The response is due to stimulation of the phospholipase C/Ca2+ pathways, but the direct mechanism responsible for the rise of intracellular Ca2+ concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca2+ signaling in frog parathyroid cells and show that Ca2+-activated Cl− channels are activated by intracellular Ca2+ increase through an inositol 1,4,5-trisphophate (IP3)-independent pathway. High extracellular Ca2+ induced an outwardly-rectifying conductance in a dose-dependent manner (EC50∼6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca2+-induced and Ca2+ dialysis-induced currents reversed at the equilibrium potential of Cl− and were inhibited by niflumic acid (a specific blocker of Ca2+-activated Cl− channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca2+-induced current, suggesting the change of intracellular Cl− concentration in a few minutes. Extracellular Ca2+-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca2+-induced current. IP3 dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG), arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE) dialysis increased the conductance identical to extracellular Ca2+-induced conductance. These results indicate that high extracellular Ca2+ raises intracellular Ca2+ concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl− conductance.Elevation of extracellular Ca On the other hand, [Ca2+]o regulates the secretion of PTH from parathyroid cells through an extracellular Ca2+-sensing receptor (CaR) 2+]o inhibits the secretion, whereas low [Ca2+]o enhances the secretion. It is believed that extracellular Ca2+ binds to CaR, and as a consequence inhibits the secretion of PTH via intracellular free Ca2+ concentration ([Ca2+]i). However, the molecular mechanism by which [Ca2+]i regulates the secretion is not well elucidated.Parathyroid hormone (PTH) regulates extracellular free Ca2+ and other CaR agonists. The CaR controls various signaling pathways 3) and diacylglycerol (DAG) and promoting rapid release of Ca2+ from its intracellular stores 2 (PLA2) and D by high [Ca2+]o are probably indirect, through the mediation of PLC-dependent activation of protein kinase C The CaR belongs to the family C of G protein-coupled receptors (GPCRs) and has a large extracellular domain that binds external CaDAG can be utilized for 2-arachidonoylglycerol (2-AG) generation 2 (cPLA2) The mitogen-activated protein kinase (MAP kinase) pathways are found in bovine parathyroid cells 2+]o+ channels. Other studies suggested the presence of voltage-gated Ca2+ channels in bovine, goat and human parathyroid cells 2+ channels, and that TRPC ion channels associated with Orai1 and STIM1 may increase intracellular Ca2+ concentration in the cells + channels in contrast to mammalian cells The electrophysiological studies using classical intracellular microelectrodes indicated that rodent parathyroid cells display a deep resting potential (about −70 mV), which is depolarized by increasing [Ca2+]o and CaR agonists raise [Ca2+]i in bovine parathyroid cells and inhibit PTH secretion 2+]i activates Ca2+-activated K+ channels in human parathyroid cell Increase in [Ca2+-activated Cl− channels and that these channels can be activated by an increase of [Ca2+]i through the mediation of the arachidonic acid cascade.In the present study, we report that frog parathyroid cells possess Ca2+ concentration [Ca2+]i in mammalian parathyroid cells 2+]i of frog parathyroid cells. NPS-R-467 and extracellular 10 mM Ca2+ saline solution induced a large increase in [Ca2+]i, but NPS S-467 was less effective than the R-467  relationships produced by a voltage ramp (167 mV/s) from −100 to +100 mV were almost linear in normal saline solution (data not shown).Basal properties of frog parathyroid cells have been reported in a previous paper 2+ concentration was increased from 1.8 mM to a higher concentration, frog parathyroid cells displayed a sustained inward current at a holding potential of −54 mV  to outwardly rectifying (6 mM Ca2+)  inhibited extracellular Ca2+-induced currents completely (n = 3)(Cl (−7 mV). When intracellular Cl− concentration decreased from 104 to 10 mM, the reversal potential shifted to −69±3 mM (n = 5) when ECl was set to −66 mV  to −58±3 mV in steady state (3 min) (n = 4) internal solution (ECl = −63 mV) containing 0.8 mM Ca2+ without EGTA, frog parathyroid cells displayed a gradual increase in outward-rectifying current (When the extracellular Caf −54 mV . I/V relsolution . However of Ca2+ . The appsolution . We haveng steps . Deactivng steps . OutwardmM Ca2+) .The actimM Ca2+) . Extracey (n = 3). The curo −66 mV . Prevent) (n = 4). When th current . The out current  and the  current .2+-free 10 mM Ba2+ solution without Na+ also induced a conductance increase and the currents had the reversal potential close to ECl  at −54 mV in response to extracellular 10 mM Ca2+, although repetitive recordings were impossible. In the conventional mode, the effects of drugs were estimated by the different responses of different populations of cells. When the internal Ca2+-buffer of 1 mM EGTA was replaced with 10 mM BAPTA, the extracellular 10 mM Ca2+-induced current decreased to 16% of the controls (2+-induced current had a moderate dependency on GTP (2+ (1) receptor, external 2-AG ether (50 µM) and Win55,212-2  did not induce a conductance increase. Intracellular 1-oleoyl-2-acethyl-sn-glycerol , cyclic AMP (1 mM) and m-3M3FBS  also did not elicit any response. Intracellular 20 or 50 µM RHC-80267 (an inhibitor of DAG lipase) did not inhibit extracellular Ca2+-induced current, but the conductance increase did not return to basal level after washout, suggesting that the drug has an unknown non-specific effect. Intracellular ryanodine (100 µM) and ruthenium red (30 µM) also did not inhibit extracellular Ca2+-induced current.After attaining the conventional whole-cell configuration with a standard Kcontrols . During controls . In the y on GTP . Even wh GTP  metabolism. External tetrahydrolipstatin  decreased the current magnitude to 10% of the controls (2) also decreased the current magnitude to 6% of the controls  did not induce a conductance increase in the cells, but the drug decreased extracellular Ca2+-induced current to 5% of the controls (2 (PLA2), epoxygenase (cytochrome P-450), cycloxygenase (COX) and lipoxygenase (LO). Baicalein  decreased the current magnitude to 64% of the controls and the moderate inhibition was significant (I/V) relationships in response to depolarizing steps. Ca2+-free saline solution containing 1 mM EGTA gradually decreased the arachidonic acid-induced current to 20% of the control (n = 3), suggesting that the IP3-independent Ca2+ store might be depleted by sustained action of an arachidonic acid metabolite. Intracellular dialysis of 12 (S)-HPETE at very low concentration also induced time-dependent outward currents and deactivating tail currents (50 for intracellular 12 (S)-HPETE was about 700 pM . Interna− (ECl). Both cocurrents . The appt 700 pM . Intrace2+ activates MAP kinase and phospholipase A2 via protein tyrosine kinase (PTK) as well as protein kinase C (PKC) in bovine parathyroid cells 2+-induced current decreased to 6% of the controls (+ current (the time constant of about 10 min) 2+-induced current to 31% of the controls , but the increase was independent of inositol 1,4,5-trisphosphate (IP3). Alternatively, the PLC-DAG-MAG pathway may produce arachidonic acid in frog parathyroid cells. Since ETYA  could not elicit the current response, arachidonic acid also may not be an intracellular mediator for raising [Ca2+]i. An arachidonic acid metabolite produced by the action of baicalein-sensitive 12-/15-lipoxygenase may increase [Ca2+]i. 12-lipoxygenase produces 12(S)-HPETE, while 15-lipoxygenase produces 15(S)-HETE. In the present study, 12(S)-HPETE as well as 15(S)-HETE elicited Ca2+-activated Cl− currents. In porcine parathyroid cells, high extracellular Ca2+ and arachidonic acid inhibit PTH secretion and baicalein restores the secretion 3 dialysis suggests other targets for the lipid messenger than the endoplasmic reticulum. The intra-granular Ca2+ concentration in secretory cells is lower than that in the endoplasmic reticulum but higher than that in the cytosol + channels in Aplysia2+ channels The present study shows that frog parathyroid cells can respond to high extracellular Ca2+-selective channels in plasma membrane 2+ entry through the channels increases [Ca2+]i. It is claimed that the molecular architecture of the arachidonate-regulated Ca2+-selective channel is a pentameric assembly of Orai1 and Orai3 subunits 2+-activated Cl− current 2+-activated Cl− conductance. In the present study, Ba2+ in Ca2+-free saline solution containing 1 mM EGTA also could elicit Ca2+-activated Cl− current in frog parathyroid cells. Furthermore, high extracellular Ca2+ and intracellular arachidonic acid could induce almost only niflumic acid-sensitive currents. An arachidonic acid metabolite may not induce the Ca2+ entry through Ca2+-permeable channels in the plasma membrane, but the metabolite may release Ca2+ from an unknown novel store independent of IP3. This mechanism for [Ca2+]i increase without Ca2+ entry may make the precise monitoring of [Ca2+]o possible. On the other hand, the IP3-independent Ca2+ store should be refilled by an innate mechanism. In the present experiment, the wash-out of Ca2+-free saline solution with normal saline solution for 10 minutes did not recover the arachidonic acid-induced current. Lu et al. In murine parotid and pancreatic acinar cells, arachidonic acid activates Ca50 of extracellular Ca2+ for inhibition of human PTH secretion is about 1.2 mM Xenopus oocytes injected with cRNA prepared from bovine CaR displayed an EC50 of 5 mM extracellular Ca2+ for stimulation of Ca2+-activated Cl− current under the condition of resting 0.5 mM Ca2+2+ concentration and PTH secretion is unknown. Normal saline in the present study contained 1.8 mM Ca2+. The higher EC50 (6 mM) in this study suggests that the isolated parathyroid cells might fairly desensitize during exposure to 1.8 mM Ca2+. Bovine parathyroid cells also displayed the desensitization of the intracellular Ca2+ response during sustained extracellular Ca2+ stimulation 2+ between 2 mM and 3 mM.EC2+-activated Cl− channels carry out the important functions in several tissues including fluid secretion from exocrine gland, amplification of olfactory transduction and block of polyspermy in amphibian oocytes 2+-activated Cl− channels and inhibition of PTH secretion is unknown. In secretory glands, a low pH of secretory vesicles makes them ready to release their contents. Acidification of secretory granules is carried out by H+-ATPase that pumps H+ into the vesicular lumen + fluxes that would prevent further H+ pumping can be cancelled with charge compensation. Acidic granules principally depend on Cl− fluxes for charge neutralization. In accordance with this mechanism, procedures reducing intracellular Cl− concentration decrease influx of Cl− through granular Cl− channels, resulting in inhibition of exocytosis − permeation through the patch membrane using gramicidin instead of amphotericin B to estimate natural intracellular Cl− concentration. The reversal potentials of extracellular Ca2+-induced currents shifted from −25 mV in early stage (1 min) to −58 mV in steady state (3 min). This suggests that initial concentration of intracellular Cl− was 51 mM, and that the concentration decreased to 14 mM in a few minutes. We suppose that the decrease of intracellular Cl− concentration in parathyroid cells also may inhibit influx of Cl− through granular Cl− channels. The inhibition may lead to the reduction in priming of PTH secretory granules for release, which is constructed by Cl−-dependent proton pumping.Ca2+ regulates MAP kinase through the mediation protein kinase C (PKC) and protein tyrosine kinase (PTK) in bovine parathyroid cells, resulting in PLA2 activation. In the present study, genistein greatly decreased extracellular Ca2+-induced current. Genistein is an isoflavone that interacts with several molecules in living cells. Genistein affects voltage-gated Na+ current in the neurons through PTK-dependent and kinase-independent mechanisms + current in frog parathyroid cells to 2% of the controls (data not shown). Alternatively, genistein also may affect PLCγ, because PLCγ has Src homology, which has tyrosine kinase activity Kifor et al. 2+-sensing.In conclusion, frog parathyroid cells use a novel mechanism for extracellular CaRana catesbeiana) weighing 250–550 g were used for the experiment over the course of a year. The experiments were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University with approval of the Institutional Animal Care and Use Committee. The keeping of bullfrogs  was approved by the Ministry of the Environment of Japan . Parathyroid cells were isolated from the parathyroid glands of decapitated and pithed animals. Two pairs of the oval parathyroid glands in both sides were quickly dissected from the precordial region which lie near the ventral branchial bodies and attached to the carotid arteries 2+-free saline containing 2 mM EDTA and incubated for 10–12 min in 2 ml of same saline containing 10 mM L-cysteine and 10 units/ml papain . The glands were then rinsed with normal saline. The individual cells were dissociated by gentle trituration in normal saline. Isolated parathyroid cells displayed an oval shape, with a diameter of about 10 µm Adult bullfrogs (I/V) relationship was obtained from the current generated by the 167 mV/s voltage ramp from −100 to +100 mV. In some case, the current-voltage relationship was obtained from the current generated the 400 ms voltage-step pulses between −104 and +96 mV in 20 mV increments from a holding potential of −84 mV. Input resistance was calculated from the slope conductance generated by the voltage ramp from −104 to −54 mV. Data were analyzed with pCLAMP and Origin 7.5 and 8.0 . Unless stated otherwise, the data are presented as means ± S.E.M., significance was tested by Student's t test and a difference was considered significant if P<0.05.Voltage-clamp recording was performed in whole-cell configuration 2+]i responses. We used a light-attenuating filter for 340 nm excitation light to balance the brightness of the two images. Measurements were taken only during first 2 h after completion of loading the fura-2 due to the reduction in fura-2 level. Measurement of fluorescence ratios under a continuous perfusion with normal saline solution was carried out for 5 min to confirm the stable basal level before applying any chemical. Switching between solutions was electronically controlled.Dissociated parathyroid cells in normal saline solutions collected into the recording chamber were incubated at room temperature in 10 µM fura-2 acetoxymethyl ester  with 0.02% cremophore EL  and washed thoroughly with normal saline after 30 min. Cell adhesive, concavalin A  was coated to the bottom of the recording chamber to immobilize the cells during the experiment. A chamber containing fura-2-loaded cells was placed on the stage of an inverted microscope . A 100-W xenon lamp emitted the excitation light. The fluorescence was imaged using a silicon-intensified target (SIT) camera  and digitized to 8 bits per pixel with an Argus 50 image processor system . A PC controlled the filter and intensifier gains. Imaging data were stored on magneto-optical disks. Changes in fluorescence ratios of excitation at 340 nm and 380 nm light pulses were measured in the selected areas to determine [Ca2, 1.8; Hepes, 10; glucose, 20; pH 7.2. The pH of normal saline and other solution was adjusted by Tris base. The external Na+-free 10 mM Ba2+ solution was prepared by the replacement of Na+,K+ and Ca2+ with NMDG+ and Ba2+. The solution exchange was done by gravity flow. For stock solution, spermine tetrahydrochloride  and ruthenium red  were dissolved in deionized water. WIN 55,212-2 mesylate , phorbol 12,13-dibutyrate , U73122 , U73343 , ryanodine , 1-oleoyl-2-acetyl-sn-glycerol , arachidonic acid , RHC-80267 , tetrahydrolipstatin , 5,8,11,14-eicosatetraynoic acid , baicalein , MS-PPOH , dicrofenac , genistein , PD98059  and m-3M3FBS  were dissolved in dimethylsulphoxide (DMSO). 2-arachidonoylglycerol  dissolved in acetonitrile, 2-arachidonoylglycerol ether , 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE, 297 µM) and 15S-hydroxy-5Z,8Z,11Z,13E-eicosateraenoic acid (15(S)-HETE, 312 µM) dissolved in ethanol and methyl arachidonyl fluorophosphonate  dissolved in methyl acetate were purchased from Cayman. Samples of the stock solutions were added to normal saline solution or internal solution to give the desired final concentrations. NPS R-467 , NPS S-467 , and niflumic acid  were directly dissolved in normal saline solution. The standard K+ internal solution contained (in mM): KCl, 100; CaCl2, 0.1; MgCl2, 2; EGTA, 1; Hepes, 10; pH 7.2. In some experiments, internal K+ was replaced with Cs+ and Cl− was replaced with gluconate−. BAPTA , GTP , GDPβS , 1,4,5-IP3  and cyclic AMP  were dissolved in internal solution.Normal saline solution consisted of (in mM): NaCl, 115; KCl, 2.5; CaClAll experiments were carried out at room temperature (20–25°C).Figure S12+-induced current in frog parathyroid cells.Extracellular Ba (A)The plot of the whole-cell current/voltage (I/V) relationship produced by a voltage ramp. Extracellular cations  other than Ba2+ were replaced with NMDG+. (B) The shift of the reversal potential of extracellular Ba2+-induced current in another cell. Intracellular Cl− concentration was reduced from 104 to 10 mM. (C) Extracellular Ba2+-induced current in the condition of external Ca2+ elimination with EGTA. (D) The I/V relationship of the difference between a and b in the pen recording C. (E) Comparison of extracellular Ba2+-induced currents measured at −54 mV with and without extracellular Ca2+. The pipette solution contained amphotericin B (133 µg/ml).(EPS)Click here for additional data file.Figure S2Effects of baicalein and niflumic acid on intracellular arachidonic acid-induced currents. (A)The plot of the whole-cell currents/voltage (I/V) relationships produced by a voltage ramp of internal arachidonic acid-induced currents under the condition of high intracellular Cl− concentration (ECl = −4 mV) and the inhibition with 20 µM baicalein. (B) The similar relationships under the condition of low intracellular Cl− (ECl = −63 mV). (C) Inhibition of the internal arachidonic acid-induced currents with 100 µM niflumic acid.(EPS)Click here for additional data file.Figure S32+-induced current.Effects of genistein and PD98059 on extracellular Ca (A) The pen recording of the inhibitory effect of external genistein on extracellular Ca2+-induced current at −54 mV. (B) The plots of the whole-cell I/V relationships during control, genistein application and wash-out. The relationships were obtained from the pen recording on the upper part. (C) The pen recording of the inhibitory effect of external PD98059 on extracellular Ca2+-induced current at −54 mV. (D) The plots of the whole-cell I/V relationships during control, PD98059 application and wash-out. The relationships were obtained from the pen recording on the upper part. (E) Comparison of the effects of genistein and PD98059 on extracellular Ca2+-induced current at −54 mV. The pipette solution contained amphotericin B (133 µg/ml).(EPS)Click here for additional data file.


Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5–2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca2+ transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca2+ transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca2+ transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.Genetically encoded Ca Genetically encoded Ca2+ indicators (GECIs)  are a recent alternative to chemically synthesized fluorescent Ca2+ indicators, such as Fura-2 and Oregon Green BAPTA-1 in vitro and in vivo decreases as the age of preparation increases 2+ imaging in viable cells in various model animals, such as mouse, nematode, fly and zebrafish Monitoring activities of individual neurons is crucial for the understanding of neuronal circuit dynamics. For this purpose, Ca2+ indicators, it is necessary to devise means such as to use different laser powers for Ca2+ imaging and photostimulation However, popularly used green fluorescent protein (GFP)-based GECIs, such as G-CaMPs 2+ indicator, R-GECO1, in which a circularly permuted red fluorescent protein (mApple) is linked to calmodulin (CaM) and its target peptide M13 from myosin light chain kinase at its C- and N-termini, respectively  compared to R-GECO1  , which was ∼2-fold greater than R-GECO1  . Since i, n = 3) , which h R-GECO1 . The aff R-GECO1 . The Ca2wn in 2+ .F/F) was 1.5–2-fold greater than that of R-GECO1 . Trains of APs at a frequency of 50 Hz were induced by current injection into the neurons, and the AP-induced Ca2+ transients were imaged from the soma and proximal dendrites at 50 frames per second (fps) using a Nipkow-disk confocal microscope. All experiments were performed under identical experimental conditions at room temperature (25–28°C). R-CaMP1.07 detected the Ca2+ transients associated with single APs with 100% probability in 7 out of 8 tested cells. In total, R-CaMP1.07 and R-GECO1 detected Ca2+ transients in response to single APs with 95% and 70% probability, respectively. The detection probability of Ca2+ transients evoked by >2 APs was 100% for both R-CaMP1.07 and R-GECO1. The ΔF/F of Ca2+ transients evoked by single APs were 9.4±2.6% and 7.4±1.7% for R-CaMP1.07 and R-GECO1, respectively, and the signal-to-noise ratios (SNRs) were 7.2±1.9 and 5.2±1.1 at 50 fps, respectively (n = 5 each). Both indicators exhibited an almost linear increase in ΔF/F and SNRs up to 6 APs. Over the entire stimulus range, ΔF/F and SNRs of R-CaMP1.07 were consistently 1.5–2.0-fold higher than those of R-GECO1. The rise and decay time constants of the AP-induced Ca2+ transients were nearly identical between R-CaMP1.07 and R-GECO1   .per se does not cause abnormal changes in cellular electrophysiological properties or synaptic activity.We next tested electrophysiological properties of neurons expressing R-CaMP1.07. No significant differences in input resistance, membrane capacitance, or resting potential were observed between control (R-CaMP1.07-negative) and R-CaMP1.07-positive cell groups . The amp2+ transients linearly correlated with the number of APs  or in vivo, because the fluorescence response of GECIs are apt to become smaller in vivo conditions compared to in vitro conditions GECIs may inherently buffer Ca2+ indicators allows imaging with increased temporal resolution, which can separate fast individual APs in burst-spike trains. Further improvement in the kinetics of R-CaMPs will facilitate a more faithful detection of APs.The rise and decay time constants in R-CaMP1.07-expressing cells were 110 and 920 ms, respectively. These values were still larger than G-CaMPs 2+ indicators , because the excitation light for these indicators also activates ChR2 and this makes it difficult to independently manipulate ChR2 during the excitation of indicators. In this study, we demonstrated that our improved red fluorescent GECI, that was excited at a wavelength substantially distinct from the action spectra of ChR2, enabled the imaging of neuronal activity triggered by ChR2. We expect that our understanding of the roles for specific cells in the neuronal network will be promisingly facilitated by the advanced technique for simultaneous visualization and manipulation of neuronal activity with our R-CaMP1.07 and ChR2 in combination.ChR2 is widely used as a tool to control neuronal excitability in various cell types and tissues B vector (Invitrogen) containing a T7 promoter as described previously Complementary DNA (cDNA) encoding R-CaMP1.01 was synthesized by randomly mutagenizing the cDNA encoding the prototype GECI, R-GECO1 E. coli KRX (Promega) transformed with bacterial expression plasmids for red fluorescent GECIs were grown at 37°C. Protein expression was induced by the addition of 0.1% rhamnose and incubating for an additional 5 h at 20°C. The indicator proteins with N-terminal histidine tags were purified in vitro characterization. R-GECO1, which lacks the N-terminal histidine tag, was used without purification. Spectral analyses were performed as described previously Fmax/Fmin, where Fmax is the fluorescence intensity at saturating [Ca2+], and Fmin is the fluorescence intensity at nominally zero [Ca2+] with 1 mM EGTA. The Ca2+ titration experiments were performed at pH 7.2 with 10 mM solutions of K2H2EGTA and Ca2EGTA from Ca2+ Calibration Kit #1 (Invitrogen), as reported previously 2, 1 MgCl2, 10 glucose and 5 HEPES (pH 7.4), and 100 µM ATP was bath-applied for 1 min after the baseline fluorescence was obtained. The images were analyzed using the AquaCosmos version 2.0 software (Hamamatsu). The transient increase in fluorescence (ΔF/F) was calculated after subtracting the background fluorescence.HeLa cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and transfected with plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s manual. The fluorescence images of cells expressing red fluorescent GECIs were acquired using a fluorescence microscope  equipped with a CCD camera , as described previously 2 with 1 ml of 50% minimal essential medium, 25% Hanks’ balanced salt solution (HBSS), 25% horse serum (Cell Culture Laboratory) and antibiotics. The medium was changed every 3.5 days. On days 3–5 in vitro, R-CaMP1.07 or R-GECO1, under the control of the CMV promoter, were introduced into neurons via targeted single-cell electroporation. Briefly, borosilicate glass pipettes (tip resistance 5–7 MΩ) were filled with HBSS containing 1–2 µg/µl of plasmid DNA and 200 µM Alexa Fluor 488 hydrazide (Invitrogen). The tip of the pipette was placed in close proximity to the soma, and electroporation was performed with 50 rectangular pulses  at a frequency of 50 Hz All experiments were performed with the approval of the animal experiment ethics committee at the University of Tokyo  and according to the University of Tokyo guidelines for the care and use of laboratory animals. Hippocampal slices were prepared from postnatal day 7 Wistar/ST rats (SLC), as described previously 3, 3.3 KCl, 1.24 KH2PO4, 1.0 MgSO4, 1.0 CaCl2 and 10 glucose, bubbled with 95% O2 and 5% CO2. All recordings were performed at room temperature (25–28°C), unless otherwise specified. Patch-clamp recordings were collected from hippocampal CA3 pyramidal neurons using a MultiClamp 700 B amplifier and a Digidata 1440A digitizer controlled by pCLAMP10 software (Molecular Devices). Epifluorescence microscopy was used to select cells containing Alexa Fluor 488 signals with the fluorescence intensity ranging from 50–85 (arbitrary units). Borosilicate glass pipettes (5–7 MΩ) were filled with a solution containing (in mM) 135 K-gluconate, 4 KCl, 10 HEPES, 10 phosphocreatine-Na2, 0.3 Na2-GTP and 4 Mg-ATP (pH 7.2). The signals were low-pass filtered at 1–2 kHz and digitized at 20–100 kHz. Data were discarded if the access resistance changed by more than 20% during the experiments. Spikes were evoked by current injections . For Ca2+ imaging, red fluorescent GECIs were excited at 568 nm (power below objective lens 8.82 µW) with a laser diode  and visualized using a 617-nm (width 73 nm) band-pass emission filter. Images were captured at 50 fps using a Nipkow-disk confocal scanner unit , a cooled CCD camera , an upright microscope  and a water-immersion objective . For the photostimulation of ChR2-expressing-neurons, a blue light pulse  was delivered through the same confocal unit for 300–3000 ms. For data analysis, the cell bodies and proximal dendrites of the neurons were identified visually to locate regions of interest (ROIs). The fluorescence intensity in each ROI was spatially averaged. The fluorescence change was defined as ΔF/F = (tF – F0)/F0, where tF is the fluorescence intensity at time t, and F0 is the baseline averaged for 2 s before time t. The maximum ΔF/F within 1 s after the action potential initiation was used as the peak amplitude of the Ca2+ transient. The signal-to-noise (S/N) ratio was defined as the average spike signal amplitude divided by the standard deviation of the baseline fluorescence intensity in the trace. Data were collected from more than 3 consecutive trials. The rise time t1/2 was measured as the time between the onset of the spike initiation and the half-peak response. The decay time t1/2 was measured as the time of half decay of a single exponential fit of the recovery from the peak response to the baseline.Hippocampal slices were mounted in a recording chamber and perfused at a rate of 1.5–3 ml/min with aCSF containing (in mM) 127 NaCl, 26 NaHCO


Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca2+ release. Voltage-induced Ca2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca2+ current and SR Ca2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca2+ sparks' amplitude that was attributed to decreased total Ca2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca2+]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.Efficient intracellular Ca Bin1(−/−) mice die shortly after birth due to hypertrophic dilated cardiomyopathy without apparent defects in vesicle trafficking, suggesting one role for Bin1 during cardiac development Disruption of Bin1 in 2+ regulation in adult skeletal muscle. We examined the function of Bin1 by applying short hairpin (sh) RNA to silence Bin1 expression in adult flexor digitorum brevis (FDB) skeletal muscle. Using in vivo electroporation mediated-transfection Since Bin1 may have distinct functions during development and in adult tissues, the objective of this study was to characterize the specific function of Bin1 in maintenance of t-tubule structure and Ca2+ channel at the t-tubule membrane, to ryanodine receptor 1 (RyR1), a Ca2+ release channel at the sarcoplasmic reticulum (SR) 2+ transient 2+ release. To test this possibility, we measured voltage induced Ca2+ release (VICR) in these fibers. The voltage-current relationship (IV plot) showed that mean amplitude of inward Ca2+ current was altered in the shRNA-Bin1 fibers. Additionally, the mean amplitude of Ca2+ transients were reduced, further suggesting ineffective conformational coupling between DHPR and RyR. While VICR is an indicator of global Ca2+ release, Ca2+ sparks measure the opening of clustered and localized RyRs. Earlier studies revealed that dystrophic muscle or aged muscle display altered Ca2+ sparks activity when compared to young and healthy muscle, suggesting the possible physiological significance of the osmotic stress induced Ca2+ sparks 2+ spark frequency and amplitude. The reduction of Ca2+ sparks amplitude could be partially attributed to a decreased Ca2+ store within the SR, suggesting that alteration in membrane structure could directly modulate Ca2+ storage, which would lead to compromised SR Ca2+ release.Intact triad junction structure in skeletal muscle is required for effective conformational coupling of (DHPR), a voltage sensor and L-type gated CaFour different shRNA oligonucleotides targeting specific exons of the skeletal muscle of mouse Bin1 mRNA were designed to allow RNAi mediated knockdown of gene expression . These sshRNA-Bin1 against exon 8 or shRNA-control plasmids were injected into separate hindlimb paws of viable 3-6 months old mice, and then electroporated following previously established protocols Considering previous reports on the role of Bin1 in t-tubule formation during muscle development, we tested if reduced Bin1 expression could alter t-tubule structure in adult skeletal muscle. DiOC5 fluorescent dye was used to visualize t-tubules of individual transfected muscle fibers to assess the membrane structure of these fibers. Bifurcated t-tubule doublets were observed in the shRNA-control fibers  top. shRTo further analyze the disrupted t-tubule structure in the shRNA-Bin1 muscle fiber we examined the ultrastructure of transfected FDB muscle bundle using electron microscopy (EM). We found that ∼30% of the FDB muscle bundle (19 of 63 cells) transfected with shRNA-Bin1 showed swollen t-tubules structures 1, which2+ homeostasis in striated muscle. To determine whether knocking down Bin1 could alter the conformational coupling between DHPR to RyR, we measured VICR in isolated muscle fibers using a silicone-grease embedding method 2+ current characteristics of DHPR, a L-type voltage gated Ca2+ channel (2+ current in the shRNA-Bin1 fibers continuously displayed reduced inward Ca2+ current even as early as at −30 mV depolarizing pulses. The reduction of inward Ca2+ current (A/F) in the shRNA-Bin1 fibers when compared to the shRNA-control was summarized as follows: at −40 mV ; at −30 mV ; at −20 mV ; at −10 mV ; and at 0 mV . The altered inward Ca2+ current in the shRNA-Bin1 fibers could be potentially due to ineffective conformational coupling between DHPR and RyR.Proper positioning of t-tubules and SR is essential for maintenance of Ca channel  [22]. In0), which defines the peak amplitude of Ca2+ release; and 2) the rising and corresponding decay time constant (τ) of exponential functions fit to tracings of Ca2+ release. Interestingly, the mean amplitude of Ca2+ transient evoked at different depolarizing pulses also exhibited compromised Ca2+ release in the shRNA-Bin1 fibers ; at −30 mV ; at −20 mV ; at −10 mV ; at 0 mV. The reductions in the amplitude of both Ca2+ current and the shRNA- Bin1 fibers further imply that structural changes in the t-tubule membranes alter the coupling of DHPR to RyR, leading to reduced global Ca2+ release. This result is in accordance with a previous study showing that knockdown of Bin1 in cultured cardiomyocytes results in a delayed Ca2+ transient 2+ release when compared to the control (2+ release in shRNA-control fibers was similar to untransfected wild-type fibers (data not shown), indicating that 14 days are sufficient to restore the Ca2+ handling properties of electroporated FDB muscle.In quantifying the kinetic properties of VICR, we examined two parameters: 1) amplitude  generating Ca2+ spark events in skeletal muscle 2+ sparks would appear more frequently in the shRNA-Bin1 fibers. As we observed in previous studies, there were only minimal spontaneous resting Ca2+ sparks in the electroporated shRNA- Bin1 or shRNA-contol fibers (data not shown).The reduced Ca1 fibers  could di2+ sparks by perfusing hypotonic solution  that transiently swells the muscle fiber. Rapid removal of hypotonic solution by perfusion of isotonic solution  restored cell volume and generated robust peripheral Ca2+ sparks activity  of the shRNA-Bin1 fibers, however, exhibit greatly reduced Ca2+ sparks frequency (2+ sparks per minute in the shRNA-Bin1 fibers (72.46 ± 12.47) was significantly lower when compared to shRNA-control (135.6±13.0)  .2+ sparks, we also examined the full duration at half maximum (D50) which represents the time between half rising time and the corresponding decay time and amplitude (ΔF/F0) 0 and D50 of individual Ca2+ release events showed that shRNA-Bin1 Ca2+ spark events tend to be lower in amplitude and shorter in duration. The mean amplitude and duration of shRNA-Bin1  were significantly lower than the shRNA-control fibers . Additionally, shRNA-Bin1 fibers seemed to display less high amplitude and long duration Ca2+ events, further suggesting that knocking down Bin1 caused a general decrease in the quantity of Ca2+ that can be released from the SR.In quantifying the kinetic properties of Ca2+ release in the shRNA-Bin1 fibers. Disruption of intracellular membrane structures and concurrent changes in intracellular [Ca2+]i storage or an altered resting [Ca2+]i level could be potential mechanisms to explain the phenotype we observed. Measurement of resting [Ca2+]i levels using Fura-2 ratiometric dye showed similar resting [Ca2+]i levels in both shRNA-control and shRNA-Bin1 fibers. Application of 30 mM Caffeine, a RyR agonist, showed reduced mean SR Ca2+ store in the shRNA-Bin1 fibers (0.57±0.03) when compared to the shRNA-control (0.76±0.05)  compared to shRNA-control fibers (1.01±0.1)  . Further.01±0.1) . These f2+ homeostasis in the heart of adult's mice 2+]i homeostasis in adult skeletal muscle as alteration in membrane structure could possibly affect the coupling of DHPR to RyR1, and hence [Ca2+] release events. Here, we show that knocking down Bin1 expression in adult FDB results in disrupted t-tubule structure : 5′GATCCGTGCGCTGCTGGTGCCAACTTCAAGAGATTTTTTGCTAGCG3′, and 5′AATTCGCTAGCAAAAAATCTCTTGAAGTTGGTACTAGCAACGCACG3′, were synthesized BamHI and EcoRI restriction sites of the pSIREN-DNR plasmid  to generate shRNA interference probes. A separate plasmid pU6-mRFP was derived from the pShuttle vector (Clontech) by two subcloning steps. First, a red fluorescent protein (RFP) cDNA was inserted behind the cytomegalovirus promoter. Second, the U6-shRNA cassette from pSIREN-DNR was ligated into the pShuttle backbone, resulting in the generation of pU6-mRFP. This plasmid forms the backbone of the shRNA-Bin1 and shRNA-control constructs used in these experiments.Initial screening experiments for RNAi probes to suppress Bin1 containing Exon 11 were conducted using multiple short hairpin (sh)RNA oligonucleotides targeting mouse Bin1 cDNA. Plasmid containing different shRNA oligos against Bin1 was then co-transfected with HA- tagged murine Bin1 cDNA to HEK293 cells to test the probe efficacy. 96 hours post-transfection, the cells were collected and prepared for western blot analysis. These results indicated that shRNA against exon 8 was the most effective in silencing endogenous expression of Bin1 containing exon 11. The shRNA oligonucleotides against exon 8 of Bin1 (shRNA-Bin1):3–6 month old male wild-type mice from C57Bl/J6 background were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection before injection of 15 uL of 2 mg/mL hyaluronidase (Sigma #H4272) into the footpad of mouse. 60 minutes later, 20 uL (2 ug/uL) of endotoxin-free plasmid DNA of shRNA-Bin1 or shRNA-control in 0.9% saline is injected into the area of the foot surrounding FDB muscle. After 15 min to allow diffusion of the plasmid, two 13 mm steel acupuncture needles were inserted on either side of the FDB, perpendicular to the muscle. Twenty pulses of a 100 V/cm electric field were applied in 20 ms bursts with a burst interval of 100 ms between the two needles. Mice were returned to their cages and monitored every half hour until they recovered from the anesthetics. Mice that showed any sign of inflammation on their electroporated feet were excluded from all of the experiments mentioned. For all experimental results reported here, mice were sacrificed at 14 days post electroporation and FDB muscles were surgically removed. Killing of the animals and tissue preparation were conducted using protocols approved by the UMDNJ-RWJMS IACUC (protocol number I10-028-3).V 1.1) (ABR) (1∶1000).RFP expression was used as an indicator to measure the transfection efficiency. In the sample preparation for western blot analysis, a portion of the muscle bundle in the transfected FDB muscle bundle that did not express RFP expression was trimmed out from the remaining muscle mass and excluded. The transfected muscle bundle was then homogenized as previously described 2+ Tyrode buffer, (in mM) 140 NaCl, 5 KCl, 10 HEPES, 2 MgCl2 (pH 7.2), and incubated in Tyrode buffer containing 2 mg/mL type I collagenase (Sigma C-5138) for 90 minutes at 37°C. After two washes in isotonic Tyrode buffer containing 2.5 mM Ca2+ , muscle fibers were gently dissociated by several passages through a series of micropipetter tips of gradually decreasing diameter. Individual FDB muscle fibers were plated onto ΔTC3 glass-bottomed Petri dishes (Fisher Scientific) in Tyrode and used for experimentation within 6 hours.FDB muscles transfected with either shRNA-control or shRNA-Bin1 were surgically removed in 0 CaIndividual FDB fibers were loaded with 100 nM DiOC5, a membrane impermeable dye, for 20 minutes at room temperature. After washing, red transfected fibers with intact sarcolemmal membranes and regular striation patterns were selected by epi-fluorescence microscopy. T-tubule staining was observed on a BioRad Radiance-2100 confocal microscope equipped with Argon laser (479 nm), Helium-Neon laser (632 nm), and a 40X, 1.3NA oil immersion objective. X-y images were acquired at 20 s per frame (512×512 pixels). Using ImageJ software, several regions of interests (ROI) were drawn on the areas of the fibers that displayed blank DiOC5 staining  and then the total affected area in each fiber was divided by the total surface area of the fiber (large ROI drawn covering the surface area of the fiber).4 and 0.1 M cacodylate buffer (pH 7.4). Samples were processed and examined as described previously Freshly dissected electroporated FDB muscle bundle was fixed in 2.5% glutaraldehyde and 0.1 M cacodylate buffer (pH 7.4) and later post fixed in 1% OsO2 incubator. Dish containing fibers was then washed three times by replacing the media with TEA containing solution as extracellular medium  . 20 µM N-benzyl-p-toluene sulphonamide (BTS) was applied for 15 min to prevent motion artifact from muscle contraction. Fibers selected for analysis were confirmed to express RFP, in addition to having intact sarcolemmal membranes and regular striation patterns observable by phase contrast microscopy. Fiber was then silicone coated on both sides, leaving a small surface area exposed to the extracellular solution  and a 40x, 1.3 NA oil-immersion objective. Resulting xt line scans of Furo-4 fluorescence were analyzed and fit to exponential functions using custom IDL software routines that were previously described 2+ transient amplitude, 5 µM BAPTA-AM was added to the muscle fibers 20 minutes before the end of the loading period for Fluo-4 AM.This approach was adapted from the methods developed by Jacquemond and colleagues solution . An Axon2+ sparks were performed using a BioRad Radiance-2100 confocal microscope equipped with an argon laser (488 nm) and a x40, 1.3 NA oil-immersion objective. For Ca2+ spark measurements, fibers were perfused with hypotonic solution  containing (in mM) 70 NaCl, 5 KCl, 10 Hepes, 2.5 CaCl2, 2 MgCl2 (pH 7.2), for 100 seconds to induce swelling before perfusion was switched back to the initial isotonic Tyrode buffer . The osmolality of all solutions were measured using a Micro Osmometer 3300 (Advanced Instruments). Line scan images of 512 pixels in length were acquired at a sampling rate of 2 ms per line, and serial x-y images of muscle fibers were acquired at 3.08 s per frame. Image analysis of Fluo-4 fluorescence in both VICR and Ca2+ spark experiments was performed using IDL software and customer devised routines After confirming the RFP expression, individually isolated FDB muscle fibers were loaded with Fluo4-AM (10 µM) for 60 min at room temperature. Measurements of Ca2+]i level. Zero Ca2+ Tyrode solution was perfused to the fiber before either adding 30 mM Caffeine or 5 µM ionomycin (Sigma) to induce [Ca2+] store released.After confirming the RFP expression, individually isolated FDB muscle fibers were loaded with 10 µM fura-2 AM for 45 min at room temperature in isotonic Tyrode solution. 20 µM BTS was applied for 15 min to prevent motion artifact from muscle contraction. The ratio of fura-2 fluorescence at excitation wavelength of 340 and 380 nm was measured on a PTI spectrofluorometer  to assess the resting [CaResults are present as mean ± SEM as tested for statistical significance by t-test, *p<0.05. Statistical analysis was performed using Origin software. In each experimental group, fibers were isolated from 8 mice, and from each mouse n≥3 fibers were used for analysis.


To test the hypotheses that pregnancy represents a physiologic condition that is associated with dry eye symptoms, and the expression of aquaporin 4 (AQP4) and AQP5 are altered in the lacrimal gland (LG) from term pregnant rabbits.Schirmer’s test, tear break-up time (BUT), and Rose Bengal staining were used to evaluate ocular surface health. LG were obtained from term pregnant rabbits and age-matched female control rabbits and then processed for laser capture microdissection (LCM), real time RT–PCR, western blot, and immunofluorescence for the detection and quantification of mRNA and proteins of AQP4 and AQP5.AQP5 from whole LG was significantly lower than that of control rabbits, while mRNA for AQP4 was not. Levels of mRNA for AQP4 and AQP5 underwent significant changes in acini and epithelial cells from specific duct segments during pregnancy. Western blot from whole LG lysates demonstrated that expression of AQP4 was 24% more abundant in term pregnant rabbits while AQP5 was 22% less when compared to control rabbits respectively. At term pregnancy, AQP4 immunoreactivity (AQP4-IR) was increased in acini while its intensity remained the same in ducts. AQP5-IR was present in both apical and basolateral membranes of acinar cells in normal control and pregnant rabbits, while ductal cells in pregnant rabbits also showed significant amount of AQP5-IR.Pregnant rabbits demonstrated typical clinical symptoms of dry eye, including decreased Schirmer score and BUT as well as increased Rose Bengal staining of cornea. In term pregnant rabbits, mRNA for AQP4 and AQP5 during pregnancy and suggested that these changes may contribute to the altered LG secretion and dry eye symptoms during pregnancy.The data presented here demonstrated significant dry eye symptoms in pregnant rabbits. Our data also showed altered expressions of  Dry eye is one of the most frequently encountered problems for eye clinicians. It affects millions of people with most being women, especially those after menopause . Dry eyeThere is a general clinical impression that pregnant women more frequently report symptoms of dry eye than non-pregnant women. Our epidemiological study suggests that a subpopulation of pregnant women indeed experience increased symptoms of dry eye during the third trimester of their pregnancies . BecauseOur recent report in pregnant rabbits showed that basal LG fluid production from term pregnant rabbits decreased from 0.35±0.04 in control rabbits to 0.21±0.05 μl/min, while pilocarpine stimulated secretion increased from 5.23±0.4 in control rabbits to 11.4±0.56 μl/min .Like other exocrine secretions, LG fluid secretion is an osmotic process driven by the transepithelial secretion of electrolytes and water that is mediated by AQP and ion transporters ,13-19. ALG fluid is produced in two stages: production of primary fluid in the acini and modification into the final fluid during transit through the duct system before reaching the ocular surface. Most studies regarding the LG have focused on the role of acinar cells, with only a few having paid sufficient attention to the duct system ,14,18,27Although our previous reports demonstrated that pregnant rabbit showed decreased basal LG fluid production , there hTwelve pregnant and twelve normal control New Zealand White adult female rabbits  weighing about 4.0 kg were used throughout our studies. Pregnant rabbits were time dated with day zero corresponding to the date of coitus. Normal gestation in the rabbit is 31 days. Animals were narcotized with a mixture of ketamine (40 mg/ml) and xylazine (10 mg/ml) and given an overdose of Nembutal (80 mg/kg) for euthanasia. Term pregnant rabbits were sacrificed on the 29th day of pregnancy. All studies conformed to the standards and procedures for the proper care and use of animals by the US Public Health Service Policy on Humane Care and Use of Laboratory Animals.Routine diagnostic techniques used in testing humans for dry eye, i.e., Schirmer’s test, tear break-up time (BUT), and Rose Bengal staining, were used to evaluate the health status of the ocular surface of normal control rabbits, and pregnant rabbits at 2, 3, and 4 weeks of gestation.Schirmer’s test: Schirmer’s strip was inserted in the lower fornix of the eye without anesthesia for 1 min, after which the strip was removed and the length of the wetted area was measured. Every care was taken to avoid touching the cornea.BUT: to evaluate tear film stability, 2 μl of fluorescein was instilled into the lower fornix of the conjunctiva. The rabbit was allowed to blink several times to distribute the fluorescein evenly on the cornea. The time from opening of the eyes to the appearance of the first dry spot in the central cornea was measured 3 times and the mean was recorded.Rose Bengal staining: Rose Bengal stains whenever there is insufficient protection of surface epithelial cells. Rose Bengal solution (2 μl) was placed in the lower conjunctiva. Results were recorded on a cornea diagram and scored using a modified standardized grading system of 0 to 3 . The intThe technique has been described in detail in our previous publications ,6,30,31.® midiKit plus on-column DNase digestion to reduce the possibility of DNA contamination . RNA quality and quantity was evaluated using a Nanodrop ND-1000 spectrophotometer  and 5 μg samples of total RNA samples were then reverse-transcribed to cDNA only if the 260/280 ratio was above 1.9  according to manufacturer’s instructions.Total cellular RNA was isolated from RNALater-treated samples with RNeasyAQP4 and AQP5 used in this study are listed in The sequences of primers and probes of ® PreAmp Master Mix Kit (Applied Biosystems). The pooled assay mix was prepared by combining up to 50 of 20× TaqMan® Gene Expression Assays into a single tube and using nuclease-free water to dilute the pooled assays to a final concentration of 0.2×. The 50 μl pre-amplification reaction included 25 μl of 2× TaqMan® PreAmp Master Mix, 12.5 μl of 0.2× pooled assay mix, and 12.5 μl of cDNA sample. The reactions were incubated in the DNA Engine® Thermal Cycler for 10 min at 95 °C followed by 14 cycles at 95 °C for 15 s and 4 min at 60 °C and then held at 4 °C. The pre-amplification product was then diluted 1:20 with 1× TE buffer and analyzed by TaqMan® real time RT–PCR.For LCM samples, pre-amplification was performed using TaqMan® Gene Expression Master Mix (Applied Biosystems) containing the internal dye ROX as a passive reference. The PCR reaction volume was 10 μl and contained 1× TaqMan® Gene Expression Master Mix, 300 nM forward and reverse primers, 250 nM probes, and 50 ng of cDNA template. The FAM signal was measured against the 6-carboxy-X-rhodamine (ROX) signal to normalize for non-PCR-related fluorescence fluctuations. The cycle threshold (CT) value represented the refraction cycle number at which a positive amplification reaction was measured and was set at 10× the standard deviation (SD) of the mean baseline emission calculated for PCR cycles 3–15. Each sample was measured in triplicate. The difference between the CT values for each target mRNA and for the internal housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in each sample was used to calculate the abundance of target mRNA relative to the abundance of GAPDH mRNA in the same sample.Amplification was performed with an ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems) using TaqManPrimary antibodies used were purchased from Santa Cruz Biotechnology . The dilution for AQP4  was 1:50, and 1:40 for AQP5 . The secondary antibodies used were fluorescein isothiocyanate (FITC)-conjugated AffiniPure donkey anti-goat IgG  at a dilution of 1:200.Samples frozen in Optimal Cutting Temperature (OCT) compound were cut 8 μm thick and placed on slides then fixed with ready-to-use formaldehyde/zinc fixative  for 15 min. They were then washed in phosphate-buffered solution (PBS) 3× for 10 min each and blocked with donkey normal serum (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The slides were then incubated with primary antibodies at respective dilutions overnight at 4 °C. On the next day, slides were again washed 3× for 10 min in PBS and incubated with secondary antibody for 1 h at room temperature then washed 3× for 10 min in PBS and 1× for 15 min in 4 mM sodium bicarbonate. Finally, one drop of aqueous mounting medium  was placed on slides and covered with coverslip. Slides were observed with a Zeiss LSM 710 confocal laser scanning microscope . FITC-conjugated secondary antibodies were visualized by excitation at 488 nm using an argon laser. Images were analyzed with LSM image browser and PhotoShop .LG samples were homogenized in isolation buffer , and centrifuged at 2,000× g for 20 min. The supernatants were denatured in SDS–PAGE sample buffer for 20 min at 60 °C, resolved on a 4%–20% gradient SDS–PAGE gel , and then transferred onto PVDF . To assess AQP proteins, a constant amount of protein from each sample was analyzed. Membrane blots were probed with AQP4 at the dilution of 1:250, and AQP5 at 1:1,000. All blots were incubated with Alexa 680-labeled donkey anti-goat secondary antibody  and detected with an Odyssey Infrared Imaging System . Densitometry analysis of resulting gel was performed by the manufacturer’s software.t-tests were performed. Relative mRNA abundance data from LCM samples and densitometry results of western blots were subjected to ANOVA (ANOVA) with SigmaPlot 11.0 .For mRNA data from whole gland samples, unpaired In normal control rabbits, the Schirmer test score was 8.69±1.07 mm, while they were significantly decreased at each time point of gestation, i.e., 6±0.54 mm at 2 weeks, 5.63±0.77 mm at 3 weeks, and 6.59±0.45 mm at 4 weeks p<0.05, .BUT was 19.79±2.39 s in normal control rabbits and the scores were significantly reduced in pregnant rabbits to 11.75±0.51 s at 2 weeks, 14.9±0.65 at 3 weeks, and 12.43±0.31 s at 4 weeks’ pregnancy p<0.05, .Rose Bengal test was unremarkable in most corneas of normal control rabbits, but in pregnant rabbits, punctate staining started to show up in many rabbits, and the intensity and severity increased as the pregnancy progressed. At term, there was a moderate to severe staining in approximately 50% of pregnant rabbits, with punctate staining observed in every quadrant of the cornea . GradingAlthough Schirmer and BUT results for pregnant rabbits were both decreased starting at 2 weeks and continued at 3 and 4 weeks, the Rose Bengal test showed a gradual increase of staining as the pregnancy progressed from 2, 3, and 4 weeks of pregnancy. Therefore, we decided to use rabbits at 4 weeks of pregnancy, i.e., term pregnancy, for the following studies.AQP4 from whole LG of term pregnant rabbits was 0.07266±0.00714, which is not significantly different from that of normal control rabbits (0.06753±0.00798), as we reported recently [mRNA level for its 0.0673±0.00798AQP4, mRNA level for AQP5 from whole LG of term pregnant rabbits (5.51±0.33) was significantly lower than that from normal controls (7.1±0.24), as we reported [AQP5 mRNA was significantly lower in acini while its level was significantly increased in every duct segment except intralobar duct  was observed on the basolateral membranes of acini and ducts in LG of both control and pregnant rabbits, with ducts showing stronger AQP4-IR than acini . AQP4-IRAQP5-IR was found in apical and basolateral membranes of acinar cells and was distributed among acini in a “mosaic” pattern with some acini and/or acinar cells in both control and pregnant rabbits exhibiting much stronger AQP5-IR than other acini/acinar cells . MinimalDry eye has been linked to many risk factors, including altered sex hormones, especially androgens ,32-34. DWe have found that many pregnant rabbits demonstrated typical clinical symptoms of dry eye, including decreased Schirmer’s test scores and BUT, and increased Rose Bengal staining. These results corroborate our epidemiology study in pregnant women  and our AQP are membrane proteins responsible for rapid water transport across plasma membranes, following osmotic gradients generated by ion transport proteins. The detection of AQP4 and AQP5 in the rabbit LG, which confirmed our previous reports in rabbits , mouse ,39, rat AQP4 in interlobar duct and AQP5 in acini from pregnant rabbits were only 1/3 that of normal rabbits. Significant changes of AQP in LG have been suggested to play a role in dry eye [At term pregnancy, our results demonstrated that the expression patterns of AQP4 and AQP5 in the LG undergo significant changes at both gene and protein levels in both acini and specific duct segments. The largest change happened in acini and interlobar duct, where the mRNA levels of  dry eye ,8. Tsubo dry eye  reported dry eye . AQP hav dry eye . In mous dry eye .The significant increase of AQP5-IR in ductal cells of pregnant rabbits suggests increased potential for water transport across both apical and basolateral membranes. Although water flow could be bidirectional, i.e., either increased secretion or increased reabsorption from the ductal cells, the reduced basal LG final fluid secretion from pregnant animals  is indicAQP5 was about 76 fold that of AQP4 in the whole LG, suggesting that AQP5 is the dominant subtype of AQP in the LG. These data are in support of the critical role of AQP in lacrimal secretion, particularly in the ducts, which are in accordance with previous findings regarding lacrimal ducts’ active role in LG fluid production [In accordance with our previous reports in normal control rabbits  and rabboduction ,18,27. Woduction ,21. HoweIt should be noted that there are substantial discrepancies in mRNA and protein expressions of the data presented here. Several mechanisms, i.e., redistribution changes of proteins between cell membranes and intracellular stores during pregnancy ,42-45, cAlthough it has been demonstrated extensively that AQP play critical role in water transport in many epithelia, care should be taken in considering how big the role it may be playing in the LG. Unlike salivary glands, which have secretion rates as high as ~75–1,000 ml per day in humans  and AQP In summary, our studies have shown increased dry eye symptoms in pregnant rabbits. The fact that AQP4 and AQP5 expressions underwent significant changes during pregnancy suggests changes in their functional status which may potentially contribute to the changes of LG fluid secretion and dry eye symptoms during pregnancy. These data also support our previous findings that both acini and ducts contribute to LG secretion. Further functional studies are needed to identify the changes in each duct segment as well as in the acini during pregnancy.


Here we present NMR assignments and functional analysis to probe Ca2+-dependent structural changes in GCAP1 that control activation of RetGC. NMR assignments were obtained for both the Ca2+-saturated inhibitory state of GCAP1 versus a GCAP1 mutant , which lacks Ca2+ binding in EF-hand 4 and models the Ca2+-free/Mg2+-bound activator state of GCAP1. NMR chemical shifts of backbone resonances for Ca2+-saturated wild type GCAP1 are overall similar to those of EF4mut, suggesting a similar main chain structure for assigned residues in both the Ca2+-free activator and Ca2+-bound inhibitor states. This contrasts with large Ca2+-induced chemical shift differences and hence dramatic structural changes seen for other NCS proteins including recoverin and NCS-1. The largest chemical shift differences between GCAP1 and EF4mut are seen for residues in EF4 , but mutagenesis of EF4 residues  had little effect on RetGC1 activation. A few GCAP1 residues in EF-hand 1  also show large chemical shift differences, and two of the mutations (K23D and G32N) each decrease the activation of RetGC, consistent with a functional conformational change in EF1. GCAP1 residues at the domain interface  have NMR resonances that are exchange broadened, suggesting these residues may be conformationally dynamic, consistent with previous studies showing these residues are in a region essential for activating RetGC1.Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca Ca2+ targets . Previoud states . Howeverecoverin . Here, wor state . Our res2+-induced protein conformational changes in GCAP1 by examining 15N-1H HSQC spectra of Ca2+-saturated wildtype protein (2+ bound at EF2 and EF3 (and no Ca2+ bound at EF4) was shown previously to activate RetGC and therefore serves as a functional mimic of the Ca2+-free/Mg2+-bound activator state  GTP as a substrate as previously described in detail [Recombinant human RetGC1 was expressed in HEK293 cells and assayed n detail ,56. Ca/Mn detail .2+-saturated GCAP1 were described elsewhere [2+ bound at EF2 and EF3) were obtained in this study by the analysis of three-dimensional NMR data as described previously [15N-1H HSQC with 2048 (1H) x 256 (15N) data points, 3D HNCACB with1024 (1H) x 64 (15N) x 120 (13C) data points, and 3D HNCO with 2048 (1H) x 64 (15N) x 128 (13C) data points were all performed on a triple-labeled EF4mut sample using 800 MHz Bruker NMR spectrometer equipped with a triple resonance cryogenic probe. In addition, 3D HNCOCACB with 1024 (1H) x 64 (15N) x 128 (13C) data points was performed on the triple-lableled sample using a 600 MHz Bruker NMR spectrometer, equipped with a triple resonance cryogenic probe. All NMR experiments were performed at 37 °C. Spectra were processed using NMRPipe software package [NMR experiments and backbone assignments for Calsewhere . Backboneviously . 2D 15N- package  and anal15N NMR longitudinal relaxation rates (R1) and transverse relaxation rates (R2) of Ca2+-saturated GCAP1 and EF4mut were measured using pulse schemes described previously at 60.81 MHz 15N resonance frequency [1 experiments were 0.01, 0.25, 0.5, 1.0 and 2.0 s. Relaxation delays for R2 experiments were 8, 16, 24, 32, 48, 64 and 80 ms. The 180° pulses for Car-Purcell-Meiboom-Gill (CPMG) sequence were applied every 1.0 ms. Uncertainty of R1 and R2 values were determined by Monte-Carlo error simulation using signal intensities that contain experimental noise fluctuation. Model-free parameters including generalized order parameters and correlation times for internal motion [requency . Relaxatl motion –67 were e/Vo versus the log of the molecular weights of standard proteins: β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). Vo is a void volume obtained using blue dextrane (2000 kDa) and Ve is a volume of elution. Determination of the molar mass of GCAP1 was carried out using a multi-angle light scattering instrument (Wyatt Technologies Inc. TREOS and Optlab Rex) coupled to a Superdex 200 HR 10/30 column (Amersham) at 4 °C equilibrated in buffers containing either 10 mM phosphate (pH 7.0) or 10 mM acetate (pH 4.5). A 0.1 ml aliquot of protein (200 μM) was loaded onto the column and eluted at a flow rate of 0.5 ml/min. The molar mass of the eluted protein was calculated from the observed light scattering intensity as described previously . Apparen2+ bound at EF2 and EF3, we first used the NMR structure of Ca2+-bound recoverin (1JSA) that contains Ca2+ bound at EF2 and EF3 as a template in the modeler software, Swiss-Model [2+ bound at EF3 and not bound at EF4). The last residue of the overlaid N-terminal domain (K87) was covalently attached to residue V88 from the homology modeled structure (using the patch command in Chimera), which effectively connected the N-terminal domain of 2R2I with the C-terminal domain generated by homology modeling. The resulting structure contained a sequestered myristoyl group with Ca2+ bound at EF2 and EF3. The entire modeled structure was energy minimized using Xplor-NIH. To generate a homology modeled structure of GCAP1 with Cass-Model . The resss-Model . Therefo


To characterize Ca2+ signaling in striatal cells, spontaneous cytoplasmic Ca2+ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca2+ transients (referred to as slow Ca2+ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca2+ oscillation. Depletion of the intracellular Ca2+ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca2+ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca2+ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca2+ oscillation in both putative-neurons and astrocytes. The slow Ca2+ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca2+ oscillation may involve in the neuron-glia interaction in the striatum.The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca The endoplasmic reticular (ER) Ca2+ store is a source of [Ca2+]i elevation and is crucial for second messenger-induced intracellular Ca2+ signaling 2+ released from the ER should contribute to the modulation of neuronal signal processing in the central nervous system.The calcium ion . mGluR5 has also been suggested as a therapeutic target for Parkinson's disease 2+In the basal ganglia, the striatum receives inputs from the cortex and is thought to play a crucial role in controlling somatic motor movements, behavioral patterns, cognition, learning, and memory 2+]i oscillations in the rat striatum 2+ oscillations were not induced by action potentials, but induced by Ca2+ release from ER. In this previous study, we did not determine whether the spontaneous [Ca2+]i transients occurred in neurons or astrocytes, nor did we identify the induction mechanism of the Ca2+release from ER. Therefore, in this paper, we identified the cell types exhibiting the spontaneous [Ca2+]i transients using transgenic mice expressing green fluorescent protein (GFP) in astrocytes, and determine one of the induction mechanisms. We also analyze the cellular correlations of the slow Ca2+ oscillations. The preliminary results of this work have been previously reported in an abstract form We previously reported long-lasting spontaneous i in the slices was measured using the membrane-permeant acetoxymethyl (AM) ester of Fura-2 LR  chamber on the stage of an epifluorescent upright microscope . [Ca2+]i changes were imaged with a 20×, NA 0.95 water-immersion objective (Olympus). The Fura-2 LR-loaded slices were alternately excited at wavelengths of 340 and 380 nm using a filter changer , and fluorescent signals were captured (F340 and F380) every 2 s with a cooled CCD  or an EM-CCD . All equipment was controlled by MetaFluor software . The experiments were performed under temperature control (30±1°C).i in a fraction of the glial cells or vessel related cells, ROI was put only in the somatic region with round shape. [Ca2+]i in a striatal cell was estimated by the fluorescence ratio (R  =  F340/F380) from each imaged cell 2+]i transient for every cell was determined using an algorithm that defined the onset as the frame after which the slope of R was larger than a given set threshold. Then, the baseline was set to the mean R value of three frames before the onset, and the change in the R value from the baseline was defined as ΔR. To eliminate false positives, [Ca2+]i transients with peaks that did not exceed the threshold (ΔR  = 0.005–0.01) were discarded. Some remaining false positives were deleted upon visual inspection. When we showed the traces of the time courses of R, the baselines were subtracted from the raw R trace. The baseline of each trace was obtained by spline interpolating the local minimum values of the trace using a higher order Hanning filter  Image analysis was performed with MetaFluor and custom-made programs written in MATLAB . In the measurement of Ca2+]i transients included in the slow Ca2+ oscillations, we calculated the transient rate, amplitude, duration, rise slope, and decay slope. The transient rate was defined as the number of onsets per unit time during recording period (>1000 s). The duration indicates the time from onset until the decay to threshold. The rise and decay slopes are the slopes of a regression line for the data points in 5–90% of the peak during the rise phase and in 95% to 1/e of the peak during the decay phase of the [Ca2+]i transients, respectively.To determine the properties of individual i transients within 600 s just before the application of pharmacological agents to confirm their effects.In the pharmacological experiments, we discarded any cells that did not show [Ca2+ oscillation, we used GFAP-GFP mice. First, we confirmed that the GFP-positive cells were astrocytes and that the GFP-negative cells were neurons by immunohistochemistry . Therefore, the proportion of GFP-negative cells to S-100-positive cells was 30.3±7.3% (mean ± SD). The densities of cells positive for S-100, and for NeuN, were 636±154 , and 1538±298 , respectively . Therefore, the proportions of S-100-positive cells to the all immunostained cells were 29.2±6.4% (mean ± SD). Hence, the probability of the GFP-negative cells being astrocytes (S-100-positive cells) was 8.9±2.9%  and 95% confidence interval of this probability was 8.9±5.7%. S-100 was also expressed in the oligodendrocytes To determine whether neurons or astrocytes exhibit the slow Cahemistry . We usedhemistry . The den2+ oscillations in a field of view . Typical time courses of the slow Ca2+ oscillations are shown in 2+ transients was long (2 s)  ×10−3 Hz in astrocytes, n = 200 putative-neurons and 78 astrocytes, 15 slices, 14 mice; p = 0.0903; Mann-Whitney U test).A fluorescence image of the Fura-2 LR-loaded cells in a striatal slice of GFAP-GFP mouse is shown in 2 s) see . Therefo2+ oscillations, individual events were dissolved from the entire R trace in a record, and four parameters, the peak amplitude, duration, rise slope, and decay slope, were extracted  ×10−3, and (1.50±1.42) ×10−3 ), and those values in astrocytes were (1.32±0.77) ×10−2, 18±16 s, (1.49±1.03) ×10−3, and (1.40±1.04) ×10−3 in astrocytes , respectively. All of those four parameters were significantly different between putative-neurons and astrocytes  of the slow Ca2+ oscillations in putative-neurons as described in a later section. To determine the contribution of the Ca2+ release from the intracellular Ca2+ store to the slow Ca2+ oscillations, Ca2+ ATPases of the ER were blocked by the administration of 2 µM thapsigargin. The transient rates of the slow Ca2+ oscillations were significantly reduced by the application of thapsigargin in both putative-neurons and astrocytes , also reduced the transient rate in both types of cells (3-induced Ca2+ release from the intracellular Ca2+ store (ER) is the main origin of the slow Ca2+ oscillations.To determine the induction mechanisms of the slow Caonducted . The slo1 µM TTX , indicat3 production and is abundant in the striatum 2+ oscillations in a dose-dependent manner. When 30 µM MPEP was applied, the slow Ca2+ oscillations were almost completely blocked in astrocytes ((7.21±3.24) ×10−3 Hz for control and (0.07±0.05) ×10−3 Hz for 30 µM MPEP ; p = 0.0295 vs. control; Kruskal-Wallis test with Steel-Dwass post-hoc test), and the transient rate of the slow Ca2+ oscillations in putative-neurons was significantly reduced ((7.98±1.15) ×10−3 Hz for control and (4.16±0.97) ×10−3 Hz for 30 µM MPEP administration ; p = 0.0053 vs. control; Kruskal-Wallis test with Steel-Dwass post-hoc test). The effect of 30 µM MPEP on the slow Ca2+ oscillations in astrocytes was stronger than that in putative-neurons . Average of the transient rates of the slow Ca2+ oscillations in the condition of 10 µM MPEP administration did not change significantly compared with that in the control condition. However, 10 µM MPEP changed the some properties of the Ca2+ oscillations  ×10−2 with 10 µM MPEP administration, n = 316 events, 25 cells, 3 slices, 3 mice; mean ± SD; p = 0.0121; Kolmogorov-Smirnov test), but not in astrocytes. In the astrocytes, the decay slope was significantly decreased  ×10−4 with 10 µM MPEP administration, n = 42 events, 8 cells, 3 slices, 3 mice; mean ± SD; p = 0.0015; Kolmogorov-Smirnov test).mGluR5 is one of the receptors contributing to IPllations . The low2+ oscillations, LY367385, the specific antagonist of mGluR1, was applied. 2+ oscillations. The average values of the transient rates of the slow Ca2+ oscillations both in neurons and astrocytes did not alter with or without LY367385 significantly  ×10−3, and 1.60±1.37 ×10−3 under the control condition , and (1.25±0.77) ×10−2, 14±15 s, (1.58±0.92) ×10−3, and 1.51±0.92×10−3 with TTX administration , respectively. The distributions of the peak amplitude, duration, and rise slope of the Ca2+ transients in putative-neurons shifted toward smaller values under the TTX condition compared to the control condition  ×10−3, and (1.85±1.02) ×10−3 under the control condition  and (1.32±0.73) ×10−2, 14±9 s, (1.50±0.89) ×10−3, and (1.90±1.04) ×10−3 with TTX administration , respectively. These results indicated that blockade of action potential reduced the amplitude, the duration and the rise slope of the slow Ca2+ oscillations in only putative-neurons but not in astrocytes.To confirm the contribution of neuronal activity to the slow Ca2+ oscillations. Hence, antagonists of ionotropic glutamate receptors, 10 µM CNQX and 50 µM AP5, were applied. The distributions of those four parameters in putative-neurons and in astrocytes were shown in −2, 18±17.0 s, (1.75±1.51) ×10−3, and (1.35±1.36) ×10−3 under the control condition  and (1.56±0.90) ×10−2, 16±17 s, (1.70±1.37) ×10−3, and (1.40±1.48) ×10−3  with CNQX and AP5 administration, respectively. And, the median values of those four parameters in astrocytes were (1.64±0.71) ×10−2, 18±9 s, (1.90±1.02) ×10−3, and 1.50±1.05×10−3 under the control condition  and (1.58±0.62) ×10−2, 16±11 s, (1.70±1.05) ×10−3, and (1.60±1.19) ×10−3 with CNQX and AP5 administration , respectively. The distributions of those four parameters of the Ca2+ transients, except for a slight difference in the duration in putative-neurons  in response to activation of mGluR5 and released ATP activates adenosine or purinergic receptors in neurons 2+ signaling 2+ oscillations in neurons. The second possibility was that some kind of metabotropic receptors other than mGluR5 concerned the Ca2+ oscillations in putative-neurons. However, as described above, we could not find striking evidence for the contribution of another receptors on the slow Ca2+ oscillations in the putative-neurons. Verification of the mechanisms of the neuronal slow Ca2+ oscillations will require further investigation.The administration of MPEP almost completely blocked the slow Ca-neurons . There w2+ oscillations in both putative-neurons and astrocytes, cellular correlations of the Ca2+ elevation were reduced by the application of TTX  and large conductance (BK) channels, are expressed in the MSNs in striatum, and their currents represent between 30% and 50% of the sustained outward current 3R-dependent Ca2+ release from intracellular Ca2+ stores suppressed MSN firing via Ca2+-activated K+ channels 2+ oscillations may affect the firing rate via Ca2+-activated K+ channels 2+ oscillations reported herein may modulate the firing properties of MSNs on an intermediate time scale, one that is longer than an action potential and shorter than a circadian rhythm.Ca2+ oscillations  , duration , rise slope , and decay slope  of the Ca2+ transients in cumulative probability plots for putative-neurons and astrocytes, respectively. The solid and dashed lines indicate the distribution of each parameter for the Ca2+ transients under the control condition and during the administration of 10 µM MPEP, respectively. P- values from the Kolmogorov-Smirnov test shown in the plots. N. S.: no significant difference.(PDF)Click here for additional data file.Figure S22+ oscillations.Blocking mGluR1 did not alter the transient rate of the slow Ca A, Typical time courses of the slow Ca2+ oscillations during the administration of 50 µM LY367385 in putative-neurons and astrocytes. Horizontal bars under the time courses indicate the application period of the agents. Scale bar, 200 s, ΔR  = 0.02. B, Transient rates of the slow Ca2+ oscillations during the administration of 50 µM LY367385 in putative-neurons and astrocytes. The number of cells recorded is 26 putative-neurons and 9 astrocytes . The average values of the transient rates of the slow Ca2+ oscillations in putative-neurons were (7.59±0.95)×10−3 Hz under the control condition, and (6.94±0.76) ×10−3 Hz with LY367385 administration. The average values of the transient rates of the slow Ca2+ oscillations in astrocytes were (2.84±0.68) ×10−3 Hz under the control condition, and (3.61±0.53) ×10−3 Hz with LY367385 administration. The average values of the transient rates of the slow Ca2+ oscillations both in neurons and astrocytes did not alter with or without LY367385 significantly . N. S.: no significant difference.(PDF)Click here for additional data file.