Cone terminals have many ribbon-style active zones at which second-order neurons receive input. We asked whether there are functionally significant differences in local Ca2+ influx among ribbons in individual cones. We combined confocal Ca2+ imaging to measure Ca2+ influx at individual ribbons and patch clamp recordings to record whole-cell ICa in salamander cones. We found that the voltage for half-maximal activation (V50) of whole cell ICa in cones averaged −38.1 mV ± 3.05 mV (standard deviation [SD]), close to the cone membrane potential in darkness of ca. −40 mV. Ca2+ signals at individual ribbons varied in amplitude from one another and showed greater variability in V50 values than whole-cell ICa, suggesting that Ca2+ signals can differ significantly among ribbons within cones. After accounting for potential sources of technical variability in measurements of Ca2+ signals and for contributions from cone-to-cone differences in ICa, we found that the variability in V50 values for ribbon Ca2+ signals within individual cones showed a SD of 2.5 mV. Simulating local differences in Ca2+ channel activity at two ribbons by shifting the V50 value of ICa by ±2.5 mV (1 SD) about the mean suggests that when the membrane depolarizes to −40 mV, two ribbons could experience differences in Ca2+ influx of >45%. Further evidence that local Ca2+ changes at ribbons can be regulated independently was obtained in experiments showing that activation of inhibitory feedback from horizontal cells (HCs) to cones in paired recordings changed both amplitude and V50 of Ca2+ signals at individual ribbons. By varying the strength of synaptic output, differences in voltage dependence and amplitude of Ca2+ signals at individual ribbons shape the information transmitted from cones to downstream neurons in vision.Cone photoreceptors depolarize in darkness to release glutamate-laden synaptic vesicles. Essential to release is the synaptic ribbon, a structure that helps organize active zones by clustering vesicles near proteins that mediate exocytosis, including voltage-gated Ca Vm). To sustain this continuous and abundant output, the number of vesicles in a photoreceptor terminal exceeds that of a conventional hippocampal synapse by more than two orders of magnitude and photoreceptors possess specialized presynaptic structures called ribbons that organize and coordinate vesicular release  for ICa, such as those caused by inhibitory feedback from horizontal cells  is sufficient to produce differences in Ca2+ influx at individual ribbons of 45% or more at a cone’s resting membrane potential in darkness. We assessed the sensitivity of confocal Ca2+ measurements by manipulating extracellular pH to produce 3 mV shifts in voltage dependence. By directly manipulating HC membrane potential in paired whole-cell recordings with cones, we confirmed the ability of ribbons operate independently by showing that inhibitory feedback from an individual HC was capable of altering Ca2+ responses at some ribbons but not others in a cone terminal  of both sexes (18–25 cm in length). We used cones from salamander retina for these studies because the ribbons in these cells are sufficiently far apart to be distinguished by optical imaging and because the synaptic terminal sits at the base of the cell body where it can be effectively voltage-clamped. All animal care and handling protocols were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Euthanasia was conducted in accordance with AVMA Guidelines for the Euthanasia of Animals. Salamanders were kept on a 12-h dark/light cycle at 4–8°C.All experiments were performed using One to two hours after onset of the dark cycle, salamanders were euthanized by decapitation and rapidly pithed. The eyes were enucleated, the retina isolated, and vertical retinal slices (125 μm thickness) were prepared as described in detail elsewhere  or 95% O2/5% CO2  with NaOH. The bicarbonate-buffered solution contained (in mM): 101 NaCl, 2.5 KCl, 2.0 CaCl2, 0.5 MgCl2, 11 glucose and 22 NaHCO3. For both solutions, the osmolality was measured with a vapor pressure osmometer (Wescor) and if necessary adjusted to ~245 mOsm. Patch pipettes were pulled with a PP-830 or PC-10 vertical pipette puller (Narishige) from borosilicate glass pipettes  and had resistances of 9–15 MΩ. For some experiments, pipette shafts were coated with dental wax to reduce stray capacitance. The intracellular pipette solution contained (in mM): 50 CsGluconate, 40 CsGlutamate, 10 TEACl, 3.5 NaCl, 1 CaCl2, 1 MgCl2, 9.4 MgATP and 0.5 GTP-Na, 5 EGTA. After salts were dissolved, the pH was adjusted to 7.2 with CsOH . All chemical reagents were from Sigma-Aldrich unless indicated otherwise.Throughout the experiments, the slice chamber was superfused at ~1 ml/min with chilled amphibian saline solution bubbled with 100% OExperiments were performed on an upright fixed-stage microscope (Nikon E600FN) under a water-immersion objective . Cells were identified morphologically and recording electrodes were positioned with Huxley-Wall micromanipulators (Sutter Instruments). After obtaining a gigaohm seal, the patch was ruptured with gentle suction. Photoreceptor and HC recordings were conducted in voltage clamp and were performed using Alembic VE-2 (Alembic Instruments) and Axopatch 200B (Axon Instruments/Molecular Devices) amplifiers, respectively. Cone membrane currents from the Alembic were low-pass filtered at 3 kHz and HC currents from the Axopatch were filtered at 2 kHz. Some membrane currents were low-pass filtered at 200 Hz to facilitate data presentation. Signals were digitized with a Digidata 1322A  and acquired with pClamp 10 software (Molecular Devices). Series resistance was maximally compensated in every cone before recording using the Alembic amplifier, which allows stable compensation  applied from a steady holding potential of −79 mV. Voltage-dependent and Ca2+-activated K+ currents in cones were blocked by TEA in the pipette solution and hyperpolarizing-activated cation currents (Ih) were inhibited by Cs+ . For determination of V50 values, passive membrane resistance was subtracted from ramp currents using P/8 subtraction. Comparison of currents obtained in the presence of and absence of Cd2+ (0.1 mM) yielded the same current/voltage profiles for photoreceptor ICa  to ICa measured using the ramp voltage protocol (n = 6 cones), we observed the same voltage dependence with both measurements.Whole-cell I2+ imaging experiments described in this study used 175 ms depolarizing steps applied from a steady holding potential of −79 mV  with 2 s between each step. We compared results obtained when the steps were applied in both forward and reverse order . In trials where steps were applied in the forward order, ribbon Ca2+ signals yielded V50 values that were 2.06 ± 1.38 mV more positive than whole cell ICa measured in the same cones . In trials where steps were applied in the reverse order, ribbon Ca2+ signals yielded V50 values that were 2.17 ± 0.97 mV more negative than whole cell ICa . Values from the two different measurement sequences thus straddled the values for ICa. Photoreceptor ICa in salamander rods shows slow and weak Ca2+-dependent inactivation  between test voltages to speed data acquisition during paired recordings. Voltage dependence measured with the two protocols did not differ noticeably.Most of the Can = 47 cones). We excluded recordings if the current needed to voltage clamp a cone at −79 mV exceeded 250 pA or if the series resistance prior to compensation exceeded 60 MΩ. When multiple recordings within a cell were compared or averaged , only trials with holding currents within 50 pA of one another were compared. When multiple stimulus trials were conducted within a cell, we waited at least 1 min between trials. We corrected membrane voltage values for a measured liquid junction potential by subtracting 9 mV.Whole cell membrane resistance and capacitance in cones averaged 192 ± 88.9 MΩ and 80 ± 16.4 pF, respectively  or Oregon Green 488 BAPTA-6F  were added to the patch pipette solution at 400 μM. Images (57 ms/frame) were acquired during the voltage step protocol described above and analyzed using Nikon Elements 4.5 and Microsoft Excel software. Fluorescence values were measured as the mean pixel intensity within the region of interest (ROI). Baseline Ca2+ signal fluorescence was calculated by averaging signals during the first 1.3 s of the imaging trial when the photoreceptor was voltage clamped steadily at −79 mV. Fluorescence changes (∆F) evoked by depolarizing voltage steps were compared to baseline fluorescence (F) and the resultant ∆F/F ratios were normalized to the largest fluorescence change in each trial before using nonlinear regression to determine V50 values  in two consecutive trials.Confocal imaging was performed with Nikon Elements software using a laser confocal scanhead (Perkin Elmer Ultraview LCI) equipped with a cooled CCD camera (Orca ER) mounted on the Nikon E600FN. Excitation at 488 or 568 nm was delivered from an argon/krypton laser and emission was collected at 525 or 600 nm, respectively, by a cooled CCD camera (Hamamatsu OrcaER). Filters were controlled using a Sutter Lambda 10–2 filter wheel and controller. The objective was controlled using a E662 z-axis controller (Physik Instrumente). Cell-impermeant CaWe measured the optical resolution of our system using fluorescent microbeads. The full width half maxima  of Gaussian functions fit to the fluorescence profiles in the x-y plane averaged 318 and 455 nm for 525 and 600 nm emission, respectively. To measure resolution in the z-axis, we acquired a z-stack and measured the fluorescence profile as a function of depth. The FWHM in the z-axis averaged 1.45 and 1.46 μm for 525 and 600 nm emission, respectively.50 values and construct ICa activation curves (G/Gmax), Ca2+ responses were fit with Boltzmann functions adjusted for Ca2+ driving force according to the equationActivation profile data from Clampfit, Nikon Elements, and Microsoft Excel was imported to GraphPad Prism to perform nonlinear regression, for statistical analyses, and to generate figures. To calculate VVrev = +41 mV and k is the slope factor of the voltage-dependent activation process. Vrev was the only constrained parameter. Distribution normality was assessed by D’Agostino-Pearson Test. Data is reported as mean ± SD unless indicated otherwise.where 2+ influx at individual ribbon sites, we used spinning disk confocal microscopy while simultaneously controlling the membrane voltage of individual cones by voltage clamp. We introduced a low-affinity Ca2+ indicator, Oregon Green BAPTA-5N , into photoreceptors through the patch pipette and then evoked Ca2+ entry by delivering a series of depolarizing voltage steps. Ca2+ signals elicited in this way were typically constrained to hotspots near ribbons and only well-localized Ca2+ hotspots were analyzed . In this cell, we also labeled ribbons by introducing a RIBEYE-binding peptide conjugated to tetramethylrhodamine (TAMRA) through the patch pipette  elicited at each stimulation step was normalized to the maximum ∆F/F signal within each trial. The change in ∆F/F as a function of voltage was then fit with a Boltzmann function modified for Ca2+ driving force. Each ribbon site was analyzed independently. Figure 50) values differing by 3.67 and 3.15 mV in trials 1 and 2, respectively. In trial 1, the V50 value for ribbon 1 fell outside the 95% confidence interval for ribbon 2. The voltage dependence of ribbon 3 differed more between the two trials, with the difference in V50 between ribbons 1 and 3 increasing from 0.32 mV to 2.70 mV between trials 1 and 2. In trial 2, the V50 value for ribbon 1 fell outside the 95% confidence interval for ribbon 3 and vice versa. Observations from eight other cones also showed reproducible differences in V50 values between ribbons in the same cone suggesting that the voltage dependence of ribbon Ca2+ responses may genuinely differ from one another. In the experiments described below, we quantified these V50 differences after assessing various factors that might confound measurement accuracy.Figure 2+ changes at individual ribbons, we drew regions of interest (ROIs) around each ribbon-associated hotspot, tightly delineating regions with clear and distinct Ca2+ increases. In experiments where we also included a fluorescent RIBEYE-binding peptide in the patch pipette to label ribbons  as we varied ROI size around the ribbon . Ca2+ measurements and z-stack images of RIBEYE fluorescence were typically acquired a few minutes apart from one another and small movements of the recording pipette or tissue during that time may have introduced additional displacements between the two measurements. Nonetheless, Ca2+ measurement planes and the brightest RIBEYE fluorescence planes differed by only 0.38 ± 0.60 μm  above the same beads imaged with 568 nm excitation/600 nm emission. Taking these optical differences into account reduces the difference between measured planes to 0.31 ± 0.60 μm. This is less than the axial resolution of the 60×, 1.0 NA objective measured at 525 nm emission (1.45 μm). This shows good agreement between planes chosen for Ca2+ signal measurements and ribbon planes identified by RIBEYE peptide labeling.We also examined the impact of confocal plane on measurements of ribbon Cam Figure . When we2+ signals were measured at or very near the actual ribbon plane, but we also considered how imperfect positioning of the focal plane of measurement might impact a ribbon’s calculated V50 value. To do so, we measured Ca2+ responses of a ribbon at one plane and then moved the objective up or down to make a second measurement. Fluorescent peptide was not included in these experiments and so we defined the ribbon plane post hoc as the plane showing the strongest Ca2+ response (largest ∆F/F). As illustrated in Figure 50 shift that averaged +0.7 ± 2.9 mV (p = 0.23) for 25 ribbons in 12 cones. A ±2.3 μm change in focal plane caused a larger, statistically significant positive shift in V50 of +1.6 ± 2.5 mV (p = 0.04). The ability of ±2.3 μm changes in focal plane to produce significant positive shifts in V50 values is likely a consequence of Ca2+ buffering and dye properties, such that greater Ca2+ influx is required to attain levels that can be accurately detected with the low-affinity Ca2+ dye.These results show that Ca50 shifts, these results suggest that iterative z-axis positioning between Ca2+ imaging trials to select the best focal plane resulted in ~95% of Ca2+ measurements being performed in a range spanning 1.2 μm about the ribbon plane. Trial-to-trial differences between V50 values of Ca2+ measurements made at different (±1.1 μm) focal planes did not differ significantly from trial-to-trial differences between V50 values obtained at the same focal plane . This further indicates that focal plane selection had little or no impact on measurement variability. Taken together, these results show that Ca2+ signals were measured at focal planes that lay within the axial resolution limit to the true ribbon planes and that small differences among ribbon measurement focal planes did not introduce significant additional variability to V50 values.Although large errors in focal plane placement can produce significant V2+ influx magnitude among ribbons. For example, while peak ∆F/F values averaged 0.76 ± 0.77, one ribbon showed a peak ∆F/F value of 3.95, more than 4 SD above the average. This large Ca2+ increase was not due to use of a particularly small ROI nor can it be explained by focal plane positioning.Because both focal plane and ROI size can influence the amplitude of ∆F/F measurements in ribbons, we focused most of our analysis on ribbon-to-ribbon differences in voltage dependence. However, there were also genuine differences in Can = 26). The optical point spread function showed a FWHM of 0.45 μm. Therefore, after consideration of optical blurring, fluorescent measurements of ribbon length are consistent with ultrastructural measurements in light-adapted turtle cones, in which ribbon length averaged 1.2 ± 0.6 μm . Measurements of ribbon length scale roughly with total ribbon size since the base of the ribbon is longer and varies more than the other two ribbon dimensions. Cone ribbons are 35–60 nm thick and extend <350 nm into the cytoplasm, but the length of a ribbon along its base can be well over 1 μm .The length of a ribbon along its base determines the number of vesicles that can be tethered at the plasma membrane and thus determines the size of the readily releasable pool of vesicles  and the slope of the regression line did not differ significantly from zero . The finding that Ca2+ signals are strongly correlated in spatial extent, but not peak amplitude, with ribbon size suggests that the number of Ca2+ channels generally scales with ribbon size.In cochlear inner hair cells, the maximum amplitude of depolarization-evoked CaCa can be altered changes in extracellular pH  to 7.6 to induce small changes in voltage dependence of ICa activation. Consistent with earlier measurements of the effect of pH on salamander cone ICa . This 0.2 unit pH change caused a readily detectable and significant shift in the V50 of Ca2+ signals measured optically at individual ribbons in the same cones that averaged +3.3 mV . These small, reproducible changes in V50 induced by altering extracellular pH confirmed that small differences in Ca2+ signal voltage dependence could be reliably detected at individual ribbons in our experiments.The voltage-dependent activation of L-type ICa activation could be a source of post-synaptic variability in bipolar cells as well as a source of variability in V50 values when comparing ribbons in different cones. We therefore examined the variability of V50 values calculated from cone ICa activation curves measured electrophysiologically. Technical variability in measurements of ICa in different cones could arise from differences in series resistance (Rser) during whole-cell patch clamp recordings. To minimize this, we employed an amplifier  with circuitry that allows complete and stable compensation of Rser  with P/8 subtraction of passive membrane properties and then fit the normalized ICa curve with a Boltzmann function adjusted for driving force. An example of ICa analyzed in this way is shown in Figure Ca from 21 cones. Even after compensating for Rser, we found modest differences in the voltage dependence of ICa from cone to cone, with an average V50 of −38.1 ± 3.05 mV  and slope factor of 4.90 ± 1.34. The peak amplitude also differed among cones, averaging 163.6 ± 41.6 pA (n = 28).Cone-to-cone differences in the voltage dependence of I50 values obtained from repeated ICa measurements in the same cone, which produced an average variance of 1.0 mV2 (SD = 1.0 mV). The Alembic amplifier circuitry allows near complete compensation for Rser, but the Clampex membrane test evaluator suggested a small residual Rser averaging 2.1 ± 0.58 MΩ (n = 36). In 14 of the 28 cones used for ribbon analysis, we calculated the voltage error that would be introduced by Rser after accounting for both the steady holding current and the magnitude of the inward ICa at its activation midpoint. The voltage error in these recordings caused by residual, uncompensated Rser averaged 0.54 ± 0.19 mV, indicating that the true V50 for the cones in our analysis is slightly more negative than the value reported above. Additionally, this analysis shows that variability in residual Rser among cones had a negligible impact on variance (0.04 mV2) in ICa V50 values among cones.We analyzed trial-to-trial variability of VCa voltage dependence, the genuine biological variability for ICa V50 among cones would have a SD of 2.9 mV . Our recordings were largely performed in large single cones but also included some small single cones and double cones ; n = 5 small single cones, 6 large single cones, 6 principal members of double cones). The traditional view is that the membrane potential for cones in darkness (−40 mV) rests near the foot of the activation curve for ICa  from slope values for ICa due to the non-linear binding properties of the Ca2+ dye. The distributions of V50 values calculated for cone ICa . Even though multiple ribbons were often measured in a single cone, V50 values of ribbon Ca2+ signals showed slightly greater variability than V50 values of cone ICa. Thus, the voltage dependence of ribbon Ca2+ signals was more variable than would be expected if all ribbons within a cone operated identically.Cone synaptic terminals contain a dozen or more synaptic ribbons that are each potentially capable of providing distinct output channels to second-order horizontal and bipolar cells. To determine whether these channels all transmit signals with equal voltage dependence, we examined the Caa Figure  and ribbs Figure  are both50 values calculated from ribbon Ca2+ influx in cones were 3.3 mV more positive than V50 values determined from ICa , such that small Ca2+ influxes evoked by weak depolarization lie below the dye’s linear response range. To test this idea, we repeated our experiments using a slightly higher-affinity Ca2+ indicator, OGB-6F (Kd = 3 μM), and expanded the test step series to include weaker steps to −49 and −44 mV. In support of our hypothesis, the difference in V50 between Ca2+ signals and ICa was reduced to 2.3 mV when using OGB-6F (n = 25 ribbons in 11 cones). However, Ca2+ hotspots were less tightly constrained with this higher-affinity dye, so we used OGB-5N for experiments examining ribbon-to-ribbon differences in Ca2+ signals.The Va Figure . We hypo50 values for ribbon-associated Ca2+ signals  biological cone-to-cone variability in ICa activation; (3) technical variability in optical measurements of Ca2+ signals; and (4) biological ribbon-to-ribbon variability in ribbon-associated Ca2+ signals. The combined impact of sources 1 and 2  was captured by measurements of variability among whole cell ICa in different cones . To avoid weighting some cells more heavily than others, we repeated this comparison after choosing a single ribbon from each cell. Selecting a single ribbon from each cone to minimize the difference between ribbon V50 and ICa V50 (and thus maximize the correlation) improved the correlation to R2 = 0.228 . Selecting the one ribbon from each cone that maximized the difference between ribbon V50 and ICa V50 also slightly improved the correlation . These coefficients of determination indicate that 16.4%–22.8% of ribbon-to-ribbon variance can be attributed to variance in ICa among cones. Thus, while the amount of variance among V50 values for ribbon Ca2+ measurements was not significantly greater than the variance among V50 values for ICa , the sources of variance in the two samples were largely independent.We assessed the amount of variance in Ca2+ signals (source 3). For each ribbon, we fit the voltage-dependent ∆F/F profiles with a Boltzmann function adjusted for driving force. We acquired up to five replicate data points for each depolarizing step in some experiments, and when such replicates were available, we fit the entire sample as one. The standard errors for V50 values obtained by nonlinear regression fits to ribbon Ca2+ signals provide a way to quantify uncertainties in V50 contributed by optical Ca2+ measurement variability. From the standard errors of Boltzmann fits to Ca2+ imaging data, we found that measurement uncertainty contributed an average variance of 3.61 mV2 .We also assessed technical variability in optical measurements of CaCa (sources 1 and 2). Subtracting this contribution of cone-to-cone variance reduces ribbon-to-ribbon variance from 12.18 mV2 to 9.50–10.23 mV2. Subtracting the additional variance introduced by uncertainties in fitting Ca2+ responses (source 3: 3.61 mV2) leaves an estimated genuine biological ribbon-to-ribbon variance of 5.89–6.62 mV2 (SD = 2.4–2.6 mV). Employing this same analysis with data obtained from experiments with the Ca2+ dye OGB-6F yielded a similar ribbon-to-ribbon variance . This range of variability is consistent with the example in Figure 50 averaging 3.3 mV between two ribbons in the same cone and indicates that the V50 values in a population of ribbons from a single cone are likely to span a range of at least 5.0 mV (±1 SD).The genuine biological variability among ribbons was then determined by subtracting contributions from sources 1, 2 and 3 from the overall ribbon-to-ribbon variability. We found that 16.4–22.8% of the overall ribbon-to-ribbon variance arises from cone-to-cone variance in I50 values may seem modest, an activation difference of this magnitude can alter Ca2+ influx by more than 45% when the cone is at a resting membrane potential in darkness of −40 mV. This is illustrated by the theoretical Ca2+ response curves shown in Figure Ca in cones using the average best-fit parameters of the ICa curves shown in Figure 50 values varying by ±1 SD from the mean  that is 1 SD above the mean would experience 45% less Ca2+ influx than a ribbon exhibiting a more negative activation function (V50 = −40.6 mV) that is 1 SD below the mean. Extending this analysis to consider V50 values ±2 SD away from the mean indicated a difference in Ca2+ influx of 70% or more. Nearly identical differences in Ca2+ influx were obtained using average parameters of the curves fit to optical ribbon Ca2+ measurements . Before imaging, we first confirmed electrophysiologically that HC-to-cone feedback was present by holding the cone at −39 mV for 2 s to activate ICa and then hyperpolarizing the HC with a step from −39 mv to −99 mV to relieve inhibitory feedback. When feedback is present, hyperpolarizing the HC relieves feedback inhibition of cone ICa and stimulates an inward current . In this example, depolarizing the HC to activate inhibitory feedback reduced the amplitude of Ca2+ influx at one ribbon (top set of traces) but not at the other two. Presumably, dendrites from the voltage-clamped HC did not contact the two non-responsive ribbons. The Ca2+ changes at the ribbon subject to HC feedback are plotted in Figure 2+ influx at every cone test potential. By fitting Boltzmann functions to these activation profiles, we also found that the V50 shifted positively by 1.0 mV when negative feedback was activated by depolarizing the HC. In this example, we held the HC at either −79 or −9 mV while testing cone responses over the entire voltage range . Averaging multiple trials in this way reduced trial-to-trial changes in Ca2+ response amplitude.We compared ribbon Cat Figure . Figure 2+ responses at this ribbon compared to responses evoked by the preceding test step applied when the HC was held at −79 mV. Strong inhibition of cone Ca2+ responses by inhibitory feedback from the HC in this example was evident despite the fact that in separate control trials conducted without a paired HC, the second in a pair of depolarizing test steps applied to the cone sometimes evoked a larger response than the first. To quantify feedback-evoked changes in amplitude and voltage dependence, we eliminated possible order effects of this protocol by averaging two sequences in which we swapped the order in which we applied the HC voltage changes. In one sequence, the first cone test step was applied while feedback was activated by holding the HC at −9 mV and the second cone test step was applied while holding the HC at −79 mV. In the other sequence, we reversed the order of HC test potentials to activate feedback during the second of each pair of cone test steps  between the two steps Figure . In the 2+ signals. We saw feedback-evoked modulation at 16 out of 25 ribbons (64%) in 14 cone-HC pairs in which the presence of feedback was confirmed electrophysiologically . In identical control trials conducted without simultaneously voltage-clamped HCs, we saw no significant change in Ca2+ signal amplitude (p = 0.4); the difference between feedback and control conditions was significant . Depolarizing HCs from −79 mV to −9 mV also caused a significant positive shift in V50  at ribbons sensitive to feedback, which differed significantly from control trials without a voltage-clamped HC . The ability of HCs to regulate Ca2+ signals at some ribbons but not others within a single cone provides further evidence that ribbons can operate in a functionally independent manner. Modulation by HC feedback could increase or diminish intrinsic differences in ribbon Ca2+ responses seen in the absence of feedback.Not every cone ribbon showed feedback-induced changes in Ca2+ signals. After accounting for technical variability in Ca2+ signal measurements and contributions from cone-to-cone variability in ICa, we found intrinsic variability  of V50 values among ribbon-localized Ca2+ signals with a SD of ~2.5 mV. Ribbon V50 values were normally distributed, suggesting that 68% (±1 SD) of ribbons in a cone exhibit V50 values spanning a range of 5 mV. We found a similar degree of ribbon-to-ribbon variability using the higher affinity Ca2+ dye OGB-6F. Consistent with this range of V50 values, we also observed reproducible V50 differences of up to 3.5 mV between neighboring ribbons in the same cone  confirmed that we could accurately detect 3 mV changes in voltage dependence  would produce a difference in Ca2+ influx of more than 45%. Variation in the voltage dependence of whole-cell ICa would also produce cone-to-cone differences in Ca2+ influx. Because release from cones is linearly related to ICa . Fast release of membrane-associated vesicles, as might occur during a rapid decrement in light, is regulated by Ca2+ within highly localized nanodomains <100 nm from Ca2+ channels even in the presence of weak exogenous buffering (Mercer et al., 2+ acting at ribbon-associated sites located a few hundred nanometers above Ca2+ channels, and so sustained release is much more sensitive than fast release to Ca2+ buffering and global Ca2+ levels (Babai et al., 2+ entry would therefore diminish during sustained release that occurs under conditions of dim illumination when Ca2+ levels are elevated similarly throughout the terminal.Endogenous Ca2+ influx in cones caused by HC feedback involves extracellular proton levels within the synaptic cleft (Barnes and Bui, Ca—which is the summed currents from all the ribbons—would be less than 20% of the impact on Ca2+ responses at single ribbons. Instead, feedback-induced changes in the amplitude and voltage dependence of whole-cell ICa measured under similar experimental conditions in paired cone-HC recordings (Warren et al., HC activity is governed by glutamatergic inputs from presynaptic photoreceptors. Although there is controversy about the underlying mechanisms (Kramer and Davenport, 2+ signals at individual synaptic ribbons in cone photoreceptors can vary significantly from one another in both amplitude and voltage dependence. V50 values of ribbons within single cochlear inner hair cells varied with a SD of 3.2 mV (Frank et al., 2+ influx at individual cone ribbons can be rapidly and actively regulated by HC feedback in a non-random fashion. Intrinsic ribbon-to-ribbon differences in cones may also be actively regulated by cellular processes operating on a slow time scale. Alternatively, they may simply result from random differences in the expression and delivery of proteins to different parts of the cone. Regardless, ribbon-to-ribbon differences in Ca2+ signals expand the range of transformations available to cones for converting light-evoked voltage responses into synaptic release, and can thus expand the range of information provided to downstream neurons about changing light levels.Our results show that CaBoth authors designed experiments, conducted experiments, analyzed data and wrote the article.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.


Rabbits are a suitable animal model for atherosclerosis due to their sensitivity to dietary cholesterol. Moreover, rabbits have lipoprotein profiles that are more similar to humans than those of other laboratory animals. However, little is known about the transcriptomic information related to atherosclerosis in rabbits. We aimed to determine the changes in the livers of rabbits fed a normal chow diet (control) or high cholesterol diet (HCD) by histological examinations and RNA sequencing analysis. Compared with the control group, the lipid levels and small LDL subfractions in plasma were increased, and aortic atherosclerotic plaques were formed in the HCD group. Most importantly, HCD resulted in lipid accumulation and inflammation in the livers. Transcriptomic analysis of the liver showed that HCD induces 1183 differentially expressed genes (DEGs) that mainly participate in the regulation of inflammation and lipid metabolism. Furthermore, the signaling pathways involved in inflammation and lipid metabolism were enriched by KEGG pathway analysis. In addition, hepatic DEGs of the HCD group were further validated by real-time PCR. These results suggest that HCD causes liver lipid accumulation and inflammatory response. Although the relationships between these hepatic changes and atherogenesis need further investigation, these findings provide a fundamental framework for future research on human atherosclerosis using rabbit models. Many laboratory animal models have been used to study the process of atherogenesis, including mice, rabbits, pigs and nonhuman primates. In particular, mouse models are widely used due to the relatively short time frame for the progression of atherosclerosis and the relative ease of genetic manipulation2. While not all aspects of mouse atherosclerosis are identical to humans, several characteristics of lipid metabolism in rabbits make them particularly suitable for the study of human hypercholesterolemia and atherosclerosis. First, a large body of evidence suggests that high levels of plasma cholesterol, especially low-density lipoprotein (LDL)-cholesterol, result in atherosclerotic lesion formation in humans3. Unlike mice, in which high-density lipoproteins (HDLs) are the predominant plasma lipoproteins, rabbit lipoprotein profiles are LDL-rich, similar to those of humans5. Second, it has been reported that cholesteryl ester transfer protein (CETP) plays a central role in lipid metabolism and atherosclerotic processes6. Rabbits have abundant CETP activity in the plasma as do humans, whereas mice do not have an endogenous CETP gene8. Third, rabbits do not have hepatic ApoB mRNA editing activity as humans, so rabbit ApoB-48 is only present in chylomicrons as in humans. However, ApoB-48 is present in all ApoB-containing particles such as very low-density lipoproteins (VLDLs), LDLs and chylomicrons in mice9. In addition, hepatic lipase (HL), which plays a major role in lipoprotein metabolism, is bound to the vascular endothelium in rabbits and humans, whereas the majority of HL is circulating in the plasma in mice11. Therefore, rabbits provide a unique system to study the initiation and progression of atherosclerosis in humans. Despite the importance of rabbit models for the study of human hypercholesterolemia and atherosclerosis, little is known about the transcriptomic information related to atherosclerosis in rabbits.Atherosclerosis is a complex multifactorial disease of the aorta and muscular arteries and the leading cause of morbidity and mortality throughout the world13. We envision that investigation of the transcriptomics in rabbits fed a high cholesterol diet can provide a better understanding of the biochemical and molecular processes involved in the initiation and progression of atherosclerosis in this model.Next-generation sequencing platforms have made genomic and transcriptomic analyses affordable for researchers to elucidate the molecular mechanisms of human diseases. RNA sequencing (RNA-Seq) uses massively parallel sequencing to analyze the transcriptome at higher resolution than Sanger sequencing- and microarray-based methodsTherefore, in the present study, we conducted RNA-Seq analysis of livers in rabbits fed a high cholesterol diet coupled with analyses of the plasma lipids and lipoprotein subfractions, aortic atherosclerotic lesions and liver pathology.P < 0.01).Compared with the normal chow diet (control) group, the plasma levels of total cholesterol (TC) and triglycerides (TG) were increased by 19.4-fold and 1.7-fold in high cholesterol diet (HCD)-fed rabbits for 16 weeks, respectively  were dramatically increased in the HCD-fed rabbits . Data from the present study showed that high dietary cholesterol increased the plasma levels of TC and TG, and increased plasma TC levels were mainly caused by elevated small LDL particles in rabbits.As shown in Fig. its Fig. . It has en face lesions in the HCD group were remarkable and distributed from the aortic arch, thoracic aorta to abdominal aorta with predominance in the aortic arch  proliferation also plays a crucial role in the progression of atherosclerosis24. Immunohistochemical staining showed that in addition to macrophages, the lesions also contained many SMCs  and Elastica van Gieson (EVG) staining. We found that the intimal lesions were mainly composed of macrophage-derived foam cells, whereas no lesions were found in the control group Fig. . It is gMCs Fig. . Thus, a26. These results indicated that liver plays a key role in the inflammatory response evoked by dietary constituents. We found that there were significant steatosis, ballooning and inflammation in the HCD group compared with the control group   were rapidly increased after consumption of an excess amount of dietary cholesterol, thereby promoting the onset of early aortic lesion formationoup Fig. . In addi01) Fig. . The res01) Fig. . These r30. To further emphasize the relationship with lipoprotein profiles and aortic atherosclerosis, in the current study, we focused on the hepatic gene expression changes and liver pathology in the setting of hepatic steatosis induced by cholesterol diet in the Japanese White rabbits. The transcriptomic analysis revealed that there were 1,183 differentially expressed genes (DEGs) compared with the control rabbits. Of them, 984 genes were up-regulated and 199 genes were down-regulated compared with the control group  is likely a target for SREBPs, which has been suggested to regulate the genes involved in cholesterol biosynthesis32. Our results showed that the DHCR24 gene was down-regulated 2.1-fold in HCD-fed rabbits. Furthermore, HCD induced many genes that mediate the inflammatory response in the liver. These genes include proteases , chemokines , adhesion molecules  and cardiovascular risk factors . It is generally accepted that atherosclerosis is a multifactorial and persistent condition and is regarded as a form of chronic inflammation induced by lipid accumulation3. Other studies showed that there is a link between cholesterol and inflammation in ApoE−/− mice and ApoE*3Leiden mice fed with high cholesterol33. Taken together, these findings suggest that hepatic lipid metabolism and inflammation may be closely associated with the initiation and progression of atherosclerosis in rabbits.To gain insight into the complex traits underlying the pathophysiological response of the liver to dietary cholesterol, transcriptomic analysis was performed by RNA-Seq. In the previous study, we identified the gene mutations or modifiers through whole-genomic sequencing and deep transcriptome sequencing of LDL receptor deficient (WHHL) rabbits and cholesterol-fed New Zealand White rabbits. The previous study mainly addressed transcriptional changes in the WHHL rabbits, while cholesterol-fed New Zealand White rabbits were used as a referencever Fig. . As show35. Vergnes et al. also discovered that the hepatic inflammatory response was evoked by high cholesterol feeding in LDLR−/− mice36. These above studies suggest that the uptake of dietary cholesterol leads to the inflammatory response of the liver in rabbits.The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis provided additional possible functional information showing the pathways relevant to DEGs during atherogenesis. In concordance with our DEG results, analysis of the KEGG pathway showed that these DEGs were mainly contained in lipid metabolism- and inflammation-related signaling pathways including PI3K-Akt, MAPK, ABC transporters, FoxO, PPAR, AMPK, NF-κB, mTOR, tumor necrosis factor (TNF), Toll-like receptor and cGMP-PKG signaling pathways. Significantly, HCD activated specific inflammatory pathways, such as NF-κB, Toll-like receptor and TNF signaling pathways in rabbits Fig. . As show3. To validate the DEGs in the liver, real-time PCR was carried out. As shown in Fig. 38. These results indicate that dietary cholesterol can be an important trigger and a possible source of the inflammatory component.Both hypercholesterolemia and inflammatory response are well-known risk factors for the development of atherosclerosis and have been shown to play a causal role in the progression of atherosclerotic plaquesThe correlation was calculated to compare the expression levels between RNA-Seq and real-time PCR. Fold changes in DEGs between the two techniques were significantly correlated in the depot (Pearson’s R = 0.88) . Eventually, twenty rabbits were selected and subsequently divided into two groups: normal chow diet group and cholesterol diet group fed a 0.3% cholesterol diet. All rabbits were given a restricted diet of 100 g/day for 16 weeks. Rabbits were individually housed in metal cages in air-conditioned rooms under a 12 h light/12 h dark cycle and were given free access to water throughout the experiments. The experimental protocol was in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and was approved by the Laboratory Animal Care Committee of Xi’an Jiaotong University.Male Japanese White rabbits (2.5–3.0 kg) were provided by the Laboratory Animal Center of Xi’an Jiaotong University. In the current study, we aimed to determine the influence of hypercholesterolemia on transcriptomic change and liver pathology that may be related to atherosclerosis in rabbits. It is well known that rabbits are sensitive to dietary cholesterol and rapidly develop severe hypercholesterolemia leading to atherosclerosis41.Blood samples were collected from the ear artery of rabbits after fasting for 16 h. The plasma levels of TC and TG were measured using commercial kits . Plasma samples were also used for lipoprotein subfraction analysis. The analysis was performed electrophoretically using high-resolution 3% polyacrylamide gel tubes and the Lipoprint LDL System  according to the manufacturer’s instructions as previously described42. Then, the aortic arch was cut into 10 sections. The sections were embedded in paraffin and cut into 5-μm thick serial sections, stained with H&E and EVG. For the evaluation of cellular components in atherosclerotic lesions, the sections were immunohistochemically stained with antibodies against RAM11 (macrophage marker)  and HHF35 (SMC marker) .Rabbits were sacrificed with an injection of phenobarbital sodium and xylazine hydrochloride. The aortic tree was isolated, opened longitudinally, fixed in 10% neutral buffered formalin, and stained with Sudan IVThe livers were fixed in 10% neutral buffered formalin and processed for embedding in paraffin. Then, specimens were cut into 5-μm thick serial sections and stained with H&E. Frozen sections of liver (5-μm thick) were stained with Oil Red O for evaluation of hepatic fatty change. The Oil Red O staining area was measured using the WinROOF image analysis software .Total RNA was extracted from the liver using RNAzol . RNA concentrations and purity were determined using Nanodrop , and integrity was verified using an Agilent 2100 Bioanalyzer . mRNA was purified from total RNA using the NEBNext® Poly(A) mRNA magnetic isolation module. A total of 6 paired-end libraries were constructed using a NEBNext UltraTM RNA Library Prep Kit following Illumina manufacturer’s recommendation. Library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumina). The libraries were sequenced on an Illumina HiSeq. 2500 platform with 100 bp paired-end reads .43. The DEGs were analyzed using DESeq software by comparing the control and HCD groups44. The significance threshold of the p-value in multiple tests was set by the false discovery rate (FDR). The threshold, the absolute values of fold change ≥ 2 and FDR < 0.05, was applied to judge the significance of gene expression. For the functional and pathway enrichment analysis, the DEGs were then mapped into the KEGG databases, and significantly enriched KEGG terms were determined by P ≤ 0.05.The Perl script was used to trim the reads with contaminated adapters, more than 20% low-quality bases , and more than 10% Ns. First, we obtained clean reads (Supplementary Table −ΔΔCT). Data were normalized to rabbit GAPDH mRNA levels.Real-time PCR was performed essentially to validate and confirm the differences in gene expression between the HCD group and the control group. Total RNA was extracted from the liver using RNAzol. cDNAs were synthesized using a RevertAid First Strand cDNA Synthesis Kit  following the manufacturer’s instructions. Real-time PCR was performed with the SYBR Green PCR master mix  using a thermal cycler dice real time system. Primers were designed with primer premier 5.0  (Supplementary Table P < 0.05.Data are presented as the mean ± SEM. Statistical analysis was performed by the two-tailed Student’s t-test using GraphPad Prism software. Differences between groups were considered statistically significant if Table S1Table S2Table S3Table S4Table S5


Extracellular ATP, together with the cognate receptors on the cell surface, ligand-gated ion channel P2X receptors and a subset of G-protein-coupled P2Y receptors, represents common autocrine and/or paracrine Ca2+ signalling mechanisms. The P2X receptor ion channels mediate extracellular Ca2+ influx, whereas stimulation of the P2Y receptors triggers intracellular Ca2+ release from the endoplasmic reticulum (ER), and activation of both type of receptors thus can elevate the cytosolic Ca2+ concentration ([Ca2+]c), albeit with different kinetics and capacity. Reduction in the ER Ca2+ level following the P2Y receptor activation can further induce store-operated Ca2+ entry as a distinct Ca2+ influx pathway that contributes in ATP-induced increase in the [Ca2+]c. Mesenchymal stem cells (MSC) are a group of multipotent stem cells that grow from adult tissues and hold promising applications in tissue engineering and cell-based therapies treating a great and diverse number of diseases. There is increasing evidence to show constitutive or evoked ATP release from stem cells themselves or mature cells in the close vicinity. In this review, we discuss the mechanisms for ATP release and clearance, the receptors and ion channels participating in ATP-induced Ca2+ signalling and the roles of such signalling mechanisms in mediating ATP-induced regulation of MSC migration.The ability of cells to migrate to the destined tissues or lesions is crucial for physiological processes from tissue morphogenesis, homeostasis and immune responses, and also for stem cell-based regenerative medicines. Cytosolic Ca There is increasing evidence to suggest that ATP-induced purinergic signalling gives rise to significant effects on stem and progenitor cell proliferation, migration and differentiation under in vitro and in vivo conditions c in NSC/NPC derived from embryonic striatum provided the first clue that stem cells can release ATP c with a half-maximum concentration (EC50) of approximately 20 μM c in human DP-MSC. The maximal Ca2+ response amplitude induced by BzATP was greater than that induced by ATP. Furthermore, BzATP/ATP-induced Ca2+ responses were attenuated by treatment with P2X7-specific siRNA [2+ signalling in human MSC. Another recent study shows mRNA and protein expression of P2X7 receptor in rat BM-MSC [2+ signalling in rat AT-MSC [2+]c, but ATP-induced increase in the [Ca2+]c was not affected by treatment with AZ10606120 [2+]c. The exact reason for such discrepancy in terms of the P2X7 receptor expression in rat BM-MSC and AT-MSC preparations remains unclear.As summarized in Table n BM-MSC , 78, 108n BM-MSC , PDL-MSCn BM-MSC  and DP-Mn BM-MSC . The susith KN62 , a humanith KN62 –119. Botnhibitor . In humaly 20 μM . ATP-indic siRNA . Collectt BM-MSC . Moreovet BM-MSC . Howevert AT-MSC . In this10606120 , a P2X7 1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14. While all the P2X receptors are exclusively activated by ATP, the P2Y receptors are activated by various nucleotides with a highly different potency or sensitivity. For example, the human P2Y receptors have the following agonist profile: P2Y1 (ADP > ATP), P2Y2 (UTP ≥ ATP), P2Y4 (UTP), P2Y6 (UDP > UTP), P2Y11 (ATP > UTP), P2Y12 (ADP > ATP), P2Y13 (ADP > ATP) and P2Y14 (UDP~UDP-glucose) c via triggering Ca2+ release from intracellular stores  and promote generation of cyclic adenosine monophosphate (cAMP) via association with the Gα,s. By contrast, the P2Y12-P2Y14 receptors  . The thiceptors; ) are linceptors; ) are cou1, P2Y2 and P2Y11 receptors represent the major ATP-sensitive human P2Y receptors that are coupled to the Gα,q/11-PLC-IP3R signalling pathway triggering intracellular Ca2+ release. Therefore, our discussion below is confined to the studies examining expression of these three receptors and their role in ATP-induced Ca2+ signalling. Two recent reviews provide further details regarding expression of the P2Y receptors and function in MSC [2+ responses to ATP in extracellular Ca2+-containing solutions. The initial transient increase in the [Ca2+]c largely remained in extracellular Ca2+-free solutions, indicating ATP-induced Ca2+ release from intracellular stores. Such observations provide unambiguous evidence to show the expression of ATP-sensitive P2Y receptors that are coupled to the Gα,q/11-PLC-IP3R signalling pathway. An early study by Kawano et al. showed that the spontaneous Ca2+ oscillations were prevented in a large proportion of human BM-MSC by treatment with BzATP, adenosine 3′-phosphate 5′-phosphosulfate or pyridoxal phosphate-6-azo  (PPADS). These observations led to the proposal that activation of the P2Y1 receptor is an essential step in an autocrine/paracrine feedback mechanism sustaining the spontaneous Ca2+ oscillations, namely, activation of the P2Y1 receptor gives rise to sequential activation of the Gα,q/11-PLC-IP3R signaling pathway, IP3R-mediated Ca2+ release from the ER, and induction of the store-operated Ca2+ entry (discussed below in more detail), and an increase in the [Ca2+]c triggers ATP release through the Cx hemi-channels c in these cells [2+]c in human AT-MSC, and expression of the P2Y1 receptor in these cells was detected at both mRNA and protein levels, using RT-PCR and western blotting, respectively [1 receptor was involved in ATP-induced Ca2+ signalling in human AT-MSC [2+]c and the Ca2+ responses induced by both ADP and ATP were reduced by siRNA-mediated knockdown of the P2Y1 receptor expression, indicating the expression of functional P2Y1 receptor and its contribution in ATP-induced Ca2+ signaling c were induced by ATP and UTP, but not ADP and UDP, in both extracellular Ca2+-containing and Ca2+-free solutions. Such an agonist profile is most consistent with the expression of functional P2Y2 receptor in rat BM-MSC c in the extracellular Ca2+-containing solutions, and both the transient and sustained components were prohibited by treatment with NF157, a P2Y11 receptor selective antagonist, or siRNA-mediated genetic depletion of the P2Y11 receptor expression [2+]c is mediated by the P2Y11-Gα,s-AC-cAMP signalling pathway that leads to extracellular Ca2+ influx and intracellular Ca2+ release mediated by the L-type voltage-gated Ca2+ channel in the plasma membrane and the ryanodine receptor in the ER, respectively c in the extracellular Ca2+-free solutions, or in the extracellular Ca2+-containing solutions in the presence of the P2X7 receptor inhibitor KN62 [11 receptor. Knockdown with siRNA of the P2Y11 receptor expression reduced ATP-induced increase in the [Ca2+]c, supporting a significant role for the P2Y11 receptor in mediating ATP-induced Ca2+ signalling c in the extracellular Ca2+-containing solutions, providing strong evidence to show the store-operated Ca2+ entry to be important part of ATP-induced Ca2+ signalling mechanism. Furthermore, we have documented the mRNA transcripts for Orai1, Stim1 and Stim2 in human DP-MSC by RT-PCR. Knockdown using siRNA of the expression of Orai1 or Stim1, but Stim2, reduced TG-induced store-operated Ca2+ entry and also ATP-induced increase in the [Ca2+]c in the extracellular Ca2+-containing solutions [2+ entry as part of ATP-induced Ca2+ signalling mechanism in human DP-MSC (Fig. It has been proposed that the store-operated CaSC Table . As ment release , 138. Inm the ER . Howeverolutions . These r2 receptors [2+ signalling mechanism give rise considerable effects on stem and progenitor cell migration [Studies have shown that extracellular ATP induces or stimulates the migration of mature cells, for example, epithelial cells , 140 andeceptors , 146–151eceptors , 87, 88 igration , 91, 15211 receptor selective antagonist, and also by treatment with 2′,3′-dideoxyadenosine, an AC inhibitor [11-Gα,s-AC-cAMP signalling pathway in stimulation of cell migration by β-NAD. In a recent study, we have shown using time-lapse imaging in combination with the scratch-induced wound healing assay that ATP facilitated human DP-MSC migration [1 or P2Y11 receptor. Such ATP-induced increase in cell migration was also suppressed by treatment with 2-APB at a concentration that preferentially inhibits the store-operated Ca2+ entry and, more specifically, by siRNA-mediated reduction of the expression of Orai1, Stim1 or both [2+ signalling mechanisms discussed above, namely, the P2X7, P2Y1 and P2Y11 receptors and the Orai1/Stim1 channel, participate in ATP-induced stimulation of human DP-MSC migration (Fig. Evidence is also emerging to support that extracellular ATP regulates human MSC migration under in vitro conditions and also their homing capability in vivo , 60, 109It is worth making a special note of the study by Ferrari et al. that showed that pretreatment with ATP significantly improved the homing ability of human BMS-MSC after they were planted into immunocompromised mice . Such a 2+ signalling in human MSC. Evidence has emerged to show an important role for such Ca2+ signalling mechanisms in extracellular ATP-induced regulation of MSC migration, but more studies are clearly required to provide a more detailed or mechanistic insight. As introduced above, cell migrates in a complex but well-orchestrated process that is often described in three major steps, establishment of a rear-to-front polarity, protrusion and formation of focal adhesions at the front or leading edge, and retraction from the rear edge [2+ signalling regulates polarization [2+ signalling. As briefly mentioned above, it is well established that PKC, MAPK and other Ca2+-dependent signalling proteins are important regulators of the cytoskeletal proteins coordinating cell migration [2+ activates or modulates these signalling molecules in MSC. For example, internal Ca2+ release triggered by activation of the Gα,q-PLC-IP3R signalling pathway activates the PKC-ERK1/2 signalling pathway in ACh-induced rat BM-MSC migration [2+-dependent signalling pathways are involved in ATP-induced regulation of MSC migration. Finally, the cell migration or the homing capacity of in vitro expanded MSC cultures to the lesion sites is limited but critical for development of regenerative medicines, particularly cell-based therapy. Evidently, more in vivo studies are required to examine whether the improved understanding of the Ca2+ signalling mechanisms underlying ATP-induced regulation of cell migration can be harnessed to improve the low or poor homing capacity of MSC and thereby the efficacy of promising applications of MSC-based tissue engineering and therapies.Recent studies have made, as discussed above, significant advances in understanding the molecular mechanisms underlying ATP-induced Caear edge . There irization , 70 and rization . Howeverigration , 7–9. Stigration . There iigration  or the pigration . Further


Both are present in the bone marrow of patients with bone marrow disorders, but thus far there are no studies that assess the capability of p,p′-DDE to affect myeloid cells. The aim of this study was to determine the effect of p,p′-DDE on promyelocytic cell differentiation and intracellular pathways related to this event. p,p′-DDE induced morphological changes compatible with promyelocytic differentiation in a concentration-dependent manner. The p,p′-DDE effect on [Ca2+]i, C/EBPβ protein levels, PKCα and p38 activation, and the role of oxidative stress or PLA2 was assayed. Exposure to 1.9 μg/mL of p,p′-DDE increased [Ca2+]i, PKCα, p38, and C/EBPβ protein levels; the increase of nuclear C/EBPβ protein was dependent on p38. PKCα phosphorylation was dependent on PLA2 and p,p′-DDE-induced oxidative stress. p38 phosphorylation induced by p,p′-DDE was dependent on PLA2, PKC activation, and oxidative stress. These effects of p,p′-DDE at concentrations found in human bone marrow may induce alterations in immature myeloid cells and could affect their cellular homeostasis. In order to establish the risk from exposure to p,p′-DDE on the development of bone marrow disorders in humans, these effects deserve further study.Dichlorodiphenyldichloroethylene ( Twelve hours before harvesting, 0.5 μCi/well of [3H]-thymidine (Amersham Life Science) was added, and cells were harvested using an automatic Skatron combi cell harvester  onto fiberglass filters. The incorporation of [3H]-thymidine was determined using a beta-plate scintillation counter . Three independent experiments were performed in triplicate.Cell proliferation was measured by a i was measured by fluorescence-activated cell sorting (FACS) using Fluo-3-loaded cells . HL-60 cells were resuspended to a density of 2.5 × 105/mL in PBS containing 13 μM Fluo-3AM and 8% pluronic acid F-127  and were incubated for 25 min at 37°C. For treatments with BAPTA-AM , cells were incubated for 30 min with the Ca2+ chelator before incubation with Fluo-3AM. Extracellular Fluo-3AM was removed by washing the cells twice. Finally, the cells were adjusted to a density of 6.25 × 104/mL and resuspended in Ca2+-free buffer to evaluate the role of [Ca2+]i. Ca2+ signal was measured with the flow cytometer for 20 seconds to establish a baseline Ca2+ level. Subsequently, cells were exposed to different experimental conditions, and the Ca2+ signal was measured for up to 3 min. Ionomycin (0.1 μg/μL) and phorbol myristate acetate (PMA) (10 nM)  were used to verify the specificity of the signal. Assessment of [Ca2+]i was performed at room temperature by flow cytometry  using the CellQuestPro software (BD Bioscience) and analysed with FlowJo v7/8 .i was evaluated. At 1.9 μg/mL of p,p′-DDE and in absence of extracellular Ca2+, [Ca2+]i increased twofold over baseline , a transient decrease in cells seems to occur at 96 h; however cultures compensated the effect and reached the steady state after this time. In fact, because results did not show an effect of 1.9 μg/mL p,p′DDE on proliferation at 96 h, we considered that the effect observed in MTT assays at this time is a consequence of a temporal decrease of metabolic capability of cells to reduce of MTT instead of cell death. Morphological changes in promyelocytic HL-60 cells were observed after a five-day exposure to 0.19 and 1.9 μg/mL of p,p′-DDE, suggesting that noncytotoxic concentrations of p,p′-DDE induce incomplete differentiation. These observations agree with the ability of p,p′-DDT (at 20 μM) to induce differentiation in adipocytes 3T3-L1 and preadipocytes 3T-F442A i. Our results demonstrate that exposure to 1.9 μg/mL of p,p′-DDE raises the [Ca2+]i level, which is known to occur in other cell types as well [2+]i chelation partially, but not significantly, inhibited p,p′-DDE-induced PKCα activation. Instead, our data revealed that the activation of PKCα induced by p,p′-DDE is ROS-dependent. Previous studies have reported that p,p′-DDE is capable of inducing ROS in blood mononuclear cells at concentrations of 60 and 80 μg/mL [α, like PLA2 [α activation induced by p,p′-DDE was also PLA2-dependent.To determine the pathway through which as well , 60–62. 80 μg/mL . Becauseike PLA2 , 65, the trans-nonachlor, induced PLA2 and PKCα activity in HL-60 promyelocytes, human THP-1 monocytes, and murine J774A.1 macrophages [ p,p′-DDE since inhibition of PLA2 prevents PKCα activation which interferes with the p38 phosphorylation induced by p,p′-DDE. This situation is analogous to that described in monocytes [It has recently been reported that other organochlorine pesticides, such as Aldrin androphages . It is ionocytes . p,p′-DDE exposure in the concentration range found in human bone marrow [α activation, whereas ROS and PKCα induction are the most important stimuli for p38 activation and consequently increase C/EBPβ protein levels in the nucleus of HL-60 cells exposed to p,p′-DDE ( p,p′-DDE in human bone marrow and suggest that exposure to this pesticide could potentially lead to the development of bone marrow disorders in human. Further studies are necessary to conclusively support this hypothesis.In summary, this study supports the fact thate marrow –7 is capp,p′-DDE . These f


ATP channels, and by a multi-cellular computational model of islet electrical activity. Here we examined the mechanisms by which gap junction coupling contributes to islet dysfunction in NDM. We first verified the computational model against [Ca2+] and insulin secretion measurements in islets expressing ATP-insensitive KATP channels under different levels of gap junction coupling. We then applied this model to predict how different KATP channel mutations found in NDM suppress [Ca2+], and the role of gap junction coupling in this suppression. We further extended the model to account for stochastic noise and insulin secretion dynamics. We found experimentally and in the islet model that reductions in gap junction coupling allow progressively greater glucose-stimulated [Ca2+] and insulin secretion following expression of ATP-insensitive KATP channels. The model demonstrated good correspondence between suppression of [Ca2+] and clinical presentation of different NDM mutations. Significant recoveries in [Ca2+] and insulin secretion were predicted for many mutations upon reductions in gap junction coupling, where stochastic noise played a significant role in the recoveries. These findings provide new understanding how the islet functions as a multicellular system and for the role of gap junction channels in exacerbating the effects of decreased cellular excitability. They further suggest novel therapeutic options for NDM and other monogenic forms of diabetes.Diabetes is caused by dysfunction to β-cells in the islets of Langerhans, disrupting insulin secretion and glucose homeostasis. Gap junction-mediated electrical coupling between β-cells in the islet plays a major role in coordinating a pulsatile secretory response at elevated glucose and suppressing insulin secretion at basal glucose. Previously, we demonstrated that a critical number of inexcitable cells can rapidly suppress the overall islet response, as a result of gap junction coupling. This was demonstrated in a murine model of Neonatal Diabetes Mellitus (NDM) involving expression of ATP-insensitive K Diabetes is a disease reaching a global epidemic, which results from dysfunction to the islets of Langerhans in the pancreas and their ability to secrete the hormone insulin to regulate glucose homeostasis. Islets are multicellular structures that show extensive coupling between heterogeneous cellular units; and central to the causes of diabetes is a dysfunction to these cellular units and their interactions. Understanding the inter-relationship between structure and function is challenging in biological systems, but is crucial to the cause of disease and discovering therapeutic targets. With the goal of further characterizing the islet of Langerhans and its excitable behavior, we examined the role of important channels in the islet where dysfunction is linked to or causes diabetes. Advances in our ability to computationally model perturbations in physiological systems has allowed for the testing of hypothesis quickly, in systems that are not experimentally accessible. Using an experimentally validated model and modeling human mutations, we discover that monogenic forms of diabetes may be remedied by a reduction in electrical coupling between cells; either alone or in conjunction with pharmacological intervention. Knowledge of biological systems in general is also helped by these findings, in that small changes to cellular elements may lead to major disruptions in the overall system. This may then be overcome by allowing the system components to function independently in the presence of dysfunction to individual cells. Multi-cellular biological systems are composed of cellular elements with distinct characteristics, which function collectively as a result of dynamic interactions. While the function of a multicellular system is dependent on the characteristics of its constituent cells, understanding such systems is complicated by the action of cellular coupling and system architecture. Furthermore, cellular heterogeneity and noise complicate assessment of the function of individual cells. As a result, changes in the behavior of individual cells can often lead to unexpected changes in the system behavior. Many diseases, both acute and chronic, arise through genetic variations that impact molecular and cellular function. Given the complexities of multi-cellular systems, effectively predicting how molecular and cellular dysfunction lead to tissue and organ dysfunction and cause disease is challenging. One approach to describe dynamic multicellular systems is using network theory, which distinguishes network structure and cellular behavior to understand how distinct functions can emerge from coupled systems ,2.ATP) channels. The resulting membrane depolarization activates bursts of action potentials and elevates intracellular free-calcium activity ([Ca2+]) in the form of oscillations which triggers pulses of insulin granule exocytosis [2+] oscillations are coordinated under elevated glucose and uniformly silent under basal glucose  and insulin secretion was observed. This also prevented hyperglycemia and diabetes as a result of ATP-insensitive mutant KATP expression [2+] and insulin release caused by mutations in KCNJ11 and ABCC8. Thus a reduction of gap junction coupling in the islet may recover islet function and blunt diabetes caused by mutations in KCNJ11 and ABCC8.Our prior studies have shown that as a result of electrical coupling, small changes in β-cell excitability (e.g. upon glucose stimulation) leads to a rapid change in global islet behavior . Similarhe islet . Specifiogeneity . Such beogeneity ,31, and ogeneity ,32, wherpression . These f2+] [2+] oscillations in uncoupled β-cells, but negligible effect in the presence of coupling [2+]  ,42. Ion 2+] ,43. Prevcoupling . In the ing [2+] , and mayATP channel expression in the presence of different levels of gap junction coupling. Using this computational model, we then examined the role of gap junction coupling in exacerbating islet dysfunction in the presence of specific KCNJ11 and ABCC8 that cause NDM. We further examined the degree to which islet dysfunction can be overcome by reducing gap junction coupling, and the relative role of cellular heterogeneity and stochastic channel noise in mediating this.In this study, we compared a computational model of islet electrical activity and insulin secretion against experimental measurements upon varied mutant K under inducible CreER control well-models human NDM  expression we imaged [Ca2+] in islets of mice expressing variable levels of Kir6.2, as indicated by co-expression of GFP, for reduced levels of Cx36 . In the presence of normal gap junction coupling (Cx36+/+) a sharp transition was observed between high [Ca2+] (many cells showing elevations) and low [Ca2+] (few cells showing elevations) as Kir6.2 expression increased (+/-) the decline was less sharp, and right-shifted to higher Kir6.2 expression (-/-) the decline in [Ca2+] was gradual  was represented by 120pS; Cx36+/- (~45% coupling) was represented by 50pS; and Cx36-/- (~0% coupling) was represented by 0pS  expression near silent behavior was observed with normal levels of gap junction coupling, and a progressive increase in [Ca2+] occurred as gap junction coupling was reduced.Previously, a computational model of islet electrical activity showed good agreement with experimental measurements of -induced suppression of [Ca2+] was linked to altered insulin secretion and glucose homoeostasis  Kir6.2 expression little decrease in [Ca2+] was observed for all Cx36 levels  or absent Cx36 (Cx36-/-) compared to normal Cx36 levels (Cx36+/+)  .2+] measurements  Kir6.2 expression there was no reduction in insulin secretion and plasma insulin for Cx36-/-, compared to large reductions for Cx36+/+  Kir6.2 expression euglycemia was observed for Cx36-/- but substantial hyperglycemia was observed for Cx36+/+ and Cx36+/-, albeit slightly less for Cx36+/-. Regression analysis showed similar strong variations in insulin secretion, plasma insulin levels and blood glucose with increasing Kir6.2 expression for Cx36+/+ and Cx36+/-, but less or absent variations for Cx36-/-  and uniform KATP activation   . A sharpATP channels resulting from mutations to KCNJ11 and ABCC8 that cause NDM. We simulated how altered ATP-inhibition kinetics  [ATP inhibition (k’1/2) [2+] upon increasing residual current (α) is equivalent to that in k’1/2) there was also a decline in [Ca2+]  ,22 and a (k’1/2) –21,48–56n [Ca2+] . In the occurred . In the coupling . In the p to ~10 . The  in the absence of electrical coupling  increases for a number of NDM mutations. We characterized how changes in both p’0 and k’1/2 act in combination, in the presence and absence of electrical coupling  is sometimes reported. Increasing H did not affect the dependence of the transition on electrical coupling  within the range of cellular ATP levels, which are substantially higher than the half maximal ATP concentration for KATP inhibition.Other parameters characterizing changes to Kcoupling . Increascoupling . Upon hicoupling . At highcoupling . A changcoupling , althougATP over-activity is increased in the islet  that indicates the level of islet dysfunction associated with each mutation. In each mutation we further tested whether a recovery in [Ca2+] could be achieved by eliminating electrical coupling. All mutations in which ATP-sensitivity is characterized .When including reported coupling , S3 Fig.αest values were included, most NDM mutations showed complete suppression of [Ca2+]  and those with DEND features (F132L) (α) is important to describe the action of KCNJ11 and ABCC8 mutations; and in many cases a recovery in islet function is predicted following a modulation of gap junction electrical coupling.When f [Ca2+] , S4 Fig,ATP function and testing the effects of stochastic channel noise at basal and stimulatory glucose. In the presence of electrical coupling, including noise resulted in no change in [Ca2+] at basal and elevated glucose , those mutations that showed recoveries in [Ca2+] , α , k’1/2 (ATP concentration at 50% KATP inhibition), p’0 (open channel probability), the transition in the presence of coupling occurs where 20–40% of cells are inactive in the absence of coupling.Through computational modeling we find similar sharp transitions to global suppression when parameters that describe the kinetics of Klet Figs  and 4. Piazoxide . These r expression there was more variability between Cx36+/- animals compared to Cx36+/+ animals  and clinical diabetes severity (discussed below). However, determining whether phase transitions occur upon increasing KATP over-activity in human islets and their dependence on gap junction coupling, as well as testing whether a human β-cell based islet model can recapitulate these findings is needed.Studies here have only considered mouse islets. However there are some differences between mouse and human islets that need considering to fully translate these findings to human islet function. The architecture of human islets is different from that of mouse islets –64, and 2+] dynamics and increase cell death in interconnected cells [2+] dysregulation and reduces cell death  and insulin upon elevated Kir6.2 expression, and its recovery upon reduced gap junction coupling  hormone action, which would elevate Caecretion . Incretiimulated ; but ampimulated ,79. We pα, which can explain this discrepancy. The kinetics of sulfonylurea inhibition of ATP-insensitive KATP channels is complex and not fully understood  and insulin secretion in electrically isolated cells. While stochastic noise affects β-cell [Ca2+] oscillations [2+] has not been examined significantly. We demonstrated noise can affect [Ca2+] levels  and Institutional Animal Care and Use Committee (IACUC).); β-cell specific, inducible Cre (Pdx-CreER); and Connexin36 knockout (Cx36-/-) mouse models have been previously described  mice were crossed with Cx36 knockout mice to yield all combinations of mice studied. Daily injections of tamoxifen  administered IP in 8–16 week old mice induced the variable Kir6.2 expression. Mice lacking Pdx-CreER or Rosa26- Kir6.2 were used as 0% expression controls.The Kir6.2 subunit mutation with GFP tag . Reported levels were averaged over days 27–29 after tamoxifen induction. Plasma insulin was measured at day 29 after tamoxifen induction from blood samples, centrifuged for 15 minutes at 13,900rpm, then assayed using mouse ultrasensitive insulin ELISA (Alpco).2 for 24–48 hours prior to study.Islets were isolated from mice under Ketamine/Xylazine anesthesia by collagenase injection through the pancreatic duct, and animals euthanized via exsanguination and cervical dislocation. Islets were handpicked after the pancreas was harvested and digested, and were maintained in RPMI medium at 11mM glucose plus 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, at 37°C under humidified 5% CO3, 5.8mM KCl, 1.2mM KH2PO4, 2.5mM CaCl2, 1.2mM MgSO4, 10mM HEPES, 0.1% BSA, pH 7.4), and then for 60 minutes at 2mM or 20mM glucose. The medium was sampled for secretion, and islets sampled for content by lysing in 1% TritonX-100 and frozen overnight at -20C. Samples were assayed using mouse ultrasensitive ELISA.Islets  were incubated first at 2mM glucose in Krebs-Ringer Buffer  for 45 minutes at room temperature, and were held in polymdimethylsiloxane PDMS microfluidic devices  in rodent islets was modeled as previously and in accordance with experimental data  -expressing cells. Simulations for Pmut were run with glucose elevated to 20mM.Expression of Kir6.2tal data ,27, by mpoK(ATP) in all cells according to:α is variable and increases with diazoxide treatment concentration. Simulations for α were run with glucose elevated to 11mM and Pmut = 100%.Diazoxide application was modeled as previously  by modifKCNJ11 and ABCC8 mutations were modeled by modifying the open probability poK(ATP) in all cells according to:k’1/2 represents the relative increase in half maximal ATP concentration, and H is the Hill coefficient. Unless noted H = 1 and was not varied when modeling mutations in order to maintain consistency. p’0 represents the relative increase/decrease in open channel conductance. α represents the fraction of current remaining at saturating ATP concentrations, equivalent to α in α values (αest) for a mutation, data reporting the fractional KATP current remaining at 3mM ATP  dependence to give a first phase release of ~20 granules/min per β-cell, and second phase rate of ~5 granules/min per β-cell , a quiescent reference cell was selected manually from an area where no significant intensity fluctuations occurred over the duration of the experiment. The variance for each pixel time-course of the image was calculated to examine which pixels displayed the greatest fluctuations in time. The variance of the manually selected quiescent reference cell was used to generate a threshold to compare intensity fluctuations of all other pixels: any pixels having a time-course variance > 2 standard deviations above that of the quiescent reference cell were counted as ‘active’. Time courses with significant motion artifacts were excluded from analysis, and photobleaching was handled by applying a linear fit. The percentage of cells active is calculated based on the number of pixels calculated to be ‘active’, normalized to the number of pixels in the area of the islet. To calculate GFP+ regions, the mean fluorescence intensity was calculated in GFP- control islets cells and used to generate an intensity threshold: any pixel having a GFP intensity greater than this threshold was considered GFP+. GFP+ area was expressed as a % of the total islet area. In islets where lack of nuclear GFP was observed, nuclear areas were manually included in the GFP+ area. For each level of Cx36 expression, the average activity over all control islets was used to normalize the maximum activity of simulation fits.Custom MATLAB scripts were used to analyze all images acquired from calcium imaging . First, 2+], a threshold of 0.165μM was applied, and cells showing [Ca2+] fluctuations that exceeded this threshold were considered active. The [Ca2+] duty cycle for each cell was represented by the fraction of time the [Ca2+] time course exceeded this threshold. The duty cycle was reported as the mean duty cycle over all cells in the islet, where a silent cell has a duty cycle of 0. The same analysis methods were used when noise was added to the model.All analysis was performed with custom MATLAB routines on the output for each simulation. For all time courses, the first ~200 time points were excluded. To calculate the fraction of cells showing elevated [CaThe total number of granules secreted was calculated according to 2+] imaging results; or insulin secretion, plasma insulin and blood glucose results. Linear regression between [Ca2+] imaging results, insulin secretion, plasma insulin and blood glucose against % GFP was grouped across Cx36+/+,Cx36+/-,Cx36-/- conditions. All statistical analysis was performed in Prism (Graphpad).Student’s t-test was utilized to test for significant differences between simulation results;  elevations, verses % GFP expression , for islets isolated from Cx36+/+ mice or measured from Cx36+/+ mice. B). As in A for Cx36+/- mice. C). As in A for Cx36-/- mice. Linear regression ± 95% CI indicated by grey lines.(PDF)Click here for additional data file.S2 FigA). Islet activity for simulations at the indicated gcoup, as represented by the fraction of cells showing significant [Ca2+] elevations, plotted against intrinsic cellular activity, as indicated by fraction of cells showing significant [Ca2+] elevations for simulations at gcoup = 0pS. Displayed are results from simulations with increasing k’1/2 at 11mM glucose, Pmut = 1. B). As in A for results from simulations with increasing α at 11mM glucose, Pmut = 1. C). As in A for results from simulations with increasing Pmut at 20mM glucose.(PDF)Click here for additional data file.S3 FigA). Fraction of cells showing significant [Ca2+] elevations for simulations that include mutant KATP channel activity, for gcoup = 120pS (black) and gcoup = 0pS (red). Simulations include all characterized mutations indicated where reported k’1/2 changes and reported α (if any) are accounted for. B). Mean [Ca2+] duty cycle for simulations that include mutant KATP channel activity as in A. All simulations were run at 11mM glucose, Pmut = 1. Results are arranged in order of clinical severity, with the clinical classification indicated: T2D- Click here for additional data file.S4 FigA). Fraction of cells showing significant [Ca2+] elevations for simulations that include mutant KATP channel activity, for gcoup = 120pS (black) and gcoup = 0pS (red). Simulations include all characterized mutations indicated where reported k’1/2 changes and estimated α (see B). Mean [Ca2+] duty cycle for simulations that include mutant KATP channel activity as in A. All simulations were run at 11mM glucose, Pmut = 1. Results are arranged in order of clinical severity, with the clinical classification indicated: T2D- Click here for additional data file.S5 FigA). Fraction of cells showing significant [Ca2+] elevations for simulations that include mutant KATP channel activity, with stochastic noise, for left: gcoup = 120pS (black) and gcoup = 0pS (red), and right: gcoup = 120pS (black) and gcoup = 10pS (green). Simulations include the characterized mutations indicated, where reported k’1/2 changes and estimated α (see B). Mean [Ca2+] duty cycle for simulations that include mutant KATP channel activity, with stochastic noise, as in A. All simulations were run at 11mM glucose, Pmut = 1. Results are arranged in order of clinical severity. * indicates mutations where sulfonylurea therapy is reported to be ineffective. Data is presented as mean±s.e.m. for n = 3 simulations with different random number seeds.ed α see  are acco(PDF)Click here for additional data file.S6 FigA). Insulin secretion following step increase in [Ca2+] at t = 5 min, showing biphasic response. B). Insulin secretion following step increase in glucose at t = 5 min, showing biphasic response.(PDF)Click here for additional data file.S7 FigA). Time-averaged insulin secretion over first phase of secretion (t = 0-5min.) for simulations that include mutant KATP channel activity for gcoup = 120pS and gcoup = 0pS, with stochastic channel noise. Simulations include the characterized mutations indicated where reported k’1/2 changes and reported α (if any) are accounted for. B). Time-averaged insulin secretion over second phase of secretion (t = 5-30min.) for simulation conditions in A. C). Time-course of the population of insulin granule pools averaged over a representative simulated WT islet with gcoup = 120pS and stochastic noise included, corresponding to data presented in D). Time-course of the population of different insulin granule pools, as in C, for a simulated islet that includes I296L mutation for gcoup = 120pS (black) and gcoup = 0pS (red), corresponding to data presented in Pmut = 1. Data in A,B is presented as mean for n = 2 simulations.(PDF)Click here for additional data file.S8 FigA). Representative [Ca2+] time courses for simulations upon reduced p’o for gcoup = 0pS with stochastic channel noise. B). As in A for simulations that include mutant KATP channel activity. C). Representative time-courses of insulin secretion averaged across the simulated islet for simulations in A, with normal and reduced p’o. D). As in C for simulations in B, with normal and reduced p’o. All simulations were run at 11mM glucose, Pmut = 1.(PDF)Click here for additional data file.S9 FigA). Fraction of cells showing significant [Ca2+] elevations for simulations at 5.5mM glucose with gcoup = 0pS, without noise and for 10 different simulations with stochastic channel noise. B). [Ca2+] duty cycle for simulations in A.(PDF)Click here for additional data file.S1 FileThe ‘read_me.doc’ file included explains how the source code files were used.(ZIP)Click here for additional data file.