaneous calcium influx and SOCE cells were incubated with 2.5 μM Fluo-4 AM and 0.02% Pluronic F-127 in M1 media for 30 min at room temperature in the dark. Cells were washed twice with M1 before and after dye incubation and finally covered with M1 . To compare data between genotypes or assay conditions Kruskal-Wallis test for variance followed by Wilcoxon post hoc test was performed.Images acquired through Andor iQ 2.4.2 on Olympus IX81-ZDC2 were analyzed with ImageJ 1.43m . Each cell was marked separately and mean fluorescence intensity was calculated in arbitrary units. Arbitrary units of fluorescence for each cell were converted to ΔF/FDrosophila pupae with TRIzol reagent  following manufacturer’s instructions. RNA was dissolved in nuclease free water and quantified using a nanodrop machine  and the integrity was checked on a 1.5% TAE gel. Approximately 200 ng RNA was used for cDNA preparation by Reverse Transcriptase as described in Pathak et al.  for SYBR assay I dTTP . For each genotype, three biological replicates and two technical replicates were included in the experiment. rp49 was used as internal control. A melt curve was performed after the assay to check for specificity of the reaction. The fold change of gene expression in test genotype relative to control was determined by the comparative DDCt, where DDCt = (Cttarget − Ctp49)test − (Cttarget − Ctrp49)control.Total RNA was isolated from 5 to 10 dissected CNS from suitably aged k et al. . QuantitPupal CNS lysates were extracted in 50 mM Tris (pH 8), 150 mM NaCl, 1 mM EGTA and 1% (v/v) Triton X-100 with 1 mM PMSF, 0.5 μM E64 Calpain inhibitor and 100 μg/ml leupeptin. Protein extract was boiled for 5 min at 95°C in 125 mM Tris pH 6.8, 5% (v/v) Glycerol, 0.25% (w/v) SDS, 2% (v/v) β-mercaptoethanol, 10% (w/v) Bromophenol blue and resolved in 8% SDS polyacrylamide gel electrophoresis. Proteins were detected with 1:5000 anti-TRP .Flies were anesthetized in ice before tethering to a tungsten wire between the head and thorax and allowed to recover for 30 min before recording flight. Flight was stimulated with a puff of air and recorded for 30 s.3-mediated Ca2+ signaling in spontaneous Ca2+ influx and SOCE in Drosophila pupal neurons, primary neurons from the central nervous systems of 48–50 h pupae were cultured for 16–18 h and Ca2+ signals of single neurons were recorded. In presence of 2 mM extracellular CaCl2 wild type and itpr mutant neurons displayed spontaneous Ca2+ signals in culture  as compared with wild type  and after application of either 50 μM lanthanum chloride  displayed reduced SOCE after passive depletion of intracellular store-Ca2+ with 10 μM thapsigargin  followed by measurement of SOCE by replenishing 2 mM extracellular CaCl2. Because itpr mutant pupal neurons displayed higher spontaneous Ca2+ influx as compared to wild type in presence of extracellular Ca2+, SOCE was determined by subtracting spontaneous Ca2+ influx from Ca2+ influx induced by store-Ca2+ depletion with thapsigargin  reduced in itpr mutant pupal neurons  reduced SOCE in pupal neurons , TRP channels  and NCX  in extracts derived from 48 h to 50 h pupal central nervous systems. The choice of candidates investigated was based on existing literature  have been attributed to PLTX-II sensitive P/Q-type VGCC, cac  expression is indirectly modulated by SOCE, through ERK1/2, in neuronal PC12  in mRNA levels of trp was observed in itpr mutant neurons, whereas expression levels of trpl and calx remained unchanged  down regulation of cac and Slowpoke was observed in dSTIM knocked down neurons whereas expression levels of Shaker, Ca-β, Ca-α1D and trp, remained unchanged. These data suggest that the expression of cac and slo are sensitive to SOCE. The reduced expression of Slowpoke possibly reports reduced activity of the P/Q-type VGCC encoded by cac, as reported earlier for cac mutant neurons  and Ca-α1D (51491) resulted in 100% lethality in late stage 3rd instar larvae and thus these animals could not be tested for flight. Pan-neuronal knock-down of Ca-β (102188) and Ca-α1T  did not affect either viability or flight. We also tested flight after knock-down of these VGCC subunits in specific neuronal subsets. The RNAi lines used were based on their efficacy as described in published literature  was significantly higher, whereas SOCE was lower as compared to wild-type dopaminergic neurons  didn’t result in flight deficits  in dopaminergic neurons reduced SOCE  does not induce higher spontaneous influx. Compromised IP3R function and SOCE in Drosophila neurons leads to defects in flight motor coordination ; a deletion mutation of ITPR1 in human causes SCA15/16  to GH. SC was supported by senior research fellowship from Council of Scientific and Industrial Research (CSIR), India and a bridging postdoctoral fellowship from NCBS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


What, When, and Whether model (www-model) which postulates that the content, the timing and the possibility of generating an action can be partially independent both at the cognitive level and at the level of their neural implementation. The original proposal was based on a limited number of studies, which were reviewed with a discursive approach. To assess whether the model stands in front of the more recently published data, we performed a systematic review of the literature with a meta-analytic method based on a hierarchical clustering (HC) algorithm. We identified 15 PET/fMRI studies well-suited for this quest. HC revealed the existence of a rostro-caudal gradient within the medial prefrontal cortex, with the more anterior regions (the anterior cingulum) involved in more abstract decisions of whether to execute an action and the more posterior ones (the middle cingulum or the SMA) recruited in specifying the content and the timing components of actions. However, in contrast with the original www-model, this dissociation involves also brain regions well outside the median wall of the frontal lobe, in a component specific manner: the supramarginal gyrus for the what component, the pallidum and the thalamus for the when component, the putamen and the insula for the whether component. We then calculated co-activation maps on the three component-specific www clusters of the medial wall of the frontal/limbic lobe: to this end, we used the activation likelihood approach that we applied on the imaging studies on action contained in the BrainMap.org database. This analysis confirmed the main findings of the HC analyses. However, the BrainMap.org data analyses also showed that the aforementioned segregations are generated by paradigms in which subjects act in response to conditional stimuli rather than while driven by their own intentions. We conclude that the available data confirm that the neural underpinnings of intentionality can be fractionated in discrete components that are partially independent. We also suggest that intentionality manifests itself in discrete components through the boosting of general purpose action-related regions specialized for different aspects of action selection and inhibition.In their attempt to define discrete subcomponents of intentionality, Brass and Haggard  proposed Motor control has been the object of interest of many disciplines, including psychology, cognitive neuroscience, and, since the earliest days, philosophy, particularly when the object of enquiry are the conscious aspects of motor control and intentionality , the timing (when component) and the possibility of being executed or inhibited (whether component). However, in reviewing the then available neuroimaging literature, they concluded that there was no sufficient evidence that such components are represented in discrete brain circuits. In their unitary brain model of intentionality, they proposed a “system” for intentional actions located in the pre-frontal cortex, anterior cingulate cortex, and supplementary motor area (SMA), with subcortical inputs coming from the striatum and through the thalamus: in their views, these systems are responsible for the all three features of intentional action www-model was based on a set of experiments that we review here briefly together with more recent observations.Ten years later, Brass and Haggard  re-assesWhat component has been mostly investigated by using fMRI procedures similar to the “Free selection paradigm”  to identify component specific clusters. As the reader will see, the specific literature available is barely sufficient to make statistical inferences on the significance of the clusters identified. However, after the initial hierarchical clustering procedure, we interrogated the vast BrainMap.org database and generated co-activation maps based on the main component-specific medial wall clusters of the frontal lobe. This permitted the desired statistical assessment of the anatomical dissociations initially identified by hierarchical clustering.These were all lingering questions on the Our meta-analysis was based on neuroimaging articles investigating the neurofunctional correlates of intentional action using PET or fMRI in adult subjects.http://www.ncbi.nlm.nih.gov/pubmed/). The search keys were: “Intentional action & fMRI” and “Intentional action & PET.” These queries returned 27 neuroimaging articles investigating the neurofunctional correlates of intentional actions. We included only studies that did satisfy the following inclusion criteria: (1) sample population composed of normal adult subjects; (2) imaging technique: PET or fMRI; (3) data reported using stereotactic coordinates; (4) comparison between intentional actions and stimulus-driven actions. As a consequence of these inclusion criteria, 12 studies were excluded from the analysis. Among the 15 studies that satisfied our inclusion criteria, 6 studies investigated the What component, 3 studies the When component, and 4 studies the Whether component. Finally, 2 studies investigated both the What and the When components. These studies were then classified on the basis of the examined component (see Supplementary Table Candidate studies were selected through the PubMed database (riteria:  sample psimple effects and (ii) interaction effects. The simple effects were: “Intention driven trials” and “Stimulus driven trials.” The interaction effectsFor the suitable studies, in the meta-analysis we used data derived from (i) within group 71 peaks associated with the What component, 42 peaks with the When component and 37 peaks with the Whether component of intentional actions.To summarize, the data selection led us to analyse 150 stereotactic activation loci, For each activation peak, we recorded all relevant information about the statistical comparison that generated it, the nature of the experimental task and the investigated component.– t-values or z-scores;– Sample size;– Average age of the subjects;– Stereotactic template (MNI or Tailarach and Tournoux template);– Whole brain or region-of-interest analysis;– Scanning Technique (PET or fMRI);– Statistical thresholds and nature of the correction for multiple comparisons.We therefore determined a list of classification criteria to characterize each peak of activation included in the dataset:www.brainmap.org).In order to combine the data coming from studies based on different stereotactic spaces, the stereotactic coordinates of studies in which activation peaks were reported in terms of the Talairach and Tournoux  atlas weWe first performed a component specific hierarchical clustering analyses (HC) of the activation peaks: the analysis allowed us to extract the principal clusters of regional effects from the database , and whose root represents one large cluster including all the activation peaks submitted to the algorithm. Each level of the tree reports the clusters created by the algorithm at a specific processing step, as it progresses from individual activation peaks at the lowest level to the all-inclusive final cluster at the top of the tree.” The procedure was continued until the average standard deviation around the cluster centroids of the individual peaks, in the x, y, and z directions, remained below 5.0 mm. This measure roughly mimics the spatial resolution of fMRI studies. As hierarchical clustering may be sensitive to the order in which the individual data are processed, and may generate alternative clustering trees when integers are used  on the one hand we wanted to test the hypothesis that co-activation maps generated starting from the three main clusters of the medial wall of the frontal lobe and of the cingulate cortex can produce similarly segregated results as in the specific hierarchical clustering, once the three maps were formally compared (2) further, we wanted to test the hypothesis on whether the brain regions outside the frontal lobe would co-vary with the original seeds in a similar manner (3) by analyzing the composition of the BrainMap.org data-base experiments, that contributed to the identification of medial-wall clusters, we wanted to learn to what extent these could be associated, even if loosely, to intentional action and its subcomponents at the center of our quest.As an initial step, three separate co-activation maps were calculated for each of the three seed clusters identified by the HC procedure. Subsequently, each co-activation map was compared with the other two lumped together.Furthermore, once a component specific cluster was identified in the BrainMap.org database, we explored its composition as far as the generative paradigms was concerned. We paid particular attention to the relative contribution of go/no-go paradigms as these are closer to aspects of motor control entailed by the intentional/conditional dichotomy, particularly for the whether component.what component the data clustered in the right middle cingulum, the right frontal middle gyrus, the right supramarginal gyrus and the left inferior triangular frontal gyrus .Figure what seed alone) most of the regions implicated by the HC analysis were also identified by the co-activation maps analysis  was used.Each individual map was then compared with a combination of the other two maps: for example, the These analyses led to identification of regions that genuinely dissociate on the basis of their co-activation with the seeds used.what, when, and whether components. Together with these brain regions, there were co-segregations of component-specific regions, particularly for the lenticular nuclei for the when component , suggesting the existence of a rostro-caudal gradient within the medial prefrontal cortex, with the more anterior regions involved in more abstract decisions of whether to execute an action and the more posterior ones recruited in specifying the content and, yet more dorsally, the timing components of actions.what component) the data clustered at the level of the right middle cingulum; the middle cingulum has been previously associated to conflict processing and conflict monitoring , as strange as it might seemphrenologically in terms of minute segregated regions exclusively involved in intentionality; on the other hand, the same observation suggests that the what, when, and whether segregation for action most likely exists beyond the concept of intentionality.In returning to the crucial matter of our contention here, the fact that the medial wall seeds, on which the co-activation maps analyses were based, segregate in an identical manner to what is revealed by the hierarchical clustering performed on “intentional” experiments, suggests that the specificity of intentionality and its subcomponents cannot be sought www action regions at some microscopical level, perhaps thanks to the boosting effect of some ascending modulatory attentional pathways for tasks in which subjects have to decide how and whether to act by themselves, rather than in reaction to a conditional stimulus. Another possibility is that the relative weights of the connections between the regions involved in the different aspects of intentionality change during intentional action, something that needs to be explored with effective connectivity techniques and that goes beyond the potentials of meta-analyses based on data generated by univariate analyses.However, it cannot be denied that the presence of an intentional stance during the paradigms meta-analyzed here induced stronger activity in these regions in a component specific manner. This suggests, on the one hand, that intentionality is expressed in these www-model of intentionality, in order to overcome the methodological limitations of previous attempts. In particular, we are much in need of novel fMRI experiments in which the sub-components of intentionality, postulated by the above mentioned neurocognitive models, are assessed with an uniform procedure in the same group of subjects. The advantages of a uniform procedure are obvious: significant differences between the intentional tasks in the different conditions should not be confounded by factors  that may hamper the possibility of firm assignations of specific functional anatomical patterns to subcomponents of intentionality. Another unexplored issue is the characterization of intentional actions in conditions in which content, timing and the very decision on whether to act or not are explicitly and jointly manipulated. Furthermore, it remains to be discovered how general-purpose action brain regions become more active when intentionality is operating. This will clearly require analytical approaches that go beyond univariate analyses of the fMRI data. Clearly, one such ambitious model should have some predictive value for pathologies in which a disorder of intentionality and its specific components is expected. Brass and Haggard (when component in Parkinson's disease). These specific associations also remain to be demonstrated convincingly. However, in spite of all these unsolved issues, we believe that the available literature contains sufficient evidence to think that the www-model of intentionality might be a useful starting framework for future investigations.After this meta-analytic review, it is clear that further studies are needed to assess the functional anatomical foundations of the  Haggard  speculatLZ, SS, and EP reviewed the data for the meta-analyses, performed the analyses, and drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, the role of TRPC6 was investigated following ischemia/reperfusion (I/R) and oxygen-glucose deprivation (OGD). We found that TRPC6 expression was increased in wild-type (WT) mice cortical neurons following I/R and in primary neurons with OGD, and that deletion of TRPC6 reduced the I/R-induced brain infarct in mice and the OGD- /neurotoxin-induced neuronal death. Using live-cell imaging to examine intracellular Ca2+ levels ([Ca2+]i), we found that OGD induced a significant higher increase in glutamate-evoked Ca2+ influx compared to untreated control and such an increase was reduced by TRPC6 deletion. Enhancement of TRPC6 expression using AdCMV-TRPC6-GFP infection in WT neurons increased [Ca2+]i in response to glutamate application compared to AdCMV-GFP control. Inhibition of N-methyl-d-aspartic acid receptor (NMDAR) with MK801 decreased TRPC6-dependent increase of [Ca2+]i in TRPC6 infected cells, indicating that such a Ca2+ influx was NMDAR dependent. Furthermore, TRPC6-dependent Ca2+ influx was blunted by blockade of Na+ entry in TRPC6 infected cells. Finally, OGD-enhanced Ca2+ influx was reduced, but not completely blocked, in the presence of voltage-dependent Na+ channel blocker tetrodotoxin (TTX) and dl-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) blocker CNQX. Altogether, we concluded that I/R-induced brain damage was, in part, due to upregulation of TRPC6 in cortical neurons. We postulate that overexpression of TRPC6 following I/R may induce neuronal death partially through TRPC6-dependent Na+ entry which activated NMDAR, thus leading to a damaging Ca2+ overload. These findings may provide a potential target for future intervention in stroke-induced brain damage.Transient receptor potential canonical 6 (TRPC6) channels are permeable to Na TRPM7 is a well-studied protein among this subfamily which has been shown to be linked with ischemia injured neurons  subfamily is also permeable to Na+ and Ca2+, we hypothesized that ischemic stroke and OGD may increase TRPC6 expression which may, in turn, activate NMDA receptors (NMDAR) and enhance Ca2+ influx. Thus, deletion of TRPC6 would be expected to reduce ischemia-induced brain damage and OGD-/glutamate-induced cortical cell death. Our study has demonstrated that TRPC6-mediated Ca2+ influx enhanced excitotoxicity by facilitation of NMDAR activation and TRPC6 deletion attenuated ischemia-induced neuronal death.TRPC6 channels are localized at the excitatory synapses and may regulate neuronal excitability , Hank's buffer, Glutamax, and penicillin-streptomycin, Trizol, SuperScript II RNaseH reverse transcriptase and T-PER buffer were purchased from Invitrogen. GoTaq Green was obtained from Promega. Neurobasal medium, B27, Dulbecco's Modified Eagle Medium (DMEM), and bovine calf serum were purchased from Hyclone. 2, 3, 5-triphenyltetrazolium chloride (TTC), propidium iodide (PI), protease inhibitor cocktail, N-methyl-D-glucamine (NMDG) and Trypan blue were obtained from Sigma. Other reagents include: CNQX, MK801 (Molecular Probes); tetrodotoxin ; Fura-2 acetoxymethyl ester . Recombinant adenoviruses AdCMV-GFP and AdCMV-TRPC6-GFP-Myc-tag were kindly provided by Dr. Christopher B. Newgard. The sources of the primary antibodies were as following: TRPC6 , MAP2 , and Beta-Actin . The goat anti-rabbit and anti-mouse secondary antibodies conjugated to horse radish peroxidase were from Jackson Immunoresearch.−/− mice. The generation of TRPC6−/− mice has been previously described  and were housed in in the transgenic animal facility (TAF). One male WT mouse was housed in the same cage with one female WT mouse. Similarly, one male TRPC6−/− mouse was housed in the same cage with one female TRPC6−/− mouse. The pregnant mice were separated from the males and kept in a single cage as soon as they showed signs of pregnancy. The pregnant mice were checked daily. The animal protocol had been approved by UCF IACUC. All experiments were conducted in accordance with the recommendations of UCF IACUC.Wild type WT mice (C57 BL/6J) were used as the background to generate TRPC6−/− and WT control mice were maintained on a 12 h light/12 h dark cycle with food and water. Three-four month-old male mice were used for this experiment. The focal cerebral ischemia/reperfusion model was generated following that described previously . The brains were immediately removed and placed into ice-cold PBS for 15 min, and then 2-mm coronal sections were cut. The brain sections were stained with TTC (2%) in PBS at 37°C for 15 min. The stained sections were photographed, and the digitized images were used for analysis. The borders of the infarct in each brain slice were outlined, and the area quantified using an ImageJ software. Infarct volume was calculated by integration of infarct areas for all slices of each brain  and maintained at 37°C with 5% CO2. Half of the medium was changed every 3 days, and the cells were cultured for 14-16 days.Primary cultures of mouse cortical neurons were prepared from the 18-day-old fetuses of pregnant female mice as previously described  for 24 h. Control cultures were incubated for 2 h in a similar balanced salt solution containing 5.5 mM glucose under normoxic conditions.After 14–16 days of culture, the primary cortical neurons were challenged with oxygen-glucose deprivation (OGD) . An appropriate cycle number was chosen to avoid the accumulation of by-products and yield accurate replication of target DNA before the characteristic plateau phase was reached in the PCR amplification curve. PCR procedure included: pre-denatured at 95°C for 5 min; amplification at 95°C 50 s, 56°C 1 min, 72°C 1 min; and extended at 72°C for 10 min. Aliquots of the PCR product were separated using agarose gel electrophoresis and visualized with ethidium bromide staining. The following oligonucleotide primers were used: for TRPC6, sense 5′-CAG ATC ATC TCT GAA GGT CTT TAT GC-3′ and anti-sense 5′-TGT GAA TGC TTC ATT CTG TTT GCG CC-3′; for β-actin, sense 5′-TGC TAT CCC TGT ACG CCT CT-3′, anti-sense 5′-GGA GGA GCA ATG ATC TTG A-3′.Total RNA was extracted from the ipsilateral and contralateral cortices of the brain tissue using Trizol reagent following the manufacture's protocol. Reverse transcription was started with 2 μg of total RNA at 42°C for 60 min in 20 μL reaction mixture containing 20 U RNase inhibitor, 0.25 mM of each dNTP, 0.5 μg Oligo(dT)Protein from ipsilateral and contralateral cortices was extracted using T-PER tissue protein extraction buffer with protease inhibitor cocktail (Sigma). Protein concentration was determined using the BCA protein assay kit . Fifty μg of protein was separated by SDS-PAGE (8–12%) and transferred to nitrocellulose membranes. The membranes were blocked in 5% non-fat milk for 1 h at room temperature, followed by an overnight incubation at 4°C with antibodies raised against TRPC6 or β-actin. Membranes were then washed and incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized using a chemiluminescence detection kit (Amersham Biosciences).For adenovirus construction, cytomegalovirus (CMV) promoter-driven human Myc-tagged TRPC6 adenovirus was constructed by cloning cDNA constructs into the pAdTrack shuttle vector and using the Ad-Easy system to generate the recombinant adenoviruses as previously described  at room temperature for 120 min. Nuclei were stained with 1 μg/ml propidium iodide  for 10 min  staining of nuclei (red) in primary neurons from WT and TRPC62 incubator at 37°C, washed 3 times in HEPES-KRH buffer  and incubated for additional 30 min. Then, the changes in free intracellular Ca2+ concentration ([Ca2+]i) in neurons loaded with Fura-2 were recorded and measured using the Nikon Live Image System with Nikon Elements software (Nis-Elements AK 4.13). Images were taken every 10 s with an exposure time of 200 ms. Fura-2 dual excitation images were captured with a camera . The baseline Fura-2 fluorescence intensity (expressed as the ratio of 340/380 nm) was measured 1 min before glutamate application. Glutamate application (100 μM) at 60 s after initiation of recording was used to evoke Ca2+ influx. An increase in relative Fura-2 340/380 fluorescence indicates an increase in free intracellular Ca2+ concentration  of each neuron at the different time points was normalized relative to the initial intensity (F0) of that neuron at 0 s (before glutamate application). The mean dynamic changes in [Ca2+]i were determined at each time point for 3–10 neurons in each experiment.After 14–16 days of culture, these neurons were loaded with the fluorescent dye Fura-2 acetoxymethyl ester (Fura-2 AM) (3 μM) for 30 min in humidified 5% COt-test was used for comparison between two groups. Statistical comparisons were performed using ANOVA and Newman-Keuls post-hoc test for pairwise comparisons. Statistical significance was accepted at P < 0.05.Values were represented as mean ± S.E.M. Student To determine whether ischemia altered the TRPC6 expression level, we measured mRNA levels of TRPC6 in the brains following I/R. We found that mRNA expression of TRPC6 was markedly upregulated, starting at 12 h after I/R in the ipsilateral, but not in the contralateral cortices Figure . Analogo−/− mice. Two days after I/R, we measured the amount of the brain damage. We found that the infarct size was significantly decreased in the TRPC6−/− mice compared to WT mice  that had bound to the nucleotide. The dying neurons had the smaller size of nuclei (<2 μM in diameter) than the other neurons that have the normal size of nuclei (4–5 μM)  with glutamate (100 μM) and NMDA (50 μM) for 30 min, respectively. Twenty-four hours after glutamate or NMDA treatment, the cultured neurons were fixed and cell viability was assessed by PI staining (red). These results showed that glutamate and NMDA significantly increased neuronal cell death compared to normal untreated neurons. Deletion of TRPC6 significantly protected neurons from glutamate and NMDA-induced cell death  into the bath. Figures 2+]i in response to glutamate application, respectively. OGD pretreatment significantly increased [Ca2+]i in neurons in both WT and TRPC6−/− neurons compared to the [Ca2+]i level at the start of recording (0 s) where F/F0 equaled 1. Deletion of TRPC6 significantly attenuated OGD-induced [Ca2+]i increase compared to control (P < 0.01). These observations indicate that TRPC6 contributes to OGD-increased Ca2+ influx.Because Ca2+ influx and whether such an influx was dependent on AMPA channel receptors whose opening also increased the opening probability of NMDA receptor channels (see below), WT cortical neurons were infected for 4 h with AdCMV-TRPC6-GFP to overexpress TRPC6 and AdCMV-GFP as control . Twenty-four hours after infection, neurons showed GFP (green)  in TRPC6 overexpressed neurons. In the presence of CNQX, overexpression of TRPC6 still enhanced [Ca2+]i in response to glutamate application, indicating TRPC6 could mediate Ca2+ influx in response to glutamate independent of AMPA receptors  was applied to a bath containing infected neurons in the presence of MK-801 (20 μM) which could effectively block NMDAR receptor mediated-, glutamate-induced [Ca2+]i increases  or activating Na+-permeable channels  in the bath solution such that the electrical charges and osmolality across the cell membrane would not be changed  in response to glutamate application. NMDG replacement significantly, but not completely, reduced such an increase in TRPC6-infected neurons  was applied to block voltage-sensitive Na+ channels in the presence of AMPA receptor blocker CNQX (10 μM)   Figure . In addi2+ overload has been considered as one of the major mechanisms underlying ischemia-induced neuronal death. Blockers of NMDA receptors were used to reduce Ca2+ influx and showed protective effects on the brain damages in animal stoke models. Unfortunately, multiple clinical trials of glutamate antagonists failed to show effective neuroprotection and had serious side effects in stroke patients -induced cell death. To determine the cellular and molecular mechanism for OGD-induced neuronal death, OGD was found to increase, but deletion of TRPC6 reduced glutamate-evoked Ca2+ entry. TRPC6 overexpression increased glutamate-evoked Ca2+ entry in the presence of the AMPA blocker, indicating TRPC6-increased Ca2+ entry could be independent of AMPA receptor channels. In the presence of the NMDA receptor antagonist MK801, the increased glutamate-evoked Ca2+ entry in TRPC6 overexpressed neurons was largely reduced, indicating that NMDA receptors were involved. To assess whether Na+ contributed to the TRPC6-enhaced Ca2+ influx, we found that glutamate-evoked Ca2+ entry in TRPC6 overexpressed neurons was largely reduced in Na+-free bath, indicating that Na+ entry was required for TRPC6-mediated Ca2+ influx. Finally, we found that glutamate-evoked Ca2+ entry was reduced but not completely abolished in OGD-treated WT neurons after blockade of voltage-sensitive Na+ channels and AMPA receptor channels. We postulate that TRPC6 channels, as well as other channels , may contribute to TRPC6-mediated Ca2+ influx, at least in part, through NMDA receptor channels. Therefore, the present data have demonstrated that overexpression of TRPC6 may contribute to neuronal death partially through Na+ entry, leading to activation of NMDA receptors, thus causing an overload of Ca2+.For decades, Ca2+ influx  pathway and increased Ca−/− mouse model. Further investigation is needed to address these discrepancies.Consistent with the present finding, TRPC6 expression levels in the retinal ganglion cells were increased 24 h after I/R in rats (Wang et al., It should be mentioned that TRPC1 or TRPC3 are also widely expressed in the nervous system and they have structural similarities with TRPC6 (Riccio et al., 2+ influx through NMDAR, which may lead to cell death (Figure 2+ -induced neurotoxicity through NMDAR, the present work has not yet determined whether blockade of TRPC6 may have a greater protective effect than directly blocking NMDAR. Such an important question needs to be addressed in future studies.In summary, the present studies have demonstrated that TRPC6 overexpression after I/R and following OGD treatment may contribute to brain damage and to OGD- and neurotoxicity-induced cell death. Activation of TRPC6 channels may increase Cah Figure . Since TThis study was carried out in accordance with the recommendations of The Guide for the Care and Use of Laboratory Animals, 8th edition. The protocol was approved by the UCF IACUC.JC, Data collection, data analysis and interpretation, manuscript writing; ZL, Data collection, data analysis, manuscript editing; JH, Concept contribution, manuscript editing; QC, Concept contribution and Manuscript editing; LC, Data analysis; RW, Concept contribution to the manuscript, manuscript editing; SC, Design of the work, provide Figures The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the effects of cyclic stretch on vaso-reactivity of mouse aortic segments, we used the Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC). The aortic segments were clamped at frequencies of 6–600 bpm between two variable preloads, thereby mimicking dilation as upon left ventricular systole and recoiling as during diastole. The preloads corresponding to different transmural pressures were chosen to correspond to a low, normal or high amplitude of cyclic stretch. At different time intervals, cyclic stretch was interrupted, the segments were afterloaded and isometric contractions by α1-adrenergic stimulation with 2 μM phenylephrine in the absence and presence of 300 μM L-NAME (eNOS inhibitor) and/or 35 μM diltiazem (blocker of voltage-gated Ca2+ channels) were measured. As compared with static or cyclic stretch at low amplitude (<10 mN) or low frequency (0.1 Hz), cyclic stretch at physiological amplitude (>10 mN) and frequency (1–10 Hz) caused better ex vivo conservation of basal NO release with time after mounting. The relaxation of PE-precontracted segments by addition of ACh to stimulate NO release was unaffected by cyclic stretch. In the absence of basal NO release , physiological in comparison with aberrant cyclic stretch decreased the baseline tension, attenuated the phasic contraction by phenylephrine in the absence of extracellular Ca2+ and shifted the smaller tonic contraction more from a voltage-gated Ca2+ channel-mediated to a non-selective cation channel-mediated. Data highlight the need of sufficient mechanical activation of endothelial and vascular smooth muscle cells to maintain basal NO release and low intracellular Ca2+ in the smooth muscle cells in large arteries. Both phenomena may play a vital role in maintaining the high compliance of large arteries.Large, elastic arteries buffer the pressure wave originating in the left ventricle and are constantly exposed to higher amplitudes of cyclic stretch (10%) than muscular arteries (2%). As a crucial factor for endothelial and smooth muscle cell function, cyclic stretch has, however, never been studied in  In the cardiovascular system large elastic arteries such as the aorta or carotid artery are well equipped to dampen the pressure wave originating from the left ventricle. Because of their close proximity to the left ventricular ejection, elastic arteries are constantly exposed to high levels of cyclic stretch and dilate by approximately 10% with each heartbeat. This cyclic stretch is much higher than in muscular arteries, which dilate by only 2–3% , which allowed for the stretching of n = 39, 4–9 months, weight ranging from 28.5 ± 0.7 g at 4 months to 33.7 ± 0.9 g at 9 months) were housed in the animal facility of the University of Antwerp in standard cages with 12–12 h light-dark cycles and free access to regular chow and tap water. Animals were euthanized by perforating the diaphragm while under anesthesia . The thoracic aorta was carefully removed and stripped of adherent tissue. Starting at the diaphragm, the aorta was cut in 4 segments of 2 mm width. Segments were immersed in Krebs Ringer (KR) solution  containing (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaEDTA 0.025 and glucose 11.1. The zero external Ca2+ solution had the same composition except that 2.5 mM CaCl2 was omitted and 1 mM EGTA was added. To normalize external Ca2+, 3.5 mM CaCl2 was added to the zero calcium solution. To avoid any vasomotor interference due to prostanoids, 10 μM indomethacin  was present in all experiments. The studies were approved by the Ethical Committee of the University of Antwerp (2015–52 and 2016–28), and all experiments were performed conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .Male C57Bl6 mice  with damped clamps of 50 ms to each pressure , which were significantly (p < 0.001) lower for the 20–28 mN cyclic stretch, which were of comparable magnitude for 10–28 and 20–40 mN and significantly larger (p < 0.001) at 28–65 mN. With mean diameters of the mouse aortic segments at 20 mN of 1,060 ± 11 μm (n = 10), the cyclic stretch amounted 4, 12, 12, and 14% at 20–28, 10–28, 20–40 and 28–65 mN, respectively.Cyclic stretch caused diameter changes Table , n = 7–9Segments' clamp frequencies were changed from 10 to 5, 2.5, 1.25, 1, and 0.1 Hz at stretch amplitudes of 20 mN or 50 mm Hg (20–40 mN or 70–120 mm Hg), with damped clamps of 50 ms to 40 mN and variable duration to 20 mN.Contractile properties of all the segments mounted in the ROTSAC were always measured in isometric conditions by interrupting the cyclic stretch and applying an afterload of 20 mN (70 mm Hg) to the lower preload at which the segments were clamped . These contractions were compared with the isometric contractions of the segments, which were continuously conditioned without cyclic stretch. As such, the diameter of all the segments was kept constant at the lower preload for cyclically stretched segments and at its single preload for the statically stretched segments. Different amplitudes of cyclic or static stretch were not applied to a single segment but delivered to different segments. For each experimental day 4 segments of one mouse were mounted in parallel in 4 ROTSAC set-ups. Two segments were stretched to 20–28 and 20–40 mN (cyclic stretch), whereas the two other segments were stretched to 20 and 28 mN (static stretch). The next experimental day two segments were stretched to 10–28 and 28–65 mN (cyclic stretch) whereas static stretch was applied to 2 other segments (10 and 65 mN). The static data at 65 mN are not included in the presented data. These experiments were repeated for 5 or 6 mice for each experimental protocol. After conditioning with cyclic or static stretch, segments were afterloaded with 20 mN, isometric data  at a concentration of 2 μM, which is a maximally effective concentration. Both phasic (in the absence of extracellular Ca2+) and tonic contractions (after addition of 3.5 mM Ca2+) were measured. The PE-induced phasic contraction was measured 3 min after the removal of extracellular Ca2+ by switching from normal KR to KR without Ca2+ and containing 1 mM EGTA (0 Ca solution)  and amplitudes  of contraction and relaxation of these phasic contractions, respectively. PE-induced contractions were also measured in normal, Ca2+ containing KR solution and these contractions were also analyzed by a double exponential function revealing time constants  and amplitudes  of the fast and slow force component. The fast component refers to the phasic contraction by PE, whereas the slow component corresponds with the contraction due to Ca2+ influx  was inhibited with 35 μM diltiazem, causing partial relaxation of the contraction by PE  . These concentration-relaxation curves were fitted with sigmoidal concentration-response equations with variable slope (GraphPad Prism) to obtain Emax- and logEC50-values for ACh.To further study the basal release of NO in the different cyclic stretch conditions, contractions by PE were measured at 1 h time intervals after mounting the segments in the ROTSAC .All data are expressed as mean ± SEM . Statistical analysis was performed with Graphpad Prism 6 software. Dependent on the type of experiment, one and two way ANOVA with Tukey's or Dunnett's multiple comparison test was applied to the data to select significance (1-adrenoceptor agonist PE (2 μM). PE-induced phasic contractions were determined in the absence of extracellular Ca2+, whereas tonic contractions were measured upon re-addition of external Ca2+. The tonic and phasic isometric contractions after normal or high cyclic stretch  were compared with the contractions of segments which were continuously mounted in low amplitude cyclic stretch (20–28 mN) or static conditions , further called static/low.Aortic segments were conditioned with cyclic stretch of variable amplitude in the presence of 300 μM L-NAME to inhibit basal NO release from the endothelium and to focus on the effects of cyclic stretch on VSMC. Isometric contractions were measured by interrupting the cyclic stretch and stimulating the afterloaded segments with the α2+ induced a preload-dependent decrease of baseline isometric force  and amplitudes  of contraction and relaxation of these phasic contractions, respectively. The time constants of contraction or relaxation were not significantly different between the conditions . Contraction and relaxation amplitudes did not differ between 10–28, 20–40, and 28–65 mN cyclic stretch, but they were all significantly different from Aon  were significantly lower than the contractions measured after static stretch. Relatively, diltiazem inhibited about 40% of the contraction in all conditions studied, whereas 60% of the tonic contraction was due to NSCC Ca2+ influx  the contraction by PE increased with time after mounting and the increase was significant after 2 or 3 h Figure . In the 2+ influx to the total contraction  or after addition of L-NAME (10 mN)  to 5 (20–25 mN), 20 (20–40 mN), or 40 (20–60 mN) mN had no significant effect at any time interval on the log ECh Figure  or the mh Figure . Similarh Figure .fast increased more over time at 0.1 than at 1 or 10 Hz, and was significantly increased at 0.1 or 1 Hz at all-time intervals after mounting compared to the physiological frequency of 10 Hz. For the slow component or phasic contraction, the time-dependent increase was also evident and more pronounced at 0.1 Hz. At 3 and 4 h after mounting, Aslow was significantly increased at this frequency when compared with 10 Hz. When Afast was plotted against Aslow  higher at 0.1 and 1 Hz than at 10 Hz .In the following experiments aortic segments were subjected to physiological stretch between 20 and 40 mN at different frequencies: 10, 5, 2.5, 1.25, 1, and 0.1 Hz. This stretch was interrupted every hour after mounting to measure isometric contractions by 2 μM PE Figure . Becausew Figure , there w2+ influx and which leads to a lower contractile Ca2+ content of the VSMC SR; (2) lower tonic contractions when segments are contracted with the α1 adrenoceptor agonist, PE; (3) a non-significant or mild increase of contractions by PE with time after mounting, suggestive for better conservation of basal NO release, and (4) unaltered relaxation of PE-precontracted segments by addition of ACh to stimulate NO release.The present study demonstrated that contractile properties of mouse aortic segments depend on the amplitude and frequency of cyclic stretch. When compared with static stretch or low amplitude (<10 mN) or low frequency (0.1 Hz) cyclic stretch, cyclic stretch at physiological amplitudes (>10 mN) and frequencies (1–10 Hz), caused (1) a decrease of baseline contraction, which is probably due to attenuated baseline Caex-vivo are underexposed, and almost exclusively studied in cultured cells grown on elastomer-bottomed culture plates, mostly at very low stretch amplitudes (2–5%) , which is of important physiological relevance  imposes a pulsatile pressure in the aorta that generates EC and VSMC stretch  in the vessel wall. The present study investigated the consequences of lower (4%) and higher (14%) than normal (12%) cyclic stretch. Cyclic stretch together with shear stress represent the most important mechanical stimuli in elastic arteries, but both physical parameters have differential effects on both EC , the second was a tonic response produced by the activation of stretch-sensitive non-selective cationic channels (NSCC) allowing external Ca2+ to the 0Ca solution containing 2 μM PE, caused tonic contractions which were significantly smaller in segments subjected to high frequency (10 Hz) or normal/high amplitude cyclic stretch, compared to segments subjected to static or low amplitude cyclic stretch or cyclic stretch with low frequency   was plotted against the slow component of contraction  at clamp frequencies of 0.1, 1, and 10 Hz. Although the slope at 10 Hz was significantly different from zero, the slope was rather flat and significantly increased at 1 and further at 0.1 Hz. This indicates that at low frequency but normal amplitude of cyclic stretch, a higher phasic contraction is followed by a higher tonic contraction. Which stretch-dependent transduction system is sensing the amplitude and frequency of the stretch and thereby couples or uncouples SR Ca2+ release and subsequent Ca2+ influx is a topic for further investigation.After the phasic contraction, re-addition of extracellular Ca Figures , 9. In t Figures , whereas2+ influx (60%). Cyclic stretch  has been described to decrease TRPC4 protein levels in isolated rat vascular smooth muscle cells, which was related to reduced capacitive calcium entry in WKY but not in SHR .In the presence of L-NAME, hence the absence of basal NO release, the tonic contraction by PE was mainly caused by NSCC-mediated Caex vivo environment. At least, this mechanical parameter has been shown to be essential for the normal development, homeostasis and remodeling of the vasculature  and frequency (>1 Hz) were able to promote the maintenance of basal NO release or efficacy. In none of the conditions, stimulated NO release by ACh was affected, suggestive for the conservation of an intact endothelial monolayer at low or even very high amplitudes or frequencies  compared with static or low amplitude cyclic stretch (Figure It has been described that pathophysiological levels of cyclic stretch result in a reduction in endothelial NO production, the hallmark of endothelial dysfunction. As such, pathological cyclic stretch induces an EC phenotype with decreased vasodilatory capacity. Since reduced NO bioavailability has been shown to increase arterial stiffness in rodents (Isabelle et al., 1-adrenoceptor stimulation of mouse aortic segments with PE and the role of basal NO release herein. Both cyclic stretch amplitude and frequency contribute in setting the mechanical properties of isolated aortic segments. In vivo hemodynamic alterations caused by hypertension and arterial stiffness result in chronic exposure to pathological cyclic stretch, leading to phenotypic modulation of EC and VSCM and resultant arterial remodeling (Thorin-Trescases and Thorin, In summary, cyclic stretch causes significant alterations of the isometric contractions produced by αAL, SD, CV, and PF designed, performed and analyzed the experiments. AL and PF drafted the work, whereas all authors approved a final version and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JVT and handling Editor declared their shared affiliation.