It has been generally accepted that E-NTPase/E-NTPDase plays regulatory role in purinergic signalling, but other functions may yet be discovered.E-NTPase/E-NTPDase is activated by millimolar concentrations of Ca2+and Mg2+ in vivo.In this article it is proposed on the basis of published data that E-NTPase/E-NTPDase could play a role in the influx and efflux of CaAttenuation of extracellular Ca2+ influx by rat cardiac sarcoplasmic anti-E-NTPase antibodies and oligomerization studies on mammalian CD39 conclusively point towards the existence of a new channel in the membrane. Further studies on these properties of the E-NTPase/E-NTPDase may provide detailed mechanisms and identify the potential patho-physiological significance. Excitable cells exhibit an action potential, a substantial general depolarization of the plasma membrane, in response to depolarizing stimuli; influx of Ca2+ occurs via plasma membrane Ca2+ channels and/or release from sarco (endo) plasmic reticulum via ryanodine-receptor Ca2+ channels which regulate the excitation – contraction coupling i results from influx of Ca2+ across the plasma membrane and Ca2+ release from the endoplasmic reticulum. Ca2+ release from the SER depends on the binding of inositol 1,4,5-triphosphate (InsP3) to its receptor Ca2+channels, and also on Ca2+ binding to ryanodine receptor – Ca2+channels.In non-excitable cells, the increase in i play a role in ROCC opening [2+]I, increase in [cAMP]i i  and acti [cAMP]i .2+ channels must be a highly regulated event involving physical movement of channel components inclusive of the alteration in channel protein conformation; Also, an extracellular source of free energy (ΔG) could be of critical importance. This might be supplied by E-NTPase/E-NTPDase mediated hydrolysis of NTP/NDP. Co-ordination of this process might play a role in the opening of Ca2+ channels, independently of membrane depolarization or other factors.Opening of Cai. Extracellular metabolism  o/ [NDP] o hydrolysis might be utilized for opening the channel formed by the homo-oligomeric ENTPase/E-NTPDase.2+/Mg2+ transport processes, and the experimental evidence obtained in favor of the whole proposal.outlines the intracellular and extracellular factors that would influence the termination of the Ca2+/Mg2+ channel. Fig. 2+/Mg2+ channel gate. Fig. Membrane depolarization could locally alter protein conformation. This in turn could potentially induce post-translational modification in the (intracellular) monomer subunits of the E-NTPase/E-NTPDase, followed by translocation to the membrane (depending on the tissue type(s) and functional requirement(s)) Fig. . Fig. 2 NPPB (5-nitro-2-(3-phenyl propylamine benzoic acid), a potent inhibitor of CFTR (cystic fibrosis transmembrane receptor) and p-glycoprotein mediated ATP release o concentration is <1 mM and restores [Mg2+]i when the heart is perfused with Ringer buffer containing 5 × 10-7 M Mg2+. Failure of either of these two transport mechanisms may result in a rise in [Mg2+]i, impairing the contractile machinery of the myocardium i = Extracellular Nucleotide triphosphate; [NDP]0 = Extracellular Nucleotide diphosphate; ROCC = Receptor Operated Ca2+ channel; SER = Sarco (Endo) plasmic reticulum; i [cAMP]= Cytoplasmic or intracellular cAMP; PMCA = Plasma membrane Ca2+pump ATPase.VOCC = voltage-operated Ca2+ channel; LGCCS = Ligand gated non-specific cation channels. RACC = Stretch/Receptor activated non-specific Ca2+channels. SUR = Sulfonylurea Receptor Proteins. CD36 = Thrombospondin receptor on platelets. CD39 = Ecto Ca2+/Mg2+apyrase.


Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA- mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis.Stimulation of 2+-fluxes and the effect of their disturbance on chemotaxis and development of iplA- cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca2+. Chemotaxis was impaired when [Ca2+]i-transients were suppressed by the presence of a Ca2+-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca2+-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca2+-influx and liberation of stored Ca2+, respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca2+-uptake and Ca2+-release activated by fatty acids or Ca2+-ATPase inhibitors were similar to wild type. Mn2+-quenching of fura2 fluorescence allows to study Ca2+-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn2+ was necessary to observe agonist-induced Mn2+-influx. cAMP evoked a [Ca2+]i-elevation when stores were strongly loaded with Ca2+, again with a similar shift in sensitivity in the mutant. In addition, basal [Ca2+]i was significantly lower in iplA- than in wild type amoebae.We investigated Ca2+]i-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca2+-release from stores to Ca2+-entry in Dictyostelium. The iplA- strain retains the capacitative Ca2+-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure.These results support the view that [Ca Dictyostelium discoideum proceeds by an oriented migration of the amoebae towards a source of the attractant cAMP which is synthesized and released periodically by cells in the center of the aggregate. Stimulation with cAMP activates liberation of stored Ca2+ and extracellular Ca2+-entry [2+]i-transient i-elevation is necessary and release of stored Ca2+ is sufficient for oriented migration [2+]i-increase is essential for chemotaxis was called into question by analysis of a cell line where the iplA gene was disrupted by homologous recombination i-elevation were detected [iplA gene product is central to the regulation of [Ca2+]i and that its presence and thus the presence of an agonist-activated [Ca2+]i-increase is not necessary for proper chemotaxis and development. However, agents that interfere with IP3-receptor mediated signaling such as XestosponginC i was not detectable resulted in the conclusion that [Ca2+]i-changes were not necessary to accomplish chemotaxis [2+]i-gradient for a given response. If in Dictyostelium a [Ca2+]i-increase was necessary for chemotaxis, the presence of a Ca2+-chelator in the cytosol should impair orientation and/or migration. In a previous study, we had introduced the Ca2+-chelator BAPTA and its derivatives into the cytosol of wild type amoebae which indeed had inhibited chemotactic migration and reduced chemotactic orientation [2+-indicator Fura2-dextran to clamp [Ca2+]i and loaded the indicator into wild type and mutant cells in the absence of external CaCl2. The treatment affected chemotactic performance of wild type as well as iplA- amoebae. Lack of extracellular CaCl2 during the loading process induced strong rounding of the amoebae and loss of migration. Figure iplA- cells . These results show that not only in wild type but also in the iplA- cell line the ability to orient and to migrate in fact depends on an agonist-activated [Ca2+]i-elevation.The observation that aggregation occurred in the mutant cell line although a cAMP-activated increase in e thus reflects efflux of Ca2+ from storage compartments. Both, cAMP and arachidonic acid activated release of stored Ca2+ . The amount of Ca2+-efflux from stores after cAMP stimulation was 61% of that found in wild type cells  by thapsigargin. XestosponginC that inhibits Ca2+-uptake and activates Ca2+-release in wild type i level in iplA- amoebae was significantly lower than in wild type and amounted to an average of 36 ± 3 nM (mean ± s.e.m. of 8 determinations in 3 independent experiments) as compared to 50 ± 2 nM in wild type .In wild type cells treatment with EGTA augments responsiveness and cAMP-elicited [Car [Ca2+] . Howeverobserved . In accoobserved  this con2+-stores, we preincubated cells with 1 mM CaCl2 for 4 h. After this treatment a [Ca2+]i-elevation upon addition of cAMP was detected in 60% of wild type amoebae even at low extracellular [Ca2+] levels, i.e. when the buffer used to wash the cells had been supplemented with only 1 μM CaCl2 . Again, these conditions were not effective in iplA- cells; rather, the sensitivity of the mutant was shifted to higher Ca2+ concentrations as had been found with Mn2+-quenching experiments. We preincubated iplA- amoebae with 20 mM CaCl2 for 3 h; after washing thoroughly, a cAMP-induced [Ca2+]i-elevation in the presence of 1 mM CaCl2 was observed . Yet, this was a rare event (once in 31 determinations).For stronger loading of CaCl2 Fig. . Startinved Fig.  in 28% o2+]i-changes for chemotaxis has been questioned by Traynor et al. [iplA- mutant cell line favouring insignificance of [Ca2+]i for the chemotactic response. The authors had observed formation of fruiting bodies even though neither 45Ca2+-fluxes nor an agonist induced [Ca2+]i-elevation were detectable. The discrepancy between our view that a [Ca2+]i-elevation is necessary for a proper chemotactic response [2+]-regulation of the iplA- mutant in detail. In particular, we tested not only basal and cAMP-activated ion fluxes but also capacitative Ca2+-entry which is induced by emptying internal Ca2+-stores via preincubation of amoebae with EGTA i it is possible that the continued presence of 10 mM EGTA or CaCl2 for many hours affects basal levels of [Ca2+]i which in turn might mediate effects on gene expression as was shown for prolonged incubation of cells with BHQ i are controlled by the iplA gene product. Besides studying agonist-induced Ca2+-fluxes we also investigated capacitative Ca2+-entry and found that this type of influx was similar in mutant and wild type cell suspensions. We obtained equivalent results by testing Mn2+-quenching of Fura2-dextran fluorescence which showed that capacitative entry is independent of the iplA gene product.By using a Ca2+-sensitive electrode, we found that in the iplA- mutant the agonist cAMP and also AA did activate Ca2+-entry into intact cells. The difference between the data published by Traynor et al. [2+-fluxes that we observed was considerably lower than in wild type cells and detectable at low extracellular [Ca2+] only. The 45Ca2+-flux studies had been performed at 100 μM external CaCl2; so the fraction of 45Ca2+ entering the cells was presumably too low to be detected reliably. Moreover, we found cAMP- and AA-induced Ca2+-release from stores in cells with permeabilized plasma membranes. These data show that cAMP-induced Ca2+-release from stores in iplA- cells is functional. However, much like the Ca2+-influx, agonist-activated liberation from stores was smaller than in wild type amoebae. In line with these results are the findings using Mn2+-quenching to assay ion fluxes in intact single cells: higher doses of Mn2+ were necessary to detect influx.On the other hand, using the Car et al.  and our 2+-influx: one type being activated by emptying of the stores and sustaining capacitative Ca2+-entry which is unaffected in iplA- cells and the other one mediating agonist-induced Ca2+-fluxes, the latter being under the control of the iplA gene product. The view that there are two strictly separated ion channels seems unlikely as under conditions of emptied stores cAMP-activated Mn2+-quenching occurred in the mutant as well. (ii) The same channel(s) mediate capacitative and agonist-activated fluxes but upon stimulation with agonists it cannot be addressed properly when iplA is disrupted. This implies a role of the protein in the liberation of Ca2+ from the stores which is a prerequisite for the triggering of Ca2+-entry [2+-influx and the generation of a full [Ca2+]i-increase is impaired. The results of the experiments where stores were strongly loaded with Ca2+ prior to stimulation support this notion. In this situation release from stores should be augmented. Indeed, in both, wild type and mutant cells, cAMP-activated [Ca2+]i-elevations occurred at an extracellular [Ca2+]  There are two types of channels responsible for Ca2+-entry . In the see Fig.  where wi2+-entry upon liberation of Ca2+ from the stores. From our data we conclude that in Dictyostelium these signals are different when the stores are emptied by EGTA or by agonist-activated signaling cascades. Otherwise one cannot explain normal capacitative Ca2+- and Mn2+-influx induced by EGTA-treatment and a requirement for 100 fold higher ion concentrations to induce Mn2+-entry by cAMP. If indeed the iplA gene product constitutes an IP3-receptor like channel that is located on membranes of stores the physical coupling of the receptor to channels in the plasma membrane as a mechanism to activate extracellular Ca2+-entry [3-sensitive store but also other stores and thus exerts a much more general effect on the cells. Studies using microarrays should reveal whether the expression of other genes is affected by the absence of iplA and thus might give a clue how [Ca2+]i is regulated in the mutant although one type of Ca2+-store is malfunctional.An as yet unresolved issue is the mechanism that induces Ca2+-entry  should b2+ fluxes and regulation of Ca2+ homeostasis take place in the iplA- mutant and that chemotaxis and development of the mutant are sensitive to disturbance of the Ca2+ homeostasis. In wild type cells and in cells lacking the iplA gene changes in [Ca2+]i are necessary to orient and to migrate chemotactically; their abolition causes loss of chemotaxis towards a cAMP source. The iplA gene product exerts a crucial role in the control of basal [Ca2+]i and of agonist induced Ca2+-fluxes. It is not required to activate capacitative Ca2+-influx. Thus the mechanisms responsible for capacitative and agonist-activated Ca2+-fluxes are different.Our results show that CaFura2-dextran and Fura2 were purchased from MobiTec; cAMP was from Boehringer.D. discoideum wild type strain Ax2 and the iplA- cell lines HM1049 and HM1038 (kindly provided by Dr. D. Traynor) were cultured as described e in cell suspensions was recorded as described elsewhere i-imaging was performed at t7–t8 as described  with Fura2.Analysis of vesicular Caescribed . In brie5 cells/ml in H5-buffer were pipetted onto a glass coverslip and allowed to settle for 60 min. Chemotaxis was recorded on a video recorder for 30–45 min. Chemotaxis was also assayed in the presence of EGTA or CaCl2; then amoebae were incubated in the respective agents for 60 min before they were challenged with cAMP. Images were digitized and the behaviour of the cells was analyzed using a computer program written for this purpose. For determination of cell velocity, a square area of interest (AI) of variable size  was placed at the perimeter of the cell in the first image digitized at the beginning of the assay. In the next image  the program analyzed an area larger than the AI (this area was defined by adding a given number of pixels on each side of the AI) for a pattern that resembled that of the AI; when such a pattern was found then the AI was placed on this new spot. The difference between the position of the AI in the first image to that in the second image was expressed as a vector of a given length. The changes in cell shape during migration were compensated by updating the pattern within the AI for every consecutive image analyzed. Calibration of the system allowed to convert the sum of the vector lengths to the distance in μm that the cells had migrated at the end of the experiment and to calculate the velocity of the amoebae. To test the effect of the intracellular presence of a Ca2+-buffer on chemotaxis, cells were loaded with Fura2-dextran (5 mg/ml in the loading solution) by electroporation in the absence of added external Ca2+. The amount of indicator present in the cytosol is in the range of 2–5% of the concentration present during electroporation iCytosolic free Ca2,5-di-(t-butyl)-1,4-hydroquinone: BHQArachidonic acid: AAArea of interest: AI2+] in cell suspensions and participated in the design of the study. DFL participated in the recordings of extracellular [Ca2+] and the design of the study. KBR analyzed chemotaxis and differentiation of wild type and mutant cells. KH performed [Ca2+]i measurements. DM analyzed fluxes in partially purified storage compartments and was involved in the design of the study. CS participated in [Ca2+]i measurements, did Mn2+-flux studies, participated in the design of the study and wrote the manuscript.RS recorded extracellular [CaiplA- cells in H5-buffer. Images of cells at t5 were captured every 15 sec for 20 min.Spontaneous cell motility of wild type and Click here for file4–t5). Images of cells at t5 were captured every 15 sec for 20 min in the continued presence of EGTA. Cells were rounded and extended smaller pseudopods than under control conditions.Basal cell motility after preincubation of wild type and mutant amoebae with 10 mM EGTA for 60 min (tClick here for file2 for 80 min (t4–t5.3) is shown. Images of cells at t5.3 were captured every 15 sec for 20 min in the continued presence of CaCl2. The behaviour of treated cells was not different from control amoebae.Cell motility of wild type and mutant cells after preincubation with 10 mM CaClClick here for file


Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/β-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways.The Frizzled  and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/β-catenin pathway or Fz/planar cell polarity (PCP) signaling in  Differential subcellular localization of Fz1 and Fz2 receptors contributes to signaling specificity within the Wnt/Fz signaling pathways Pattern formation in multicellular organisms relies on inductive signaling events. Several evolutionarily conserved ligand–receptor combinations and associated signal transduction pathways are used again and again during development to induce tissue- and cell-type-specific responses. Thus, context-dependent signaling specificity is an important prerequisite for patterning and differentiation. Although for many signaling pathways the flow of information is largely established, the underlying signaling specificity mechanisms remain unclear.Drosophila; reviewed in Drosophila and vertebrates  that points distally, and in the eye PCP is manifest in the regular ommatidial arrangement in the anteroposterior and dorsoventral axes (reviewed in Members of the Frizzled (Fz) family of seven-pass transmembrane proteins act as receptors for the Wnt family of secreted ligands . In mosttebrates . PCP is Drosophila, Fz (for clarity we will refer to it as Fz1) and Fz2 are functionally redundant receptors for Wg, activating the canonical Wg/Arm cascade  -Fz(s) combinations are associated with either canonical or Fz/PCP signaling. However, the specificity is not simple. For example, although Wnt5a and Wnt11 cause embryonic phenotypes associated with the Fz/PCP-like pathway , coexpredppGal4 control) is localized throughout the apical–basal axis of larval imaginal disc epithelia, and extracellular Wg binds to Fz2 predominantly at the basolateral membrane . Similarly, overexpressed Fz2 . In addition, we show that the seven-pass transmembrane region contains elements that are critical for PCP signaling. Based on our data, we propose a model in which subcellular localization, possibly through the association of Fz with other membrane proteins such as coreceptors, is a critical aspect in regulating the signaling readout and specificity within the Wnt/Fz signaling pathways.dpp-Gal4 control also displays a strong enrichment in the apical region of the disc epithelium  in wing imaginal discs. Fz1 and Fz2 were subdivided into three parts: (1) the N-terminal Wnt-interacting cysteine-rich domain (CRD), (2) the remaining proximal extracellular domain and 7 transmembrane region and loop region (collectively referred to as 7-TM), and (3) the intracellular C-tail. All chimeric proteins were Myc-tagged between the CRD and 7-TM region . In sumdpp-Gal4 control in wing imaginal discs caused wing cell hairs to point away from the expression domain  phenotype ; Table 1n domain B. This in domain . Expressn domain ; Table 1n domain ; Table 1Taken together, these experiments demonstrate that (1) apical Fz1 localization correlates with higher levels of Fz/PCP signaling activities and (2) the 7-TM region of Fz1 is critical for effective PCP signaling.Next we wished to determine which part of the C-tail of Fz1 or Fz2 is responsible for the difference in subcellular localization. The protein sequences of the Fz1 and Fz2 C-tails are homologous over the first 29 amino acids (45% identity), but Fz2 is longer by an additional 61 amino acids . The apiFirst, we mutated several Fz1–1-1–specific residues to those of Fz2, or deleted conserved amino acid stretches within the Fz1–1-1 C-tail see , and wheSecond, we tested whether sequences within the extended Fz2 C-tail have an effect on localization or PCP signaling. We introduced a stop codon after the L633 residue of Fz2 (corresponding to the position of the stop codon in Fz1) in Fz1–1-2 and Fz1–2-2 chimeras A and 2E.These same chimeras were tested in the functional assay for PCP signaling activity. Strikingly, expression of Fz1–1-2S caused a phenotype very similar to that of Fz1–1-1 H, but diTo test whether the extension within the Fz2 C-tail can more generally block apical localization, we added the Fz2 extension on to Fz1–1-1 E, resembtubulin promoter [tub] ) were also tested for their ability to rescue the −fz eye and wing PCP phenotype. tub-Fz1–1-1 and tub-Fz1–1-2S  fully rescue the −fz loss-of-function (P21fz/R52fz) phenotype in both the eye and wing   . Similaron (APF) . These don (APF) , suggestE59fmi clones in larval wing imaginal discs. Fz1–1-1 is localized apically in E59fmi mutant cells in third instar imaginal discs, indistinguishable from its localization in wild-type tissue  often causes wing notching and loss of wing margin bristles , with 65% of wings showing large areas of margin bristles missing and severe wing notching  we observe very strong wing notching effects and a general reduction of the posterior wing compartment , suggesting that Fz1–1-2 contains substantial PCP signaling activity.Is the apical localization of Fz required for PCP signaling? The Fz1–1-2 chimera, which is distributed ubiquitously within the apical–basolateral membrane, only partially rescues the ling see D. In conssay see . Taken t−fz in the eye), the relative difference might be sufficient for the partial rescue.Because both GOF and loss-of-function studies indicate that the Fz1 7-TM region is critical for Fz1 function, Fz1–1-2 is expected to have Fz/PCP signaling activity, although with altered subcellular distribution. Thus, the remaining PCP signaling activity of Fz1–1-2 seen is probably due to the presence of some of this protein in apical regions. It is difficult to determine how much of Fz1–1-2 is actually localized to this membrane region. Since the immunohistochemical staining indicates that it is not excluded apically, we assume that Fz1–1-2 has enough apical localization to participate when PCP signaling is initiated. It has been suggested that wing cell orientation does not depend on absolute Fz levels, but instead depends on relative Fz/PCP activity differences in a Fz activity gradient across a field . Thus, atub-fz1–1-2 rescues the −fz phenotype better in the wing than in the eye, whereas there is no apparent difference in rescue activity between the eye and the wing for tub-fz1–1-1 or tub-fz1–1-2S. The difference could be due to the observed nonautonomous PCP signaling effects in the wing , and it36 h APF , suggestfmi dependent. This result suggests that Fmi and Fz1 get recruited to apical junctions independently. During later stages, Fmi and Fz1 then affect each other's localization through PCP signaling. At this point, it remains unclear which molecules initially recruit Fz1 into the apical junctional region.We showed here that initial apical localization of Fz1, preceding both stages of PCP signaling, is not Secreted Wg mainly binds to Fz2 at basolateral membrane regions of the wing epithelium , indirecfz, fz2 double mutant phenotype . UAS-Fz1–2-1 was constructed by combining the Fz1 CRD (Fz1 residues 1–166) with the Myc tag, the Fz2 7-TM region (amino acids 220–617), and the Fz1 C-tail (amino acids 558–585). A HindIII site (generated in vitro) was used to combine Fz1 CRD with the Fz27-TM region. An XhoI site was used to link the Fz2 7-TM region with the Fz1 C-tail. C-tail mutation constructs of Fz1 were generated through PCR-based site-directed mutagenesis . Fz1–1-2S and Fz1–2-2S were generated by introducing a stop codon after residue L633 of Fz2. Fz1–1-1C2 was generated by introducing a BsiWI site at the residues R574–T575 of Fz1 and R626–T627 of Fz2 (the RT residues remain the same by this mutagenesis). This added the Fz2 amino acids 627–694 to the Fz1 C-tail at the RT residues. The respective UAS transgenic flies were generated by standard procedures.dpp-Gal4, en-Gal4, or omb-Gal4 (tub-promoter-driven Fz chimeric constructs were generated by cloning the respective Fz1/2 constructs into the Casper4-tub vector (containing a 2.4-kb tub promoter fragment in Casper4—a kind gift from Stephen Cohen). P21fz and R52fz are null alleles of fz  in 0.1% Triton X-100 PBS for about 30 min or longer, and then mounted in 80% Glycerol PBS. Eye embedding and sectioning was performed as described by


Coordination of [Ca2+]i events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca2+]i activity in individual cells and a robust coordination of [Ca2+]i signals in neighboring astrocytes. These findings indicate potential neuron–glia communication in the intact brain.Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca Two-photon laser scanning microscopy was used to image calcium concentration changes in astrocytes in the cerebral cortex of anesthetized rats Astrocytes are nonneuronal cells of the brain with some known and hypothesized functions  in the cytosol undergoes large changes spontaneously or in response to various physiological and pharmacological manipulations, such as mechanical stimulation, membrane potential depolarization, and activation of metabotropic glutamate receptors  and/or metabotropic glutamate receptors  ester in juvenile rats in vivo. We find that [Ca2+]i dynamics in astrocytes is rather quiescent during baseline anesthesia. However, increased population bursting, brought about by attenuating γ-aminobutyric acid (GABAA) receptor-mediated neurotransmission, leads to increased magnitude [Ca2+]i surges, and the [Ca2+]i changes become more strongly coordinated in neighboring astrocytes.We have used two-photon laser scanning microscopy (2-PLSM) to monitor cytosolic CaTo examine the depth of penetration of the Fluo-4 AM, coronal brain slices (300 μm thick) were acutely prepared after the residual dye was washed off from the craniotomy. A large number of cells below the craniotomy showed fluorescence labeling . On the 2+]i transients , cells with astrocytic appearance (n = 185) were selected for long-term (10–20 min) monitoring. For quantitative studies, three states of [Ca2+]i activity were distinguished: (a) quiescent state with very slow (less than 0.025 Hz) oscillations of baseline fluorescence level, (b) [Ca2+]i spikes , and (c) [Ca2+]i plateau potentials . [Ca2+]i spikes and [Ca2+]i plateau potentials were automatically detected. In the control (baseline) condition, 11% of astrocytes had at least one spike event, and 52% had at least one plateau event in 10 min. The mean frequency of [Ca2+]i spikes among the cells that had at least one [Ca2+]i spike was 0.121 ± 0.098 per minute  and the mean frequency of [Ca2+]i plateau was 0.118 ± 0.058 per minute (mean duration: 160.4 ± 114.9 s).In our initial experiments, we made a large number of line scans (sampling rate ∼200 Hz) of dye-loaded cells to examine whether some of them were neurons. We never observed short-lasting [Ca2+]i dynamics were affected by increasing neuronal activity, we induced regularly occurring population bursts by local application of bicuculline (n = 7 rats). Large amplitude (0.69 ± 0.26 mV) synchronous field events (approximately 100 ms) occurred at relatively regular frequency (0.15 ± 0.06 Hz), associated with multiple unit discharges. No significant difference was observed in average heartbeat frequency between the control sessions and bicuculline sessions .To investigate whether the baseline values of [Ca2+]i in astrocytes (n = 214 cells). First, the incidence of [Ca2+]i spikes and plateau events was counted in the absence and presence of bicuculline-induced population bursts. Under bicuculline condition significantly more astrocytes had [Ca2+]i spikes , whereas the probability (52% versus 54%) of plateau potentials did not differ significantly. The mean duration of plateau potentials, however, was significantly longer  after bicuculline treatment. Among the cells that exhibited at least one spike or plateau event, there was not a significant difference in frequency of the event occurrences . Thus, the major difference between control and bicuculline conditions was the higher proportion of active astrocytes under bicuculline.We used two methods to evaluate the effect of neuronal activity on [Ca2+]i changes in the frequency domain. The 0ΔF/F trace was considered as a continuous process, and the power spectrum estimate was calculated with a multi-taper method for each astrocyte and averaged across cells. There was a general increase of power at all frequencies in bicuculline-treated animal. The most consistent significant increase (p < 0.05) of power appeared in the frequency range of 0.10–0.24Hz, reflecting the increased incidence of [Ca2+]i spikes. Short-term cross-correlation of neuronal field bursts and [Ca2+]i signals (± 10 s) did not show a significant time-locked relationship  and distant cell pairs (greater than 50 μm). In control conditions, the temporal correlation of [Ca2+]i signals in neighboring pairs was somewhat larger than in distant pairs, but this difference was not significant . Nevertheless, [Ca2+]i signals in astrocytes were not completely random, since the cross-correlograms had wide central peaks at the 10–100 s scale  was similar to those in the control condition. However, synchrony between local pairs  increased several-fold relative to both distant pairs under the same condition  and to local pairs in the baseline condition .In individual experiments, propagation of synchronous activity could be observed visually  but the  s scale B. In conuculline C. Correln = 1,512 cell pairs, r = 0.019, p = 0.46). In contrast, a significant negative correlation was found between the synchrony of [Ca2+]i signals in the bicuculline condition .Using a different approach, the magnitude of the zero-timelag correlation coefficient for each cell pair was plotted against distance between the cell pairs. Under control condition, no notable relationship was observed between these variables  and NMDA receptors  in close contact with small brain vessels . It has ompounds . Furtherompounds . These fMale and female rats, 12–16 d postnatal (P12– P16), of the Sprague–Dawley strain were used in these experiments. Animals were deeply anesthetized with 1.7 g/kg urethane. An outline of the craniotomy above the primary somatosensory (barrel) cortex was marked with a dental drill. A metal frame, similar to what has been described in 3, 1.1 mM NaH2PO4, 2 mM CaCl2, 1 mM MgSO4; pH adjusted to 7.4) and mixed for a further 15 min. A small volume (up to 12 μl) of the dye-containing solution was applied to the cortical surface by a micropipette. The solution was retained in place by a small piece gelfoam. The unbound dye was removed 45–60 min after the surface application of Fluo-4 AM by irrigating the exposed surface with ACSF for at least 10 min. The craniotomy was then covered with 1% agar dissolved in phosphate-buffered saline (pH 7.4), and a glass coverslip was placed on a metal frame. This arrangement allowed access for a glass recording electrode from the side. Juvenile rats (P13–P15) were used because we found in preliminary experiments that in adult animals, mostly vascular cells were loaded with the current protocol.Fluo-4 AM  was mixed with 2 μl of Pluronic  and 5 μl of dymethyl sulfoxide  for 15 min. The solution was then diluted in 18 μl of artificial cerebrospinal fluid (ACSF)  was monitored continuously. The R wave of EKG was used to monitor brain pulsation-derived movement of artifacts during imaging. Population bursts of cortical neurons  were excluded from the analysis. For display purposes, the signal was convolved with a Hanning window of order three to smooth the signal trace. Power spectra of fluorescent signals were computed using the multi-taper method (NW = 4). For the calcium event detection, 0ΔF/F signal was convolved with a Hanning window of order 15. “Spike” events were defined as transient increase of 0ΔF/F signal exceeding 20%, lasting 5–50 s. “Plateau” events were defined as sustained increase of 0ΔF/F (greater than 20%) signal longer than 50 s. Peak amplitudes of both spike and plateau events required an increase of at least 50% 0ΔF/F from the onset of events. Calcium events were automatically detected with the above detection. Cross-correlation between cell pairs was computed by normalizing the 0ΔF/F signals to unity  so that the computed values represent the correlation coefficient between the two signals at a given timelag. All numbers are indicated as mean ± standard deviation, unless otherwise noted.Fluorescence signal was quantified by measuring the mean pixel value of a manually selected somatic area for each frame of the image stack using ImageJ software. The values were exported to MatLab and the fluorescence change 2PO4, 155.17 mM NaCl, 2.96 mM NaHPO4, pH approximately 7.4), the slices were treated with S100B antibody  in Triton X-PBS (0.5% Triton X in PBS) overnight. The sections were subsequently washed three times in PBS, followed by incubation with the secondary fluorescent antibody  in Triton X-PBS solution for 2 h. Simultaneous viewing of the two image stacks allowed a systematic comparison of the extent of overlap between Fluo-4 loading and S100B immunoreactivity  because this antibody stains the somatic region of astrocytes as well as its processes . Followiactivity .Video S1The primary somatosensory cortex (P15) was stained with Fluo-4 AM in vivo and subsequently imaged in vitro. Acute slices (approximately 300 μm thickness) were cut in cold ACSF after the cells were loaded in vivo. .(49 MB AVI).Click here for additional data file.Video S2Same slice as shown in (48 MB AVI).Click here for additional data file.Video S3Detailed imaging of in vivo-loaded acute slice preparation of the primary somatosensory cortex . Z step = 1 μm; scale bar = 20 μm.(39 MB AVI).Click here for additional data file.Video S4Empty circles in layers II/III, presumed unloaded neurons (note their absence in layer I). The loaded cells have typical glial morphological appearance. Z step = 1 μm; scale bar = 50 μm.(50 MB AVI).Click here for additional data file.Video S5Acute slices (300 μm thickness) were prepared from the in vivo Fluo-4 AM-loaded somatosensory cortex. The slices were subsequently incubated in EDAC containing saline followed by formalin fixation. The loaded astrocytes were identified by examination at various depths and numbered (left). Next, the slices were processed for immunocytochemistry with astrocyte marker S100B. Depth scans (1 μm between the frames) were taken again to determine immunoreactivity of cells with S100B (right movie). An overlapping set of the cells was identified to be S100B-immunoreactive, indicating that nearly all Fluo-4 AM-loaded cells were astrocytes.(5 MB AVI).Click here for additional data file.Video S6Movie taken from a P14 rat. Image was taken with 2 Hz sampling rate for 10 min and compressed to 36 s for display purposes. Note spatial- and light-emission-stability of the recorded cells. Note also that at frames approximately 9 s and 15 s, two of the astrocytes in the middle display transient increased fluorescence. Scale bar 50 micro μ.(55 MB AVI).Click here for additional data file.


In this study we have used whole cell patch clamp recording and fura-2 Ca2+ imaging to characterise the actions of pregabalin on the electrophysiological properties of cultured dorsal root ganglion (DRG) neurones from neonatal rats. The aims of this study were to determine whether pregabalin and gabapentin had additive inhibitory effects on high voltage-activated Ca2+ channels, evaluate whether the actions of pregabalin were dependent on GABA receptors and characterise the actions of pregabalin on voltage-activated potassium currents.Gabapentin and pregabalin have wide-ranging therapeutic actions, and are structurally related to the inhibitory neurotransmitter GABA. Gabapentin, pregablin and GABA can all modulate voltage-activated Ca2+ current in cultured DRG neurones. The residual Ca2+ current recorded in the presence of pregabalin was sensitive to the L-type Ca2+ channel modulator, Bay K8644. Saturating concentrations of gabapentin failed to have additive effects when applied with pregabalin, indicating that these two compounds act on the same type(s) of voltage-activated Ca2+ channels but the majority of Ca2+ current was resistant to both drugs. The continual application of GABA, the GABAB receptor antagonist CGP52432, or intracellular photorelease of GTP-γ-S had no effect on pregabalin-induced inhibition of Ca2+ currents. Although clear inhibition of Ca2+ influx was produced by pregabalin in a population of small neurones, a significant population of larger neurones showed enhanced Ca2+ influx in response to pregabalin. The enhanced Ca2+ influx evoked by pregabalin was mimicked by partial block of K+ conductances with tetraethylammonium.Pregabalin (25 nM – 2.5 μM) inhibited 20–30% of the high voltage-activated Ca+ currents, the inhibitory effect of pregabalin was prevented with apamin. The delayed enhancement of K+ currents was attenuated by pertussis toxin and by intracellular application of a (Rp)-analogue of cAMP.Pregabalin produced biphasic effects on voltage-activated K2+ and K+ channels. The pharmacological activity of pregabalin is similar but not identical to that of gabapentin. The actions of pregabalin may involve both extracellular and intracellular drug target sites and modulation of a variety of neuronal conductances, by direct interactions, and through intracellular signalling involving protein kinase A.Pregabalin reduces excitatory properties of cultured DRG neurones by modulating voltage-activated Ca The activation of KATP channels is implicated in these responses because not only does glibenclamide, a KATP channel antagonist, attenuate the gabapentin response but also pinacidil, a KATP channel agonist, mimics the response and did not have additive effects with gabapentin [The mixed responses to pregabalin seen in the Cabapentin . Additiobapentin .+ currents observed. The inhibitory actions of both gabapentin and pregabalin on K+ currents recorded from DRG neurones may not reflect a direct action of these drugs on K+ channels. The effects of apamin, which prevented the inhibition of outward current by pregabalin indicated that small conductance Ca2+-activated K+ channels were involved in the response. This is consistent with the effects of gabapentin seen in isolated cortical neurones [2+ channels may underlie the inhibition of outward K+ current. A reduction in Ca2+ influx would lead to reduced activation of Ca2+-activated K+ channels and therefore a smaller outward current. If this indirect effect of pregabalin resulted in a disproportionate reduction in Ca2+-activated K+ conductances this may result in prolonged depolarisation. In turn a prolonged depolarisation may maintain activation of Ca2+ channels that are insensitive to pregabalin and an increase in Ca2+ influx as seen in a sub-population of larger DRG neurones in the Ca2+ imaging experiments. It is not clear why gabapentin, which produced modest inhibition of K+ currents, did not also produce enhancement of K+-evoked Ca2+ transients. It may be explained by a balance between inhibition of Ca2+ influx through voltage-activated channels and modulation of Ca2+ flux through inhibition of K+ conductance. Consistent with this hypothesis, gabapentin appears to be less effective than pregabalin at modulating K+-evoked Ca2+ transients in DRG neurones. This is supported by our previous study that showed that gabapentin did not significantly alter the peak Ca2+ transients [Mixed or biphasic responses to gabapentin and pregabalin were seen, with both inhibition and enhancement of Kneurones . The preansients .+ currents by gabapentin and pregabalin is a powerful mechanism for reducing cell excitability. The slow development of this response and its gradual decline suggested that intracellular signalling events were involved. Pregabalin produces enhancement of K+ currents when applied either inside or outside DRG neurones. The delay in the development of these responses is less with intracellular application of pregabalin, suggesting that it may have an intracellular site of action. This effect may also depend on drug uptake into cells via L α-amino acid transporters and the presence and activity of these transporters may greatly influence cell sensitivity to gabapentin and pregabalin. Although it is not clear how gabapentin and pregabalin might activate PKA, it is a candidate target site. Our previous work showed that inhibition of Ca2+ currents by gabapentin was sensitive to cAMP analogues that activated or inhibited PKA [+ currents by pregabalin. In the literature there are a number of reports of PKA altering neuronal excitability and modulating K+ conductances. PKA is involved in pain signalling, playing a role in prostaglandin-induced activation and sensitization of DRG neurones [+ conductances by prostaglandin E-2 [+ channel activity [+ and Ca2+ channels by a synthetic cannabinoid receptor agonist and the sensitivity of these responses to Wiptide, a PKA inhibitor [The delayed enhancement of voltage-activated Kited PKA . Similarneurones . PKA actneurones  and A-tyneurones  and is indin E-2 . Our resndin E-2 , large cactivity  and cannactivity . In the nhibitor . Howevernhibitor  could de2+ and K+ conductances may underlie this characteristic.An interesting feature of some actions of gabapentin and pregabalin is that their effects often outlast the period of drug delivery. Maintained intracellular signalling effects of these drugs and the delayed responses involving PKA modulation of Ca2+ channels with resulting reduced activation of Ca2+-activated K+ currents and effects on voltage-dependent ion channel availability. Although the delayed enhancement of voltage-activated K+ channels could contribute, this effect appears to develop over a longer period. These actions may contribute both to anticonvulsant effects of gabapentin and pregabalin as well as their therapeutic actions in pain disorders. It is particularly worth noting that the inhibition of Ca2+ influx is most consistently seen in small diameter cultured DRG neurones that are likely to be the pain fibres.Gabapentin has previously been shown to attenuate high frequency action potential firing both in CNS neurones  and in c+-evoked neurotransmitter release in CNS neurones [2+ channels through drug interactions with α2δ subunits, reduced activation of Ca2+-dependent conductances and modulation of both Ca2+ and K+ channels through metabotropic mechanisms that involve PKA. Interestingly, both gabapentin and pregabalin inhibit release of sensory peptides (substance P and CGRP) this may provide a mechanism of action down-stream of ion channel modulation. This effect is only observed after inflammation and may contribute to their analgesic properties of gabapentin and pregabalin [Our data adds to previous work that has assessed the actions of gabapentin and pregabalin on Kneurones  and the neurones ,9,42-44.egabalin .2+ channels in DRG neurones are targets for antinociceptive agents but the pharmacology of these channels remains to be fully exploited in pain therapy [Ca therapy ,47. Furt3 (14 mM), streptomycin (50 μg/ml) and penicillin (50 IU/ml). The cultures were maintained for up to two weeks at 37°C in humidified air with 5% CO2. Cultures were re-fed with fresh media after 5 days.One to four-day old Sprague-Dawley rats were decapitated and dorsal root ganglia removed. DRG neurones were dissociated enzymatically (0.125% collagenase for 13 minutes and 0.25% trypsin for 6 minutes) and mechanically (trituration). Primary cultures of DRG neurones were plated on lamin-polyornithine coated coverslips and bathed in Ham's F-14 culture medium  containing 10% horse serum (Gibco), low NGF , NaHCO2+ channels and other potential effectors [In some experiments DRG neurones were pre-treated with pertussis toxin  to ADP-ribosylate the α subunits of certain G-proteins. This prevents pertussis toxin-sensitive G-protein being activated through a range of G-protein coupled receptors and inhibits coupling to voltage-activated Caffectors ,48.2+ imaging were used to measure the inhibitory actions of pregabalin and gabapentin on Ca2+ entry through voltage-activated channels. Initially, multiple firing properties of a sub-population of DRG neurones were studied using a patch pipette solution containing in mM: KCl, 140; EGTA, 5; CaCl2, 0.1; MgCl2, 2.0; HEPES, 10.0; ATP, 2.0. The extracellular solution containing in mM: NaCl, 130; KCl, 3.0; CaCl2 2.0; MgCl2, 0.6; NaHCO3 1.0, HEPES 10.0 and glucose 5.0. For recording Ca2+ channel currents the patch pipettes were filled with CsCl-based solution containing in mM: 140 CsCl, 0.1 CaCl2, 5 EGTA, 2 MgCl2, 2 ATP, 10 Hepes. The pH and osmolarity of the patch pipette solutions were corrected to 7.2 and 310–320 mOsm.l-1 with Tris and sucrose. The extracellular bathing solution used to study Ca2+ currents contained in mM: 130 choline chloride, 2 CaCl2, 3 KCl, 0.6 MgCl2, 1 NaHCO3, 10 HEPES, 5 glucose, 25 tetrethylammonium chloride, 0.0025 tetrodotoxin (Sigma). The pH and osmolarity of this extracellular bathing solution was corrected to 7.4 and 320 mOsml-1 with NaOH and sucrose respectively. The recording solutions used in these experiments were designed to attenuate voltage-activated Na+ and K+ currents and isolate voltage-activated Ca2+ currents. The range of values for the series resistance under our recording conditions was from ~8 to 15 MΩ. Pregabalin and other drugs were applied to the extracellular environment by low-pressure ejection from a blunt pipette positioned about 50–100 μm away from the cell being recorded. Voltage-activated Ca2+ currents were evoked by 100 ms voltage step commands applied every 30 s.The whole cell patch clamp recording method and fura-2 Ca+ currents but NaCl-based extracellular bathing solution and KCl-based patch pipette solutions were used.Similar protocols were used to study the actions of pregabalin on voltage-activated K2+ currents intracellular flash photolysis was achieved by flashing the neurone being studied (three 200 V flashes to produce ~15μM GTP-γ-S) using a XF-10 xenon flash lamp with a UG11 bandpass filter  was included in the CsCl-based patch pipette solution. After obtaining control Caentific; )2+ and K+ currents had scaled linear leakage and capacitance currents subtracted to obtain values for the net inward Ca2+ current or net outward K+ current. Data are given as mean ± standard error of the mean (s.e.m.) values and statistical significance was determined using a paired or independent Student's t test as appropriate.All voltage-activated Ca2+ imaging in cultured DRG neurones the cultures were incubated for 1 hour in NaCl-based extracellular solution contained (in mM): NaCl, 130; KCl, 3.0; MgCl2, 0.6; CaCl2, 2.0; NaHCO3, 1.0; HEPES, 10.0; glucose, 5.0 and fura-2AM, 0.01; . The pH was adjusted with NaOH to 7.4 and the osmolarity to 310–320 mOsm with sucrose. The cells were then washed for 10–20 minutes with NaCl-based extracellular solution to remove the extracellular fura-2AM and this period allowed cytoplasmic de-esterification of the Ca2+ sensitive fluorescent dye. The cells were constantly perfused with NaCl-based extracellular solution (1–2 ml/min) and viewed under an inverted Olympus BX50WI microscope with a KAI-1001 S/N 5B7890-4201 Olympus camera attached. Some Ca2+ imaging experiments were carried out using a Ca2+-free NaCl-based solution (as standard NaCl-based extracellular solution but with no added CaCl2) or choline chloride-based solution (as standard NaCl-based extracellular solution but with choline chloride in place of NaCl). The fluorescence ratiometric images from data obtained at excitation wavelengths of 340 nm and 380 nm were viewed and analysed using OraCal pro, Merlin morphometry temporal mode . The DRG neurones were stimulated with NaCl-based extracellular solution containing high K+ (30 mM), which produced depolarisation, activation of voltage-gated Ca2+ channels and large transient increases in intracellular Ca2+. Three consistent transient increases in intracellular Ca2+ could be obtained in a single experiment on cultured DRG neurones [2+ transient amplitude, duration at 1/2 peak amplitude and total Ca2+ flux were measured. All experiments were conducted at room temperature and data are expressed as means ± s.e.m.For Caneurones . The actDRG, Dorsal root ganglion.cAMP, Cyclic adenosine monophosphateGABA, Gamma aminobutyric acidGBP, GabapentinGTP-γ-S, Guanosine 5'-o(3-thio)triphosphateCa, Calcium currentINGF, Nerve growth factorPGB, PregabalinPKA, Protein kinase APP, Pre-pulse(Rp)-cAMP, (R)-adenosine, cyclic 3', 5'-(hydrogenphosphorothioate) triethylammonium2+ imaging experiments. DM and RHS designed and conducted the experiments on K+ currents and wrote the first draft of this manuscript.All the authors of this manuscript contributed to electrophysiological and Ca


Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) that is composed of liberation of stored Ca2+ and extracellular Ca2+-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca2+]i.Differentiating 2+-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca2+ was reduced as compared to wild type. Yet, single cell [Ca2+]i-imaging of regA- amoebae revealed a cAMP-induced [Ca2+]i increase even in the absence of extracellular Ca2+. The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca2+]i in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca2+]i-transients also in nominally Ca2+-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced.We analyzed Ca2+. cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca2+]i of regA- cells. Intracellular cAMP levels control [Ca2+]i by regulating Ca2+-fluxes of stores which in turn affect Ca2+-influx, light scattering oscillations and chemotactic performance.We conclude that there is a link between the second messengers cAMP and Ca Dictyostelium discoideum amoebae form a multicellular organism by chemotactic aggregation. The signaling molecule that mediates aggregation and development is cAMP. Aggregation proceeds in a rhythmic fashion; cAMP is secreted periodically by cells in the center of the aggregate. Cells in the neighbourhood respond by an oriented inward movement and secrete cAMP themselves to relay the signal. In cell suspensions periodic synthesis and release of cAMP leads to rhythmic shape changes that cause alterations in light transmittance and spike-shaped and sinusoidal light scattering oscillations i were postulated to comprise the (or at least a part of the) master oscillator controlling oscillation patterns [2+]i-transient induced by addition of CaCl2 or calmodulin antagonists alters light scattering oscillations and can even reset the oscillation phase [2+]i-increase determines whether light scattering and the accompanying cAMP oscillations are abolished or augmented: large [Ca2+]i-transients inhibit cAMP and light scattering oscillations [2+]i-elevations enhance oscillations of both parameters i we analyzed extracellular Ca2+-fluxes in cell suspensions and studied [Ca2+]i in single amoebae. cAMP-induced Ca2+-influx in suspensions of regA- cells occurred with a similar time course as in wild type. Yet, influx was reduced by approximately 40% . Addition of the PDE inhibitor IBMX (up to 200 μM) to wild type cells affected neither the amount nor the characteristics of cAMP-activated extracellular Ca2+-fluxes.To test whether the absence of the main cAMP-specific phosphodiesterase affects regulation of [Ca40% Fig. . The los2+]i and cAMP-activated [Ca2+]i-changes of regA- to wild type cells in the absence and intracellular presence of IBMX. The inhibitor should affect the activity of both cAMP phosphodiesterases, RegA and PDE-E [2+]i was similar in both strains . After ium Fig. , Table 12+]i-regulation in regA- amoebae might be caused by a change in the characteristics of Ca2+-fluxes of internal stores. A positive influence of cAMP via PKA-mediated phosphorylation of both, IP3-receptors and ryanodine receptors on release of stored Ca2+ has been reported . In wild type amoebae a cAMP-stimulated [Ca2+]i-increase is not detectable in the presence of BAPTA; the occurrence of a transient [Ca2+]i-elevation in regA- cells indicates an augmented release of Ca2+ from stores in the mutant. Support for an effect of cAMP via PKA came from experiments where we incubated cells with the membrane permeant activator of PKA, Sp-5,6-DCl-cBIMPS [2+]i was increased in regA- cells upon treatment with 30 μM Sp-5,6-DCl-cBIMPS for 60 min ; agonist-induced [Ca2+]i-transients in nominally free Ca2+-buffer were unaltered in height  as compared to control cells. In addition, we found that preincubation of wild type amoebae with 30 μM Sp-5,6-DCl-cBIMPS reduced cAMP-activated Ca2+-influx in cell suspensions by 26 ± 8% (mean ± s.e.m. of 3 independent experiments).The effect of the increased basal cAMP concentration on the [Caview see ). We the+]i Fig. . The elel-cBIMPS ,21. Basa2+]i, either by affecting Ca2+-stores or by activating Ca2+-influx alter light scattering oscillations e in regA- cell suspensions revealed irregular [Ca2+]e oscillations, similar to the results obtained for light scattering ; in both strains chemotactic velocity was reduced. The effect was more pronounced in regA-: already in the presence of 2 mM EGTA cells chemotaxed more slowly than under control conditions  as compared to control cells; Mann Whitney rank sum test). Wild type cells were unaffected by preincubation with 5 mM EGTA for up to 1 hour whereas at 10 mM EGTA chemotaxis was reduced.It had been reported previously that ormation . In accoormation  we also 2+ was demonstrated to control light scattering oscillations by affecting the synthesis of cAMP; depending on the height of an artificial [Ca2+]i-transient the production of cAMP which in this case serves as first messenger was either augmented or blocked [2+ in Dictyostelium cells. We observed altered agonist-induced Ca2+-fluxes and [Ca2+]i-transients in the regA- mutant cell line where the absence of the main cAMP-hydrolyzing PDE led to a fourfold increased basal cAMP level. One could argue that the effect on [Ca2+]i was not a consequence of the increased basal concentration of cAMP but rather due to a potentially altered pattern of gene expression in the mutant strain. Indeed, this is possible and could result in a different signal perception and/or processing. However, we consider an alteration in gene expression unlikely to be responsible for the augmented [Ca2+]i-transients upon cAMP-stimulation since the same effect could be evoked in wild type amoebae by loading of the PDE inhibitor IBMX into the cytosol. In addition, IBMX evoked an increase in basal [Ca2+]i in both, wild type and mutant cells. In regA- the inhibitor should act on the additional cAMP-PDE (PDE-E) [2+]i might not be achieved as consistently as in the mutant since IBMX must act on both PDEs.The cytosolic concentration of Ca blocked ,4. The r (PDE-E) ,17 and t2+]i could be caused by several factors. Ca2+-flux characteristics can be changed by influencing Ca2+-channels and/or Ca2+-ATPases located on both, the plasma membrane and membranes of internal stores. When we analyzed Ca2+-fluxes with a Ca2+-sensitive electrode influx was reduced in the mutant while the rates of influx and efflux were unchanged. If the activity of the plasma membrane Ca2+-ATPase (PMCA) was altered then flux rates should be affected. Moreover, the reduced amount of Ca2+-influx precludes activation of a plasma membrane Ca2+-channel. In other cell systems activation of the PMCA and of Ca2+-channels by an increase in cAMP levels was shown [2+-channel or PMCA activity in Dictyostelium amoebae.The sensitizing effect of the increased amount of cAMP on [Caas shown -24 but oDictyostelium a cAMP-activated [Ca2+]i-elevation occurred in the extracellular presence of the Ca2+-chelator BAPTA. This argues for an alteration of Ca2+-uptake into and/or Ca2+-release from stores. An as yet unknown negative regulation of Ca2+-sequestration could cause accumulation of Ca2+ in the cytosol; until now, however, activation of SERCA-type Ca2+-ATPases was found only . On thear cells ; the samar cells . Stimulaemployed . In summdazolium  which wadazolium . Calcinereceptor . In regA2+]i to be responsible for the irregular light scattering and extracellular [Ca2+]-oscillations of regA- cells. Previously, Wessels et al. i-transients inhibit production of cAMP [2+]i-oscillations and their mutual dependence has also been shown in neurons: absence of either, cAMP or [Ca2+]i-oscillations resulted in failure of the other component to oscillate [Dictyostelium the strong decrease in peak cAMP oscillation levels affected [Ca2+]e-oscillations which were irregular. The basis is probably an influence on [Ca2+]i-oscillations. Such oscillations were suggested to occur but have not been demonstrated in single cells until now, presumably due to the small size of the amoebae and the characteristics of the wave i-regulation. In the mutant strain the threshold for generation of an agonist-induced [Ca2+]i-increase is lower than in wild type. The [Ca2+]i-elevation is not as tightly controlled and occurs even in the presence of BAPTA. The characteristics of a [Ca2+]i-increase are important for the resulting cytoskeletal rearrangements and whether pseudopods are formed correctly. Indeed, artificial induction of a small global [Ca2+]i-transient by incubation with calmidazolium caused overall pseudopod protrusion [2+]i-gradient at the rear end was shown [2+]i-pattern during chemotaxis. By contrast, a role of the [Ca2+]i-elevation for the chemotactic response was questioned by Traynor et al. [3-receptors of higher eukaryotes aggregated and differentiated almost normally but displayed no cAMP-activated global [Ca2+]i-change; yet, the existence of localized, small [Ca2+]i-transients in this particular mutant cell line that had escaped detection could not be excluded i-elevations which in turn lead to uncontrolled pseudopod extension. [Ca2+]i regulates cAMP acting as first messenger in a negative feedback loop: when the [Ca2+]i response is increased the amount of cAMP synthesized upon stimulation is low as observed in regA- cells devoid of the phosphodiesterase RegA. The low level of cAMP relay results in improper light scattering oscillations. We conclude that intracellular cAMP acts on [Ca2+]i via PKA: phosphorylation of the system responsible for release of Ca2+ from stores leads to a greater sensitivity facilitating Ca2+ liberation. The cAMP activated [Ca2+]i-increase is due to Ca2+-release from internal stores which triggers subsequent extracellular Ca2+-entry. The fraction of the [Ca2+]i-elevation that is mediated by liberation of Ca2+ is thus larger in the mutant.Abnormal basal levels of cAMP impair chemotactic performance by augmenting agonist-activated e) was measured in 2 ml of cell suspension  with a Ca2+-sensitive electrode  as described [2+]e-oscillations were measured at a cell density of 1 × 108 cells/ml.The extracellular Caescribed . [Ca2+]e2+]-imaging was done as outlined in -imaging experiment. To test the response of amoebae in the presence of BAPTA, 75–78 μl of H5-buffer was pipetted to the cells; 10 μl of 10 mM BAPTA was added during the [Ca2+]-imaging experiment and 10–12 sec later cAMP was given. When the response of cells was to be analyzed in the presence of extracellular CaCl2, H5-buffer (85–88 μl) with 1 mM CaCl2 was added to the cells 15 min before the [Ca2+]-imaging experiment to load stores  to the cells. To load cells with IBMX, they were electroporated with fura2-dextran in the presence of 250 μM of the inhibitor. The cytosolic concentration of IBMX is in the range of maximally 2–5% of the concentration present during electroporation [regA- was done at t4 and wild type [Ca2+]-imaging was done at t7–8. In another series of experiments we treated regA- cells with Sp-5,6-DCl-cBIMPS, a membrane permeant activator of PKA [2+]-imaging experiment.Cytosolic iCytosolic free CaPhosphodiesterase: PDE3-isobutyl-1-methylxanthine: IBMXcAMP-dependent protein kinase: PKA2+-ATPase: PMCAPlasma membrane Ca2+] recordings and light scattering experiments. He also determined cAMP levels and designed the study. KBR did chemotaxis experiments at different external conditions. KH carried out [Ca2+]i-measurements. CS did [Ca2+]i-imaging experiments, designed the study and wrote the manuscript. All authors read and approved the manuscript.DFL performed extracellular [Ca


Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn2+ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K+ (KATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na+ (TTX) and N-type Ca2+ channels (ω-conotoxin), but not L-type Ca2+ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca2+ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca + channels in the presence of glucose. Furthermore, we find that membrane depolarisation results in inhibition of Na+ and Ca2+ channel activity and α-cell exocytosis. Thus, we propose that elevated blood glucose reduces α-cell electrical activity and glucagon secretion by inactivating the ion channels involved in action potential firing and secretion.Glucagon is a critical regulator of glucose homeostasis. Its major action is to mobilize glucose from the liver. Glucagon secretion from α cells of the pancreatic islets of Langerhans is suppressed by elevated blood sugar, a response that is often perturbed in diabetes. Much work has focused on the regulation of α-cell glucagon secretion by neuronal factors and by paracrine factors from neighbouring cells, including the important islet hormone insulin. In contrast, we provide evidence in support of a direct effect of glucose on α cells within intact rodent and human islets. Notably, our work implicates an α-cell glucose-sensing pathway similar to that found in insulin-secreting β cells, involving closure of ATP-dependent K Elevated glucose levels reduce electrical activity and the release of glucagon via inactivation of ion channels in pancreatic islet cells. Increasing concentrations of the KATP channel activator diazoxide demonstrated that moderate activation of KATP channels (0.3–10 μM diazoxide) relieved the suppression of glucagon secretion from both mouse (50) that is much lower than what is seen under high-glucose conditions (∼2 μM versus ∼50 μM).We next examined glucagon and insulin secretion during pharmacological manipulation of Khannels) . We haveth mouse A and ratth mouse B islets.th mouse A and 2B.th mouse C with a ATP channels are expected to be open, the KATP channel antagonist tolbutamide also produced a biphasic effect on glucagon release. Augmentation of glucagon secretion was seen at tolbutamide concentrations of up to 1 μM , whereas inhibition was observed at higher concentrations  (2+]i responses to glucose (We next examined the function of α and β cells in situ by monitoring the i by 62 ± 3%  in α cells, and that this effect could be completely reversed by 2 μM diazoxide , far below the concentration necessary to block β-cell responses  to test a role for these channels in α-cell function and glucagon secretion. TTX (0.1 μg/ml) suppressed glucagon release from mouse islets by 56 ± 5%  under low-glucose conditions (+ channel-dependent mechanism. TTX has no effect on glucose-stimulated insulin secretion in mouse islets (2+]i response evoked by low-glucose concentrations (n = 18 cells in 4 islets), but had no effect on β-cell [Ca2+]i (unpublished data).The above data suggest that glucose-dependent inhibition of α-cell Kt −42 mV , contribt −42 mV . We thusnditions A. This ee islets A; again + channel activation, the opening of VDCCs allows Ca2+ into α cells, triggering the exocytosis of glucagon-containing vesicles. We applied whole-cell patch-clamp recordings to establish the α-cell Ca2+ channel complement. The integrated whole-cell Ca2+ current measured during 50-ms depolarisations from −70 mV to 0 mV amounted to 6.2 ± 0.8 pC (n = 15) under control conditions, 2.6 ± 0.8 pC  in the presence of 50 μM nifedipine, and 4.1 ± 0.5 pC  in the presence of 1 μM ω-conotoxin. Thus, L- and N-type Ca2+ channels account for 58% and 34% of the α-cell Ca2+ current, respectively.Downstream of Na2+ channel blocker ω-conotoxin inhibited the release of the hormone to a level similar to that of high glucose, tolbutamide, and TTX. Furthermore, glucose exerted no additional inhibitory action in the presence of ω-conotoxin. Thus, glucagon secretion stimulated by low-glucose levels depends principally on Ca2+ influx through N-type channels, although these only account for one third of the Ca2+ current. We confirmed this Ca2+ channel dependence by conducting high-resolution single-cell capacitance measurements of exocytosis (ocytosis B. Exocytocytosis C.2+ currents were elicited by step depolarization to 0 mV in the absence (black) or presence (red) of 1 μM ω-conotoxin following a 200-ms conditioning pulse to −70 mV (2+ current amplitude in the absence (open squares) and presence (circles) of ω-conotoxin (upper panel). The ω-conotoxin–sensitive N-type Ca2+ current component is shown in the lower panel of 0.5) at −31 ± 6 mV (n = 6). A residual, ω-conotoxin–insensitive Ca2+ current also appears to undergo voltage-dependent inactivation (2+ channels that have been reported in α cells i in single, isolated α cells, but in agreement with the conclusion of Ostenson et al. /[FuraR+20] × 128+1) of these were analysed over time with Origin v7.0220 . Prior to experimental recordings, islets were perfused for 10 min with the appropriate control solution, and Ca2+ responses were monitored periodically during this time to ensure that responses (or lack thereof) were stable prior to beginning the experimental recording. Ca2+ responses were determined by baseline subtraction and calculation of the integrated response . The slope of this response, reported here as arbitrary units (AU), was calculated for the final 60%–90% of a given treatment period to allow for equilibration of the responses.Intact islets were loaded with fluo-4-AM (1 μM) and fura red (5 μM) in 0.5 mM glucose buffer (see below) with 0.01% pluronic acid for 30 min at 37 °C. Islets were fixed with a wide-bore holding pipette within a continuously superfused and temperature-controlled (37 °C) bath on an Axioskop 2 FS-mot microscope  and Pulse software (version 8.50). Single α cells were identified by their small size and Na+ current inactivation properties [2; 1.2 MgCl2; 5 HEPES (pH 7.4 with NaOH); and 5 glucose. Except for 2SO4; 10 NaCl; 10 KCl; 1 MgCl2; and 5 HEPES (pH 7.35 with CsOH). Exocytosis was monitored as changes in α-cell capacitance using the software-based lock-in function of the Pulse software. The standard whole-cell measurements ; 3 Mg-ATP; and 25 μmol/l EGTA . Pulses were applied at low frequency (<0.05 Hz) to allow the exocytotic capacity to recover fully between the pulses.Whole-cell currents and exocytosis were recorded using an EPC-9 patch-clamp amplifier  and post-test, as appropriate.Data are presented as means and standard errors. Significance was examined by either the unpaired


Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca2+ signals involved in neurophysiological functions. New methods for in vivo imaging of Ca2+ signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca2+ imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca2+ reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca2+] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca2+ response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca2+] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca2+] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca2+ stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca2+ signalling pathways and for functional mapping of neurophysiological processes in the fly brain.Many different cells' signalling pathways are universally regulated by Ca Increases in intracellular [Ca2+] can reach very high concentrations in cellular microdomains in which Ca2+-dependent processes can be both rapidly and selectively activated. These kind of fast Ca2+ transients regulate cellular events at the synaptic terminal, such as exocytosis. However, Ca2+ oscillations or slow Ca2+ waves that propagate across cells or tissues, can also drive functions such as gene expression. The widely different dynamic properties of Ca2+ signalling forms the basis of how Ca2+ ions are able to be a universal regulator of many signal transduction pathways Homeostatic mechanisms ensure that Ca2+ signals can be characterised by optical imaging. However, the choice of Ca2+ sensitive fluorescent or bioluminescent probe will introduce a bias in optical imaging studies, because the signal output will be dependent on a number of important factors including; the Ca2+ binding affinity, the intrinsic properties of the Ca2+ induced light reaction, whether or not and where the probe is genetically targeted, the sensitivity of the technique, the size of the field of view and the pre-selection of how data will be acquired .The spatiotemporal properties of CaIn vivo Ca2+-imaging in the brain of Drosophila with genetically encoded Ca2+ sensitive fluorescent reporters, such as cameleon 2+ signals, excitation light is required and this introduces some limitations relating to photo-toxicity, photobleaching and autofluorescence. Furthermore, when fluorescent probes are used, it is necessary to pre-define the temporal parameters of each study and studies are often designed with the expectation that a Ca2+ response with certain temporal parameters will follow a given stimulus (e.g. application of NMDA or stimulation with different odors). Hence, Ca2+ signals that occur long after a given stimulus in living biological systems are not well described 2+ signals is not associated with phototoxicity and provides an excellent signal-to-noise ratio. GFP-aequorin (GA) is a Ca2+ sensitive bioluminescent photoprotein with improved light emission properties compared to aequorin alone 2+ binding 2+] to be followed over a wide dynamic time range, from milliseconds to hours Bioluminescence imaging of Cain vivo bioluminescence imaging of Ca2+ signaling in the brain of Drosophila. Whole brain imaging was undertaken on transgenic flies with targeted expression of the GA probe in specific neural structures, such as Mushrooms Bodies and ellipsoid-body . A photon counting based technique was applied to avoid the necessity to pre-select temporal parameters and to provide a wide dynamic range for detecting Ca2+ signals. In conclusion, bioluminescence imaging of Ca2+ signals permits the imaging of neuronal ensembles in deep regions of the brain and gives great flexibility to analyse the temporal parameters of Ca2+ signaling over long durations.Here, we demonstrate the use of in vivo imaging with GA was first determined by targeting the Ca2+ reporter to the MBs using the P[GAL4] line OK107 2+-responses were recorded with whole brain bioluminescence imaging after a short bath application of KCl (70 mM) . K+-depolarised Ca2+ responses in the MBs were also reduced by the L-type voltage-gated calcium channel (VGCC) blocker, verapamil (10 µM) (GA transgenic flies were developed with the P[GAL4]/UAS system  (70 mM) . At low  (10 µM) . During Drosophila, the central-complex (CC) is composed of small cells located deep inside the middle of the brain  that send projections to the eb (GA expression was therefore targeted to a subset of ring-neurons in the ellipsoid-body (eb) , using tl bodies , which wo the eb . This reo the eb .2+ within the calyx and the cell bodies  is cholinergic l bodies . Howeverin later   may therefore arise from Ca2+-oscillations, which could not be temporally resolved with the sensitivity of this technique. In mammals, intra-cellular Ca2+ release from the ER has already been reported to be involved in Ca2+-oscillations regulating gene expression and in functions such as synaptic plasticity 2+ oscillations were sensitive to the irreversible inhibitor of Ca2+ ATPase, thapsigargin 2+ levels within the endoplasmic reticulum (ER). Thapsigargin was found to significantly reduce the secondary response caused by nicotine application , encodes for a phosphodiesterase (PDE). This enzyme regulates the level of cAMP 1dnc mutant flies and the delayed secondary rise in Ca2+ induced by nicotine was found to be reduced by a mean of about 50% in the 1dnc mutant fly . This approach can also be applied to pharmacological investigations and studies on mutants.Described here is a new approach for 2+ signals could be monitored over a wide dynamic temporal range for which they are known to operate. This would incorporate Ca2+ signals that may be in control of long term cellular changes, such as gene transcription, synaptic plasticity or cellular proliferation. The spatial resolution of whole brain bioluminescence imaging is dependent on both the light scattering caused by tissue and on the resolution of the camera. Despite the moderate resolution of images acquired in these studies, the origin of the signal is known from genetic targeting and corresponding expression patterns of the reporter. However, the spatial resolution could be improved by using higher magnifications. In an integrated system like the fly brain, bioluminescence imaging could also be useful for defining the spatial and temporal parameters of Ca2+ signals linked to known functions. Once these parameters are identified, probably that higher spatial and temporal resolution of the signal could be sort with fluorescence approaches.The use of a photon-counting based technique in bioluminescence imaging eliminated the necessity to pre-select exposure times and recording durations, so that Ca2+ response in specific neuronal ensembles was identified because of the method employed, which involved continuous recording of Ca2+ signals over a long period and selection of time integrals from 50 milliseconds to 10′s of seconds offline.In flies, the MBs have been implicated in several functions, such as learning and memory 2+ oscillations having a 4 min period have previously been reported in the MBs with transgenic flies expressing aequorin alone in vivo, while Rosay et al., worked on an in-vitro brain preparation.Ca2+ rise was not related to toxicity, because the delayed secondary response could be observed after successive applications of both nicotine and acetylcholine. In physiological conditions, prolonged and sustained rises in Ca2+ are usually toxic for the cell. One explanation is that the observed secondary rise in Ca2+ after nicotine stimulation could be made up of fast Ca2+-oscillations linked to long-term changes, such as gene expression or synaptic plasticity. IP3 signalling has been identified as the driving element for cytosolic Ca2+ oscillations in some model systems. Indeed, we found that the secondary response was significantly reduced by the Ca2+-ATPase inhibitor, thapsigargin, which depletes IP3 sensitive stores of Ca2+.The delayed rise in Ca2+ was also found to be reduced in the dnc mutant by a value that is in the same range of PDE reduction that is reported for the 1dnc mutant dnc gene is highly complex dnc mutation causes abnormalities in several learning paradigms and alters synaptic transmission and growth cone motility. Repetitive exposures to nicotine have also been shown to induce a hyper-responsiveness that is dependent on cAMP signalling and known to be sensitive to the dnc mutation dnc sensitive delayed secondary Ca2+ response occurring specifically in the MB lobes may play a critical role in cellular processes (synaptic transmission and/or synaptic plasticity) contributing to memory formation.The secondary CaDrosophila brain, such as the ellipsoid-body of the CC has never been reported with other approaches. Results here show bioluminescence imaging of Ca2+ signals using GFP-aequorin can allow the functional characterisation of these deep brain structures. Using a photon counting based technique also allowed images to be analysed with variable time integrals so that the delayed secondary response could be readily identified, despite its relatively low amplitude and prolonged time course. Whole brain bioluminescence imaging can therefore reveal widely dynamic spatial and temporal parameters of the Ca2+ signalling system in the living organism. More sensitive techniques (e.g. fluorescence imaging) could then be applied to obtain greater spatiotemporal resolution of the Ca2+-signal in structures, such as the mushroom bodies. Anatomo-functional mapping of the Drosophila brain using this in-vivo genetic approach will therefore provide important information for studies on complex neural pathways, involved in learning and memory, locomotor activity, circadian rhythms and olfaction.To date, functional imaging on structures located deep in the Drosophila melanogaster lines were maintained at 24°C on standard food medium. P[GAL4] OK107 line and 1dunce mutant was obtained from the Bloomington Drosophila Stock Centre, C232 by D. Armstrong and K. Kaiser. All flies were backcrossed six fold with Canton-S to standardize the genetic background (Cantonization).All The GFP-aequorin (GA) insert of pG5A 2, 2 mM CaCl2, 36 mM Sucrose, 5 mM Hepes-NaOH, pH 7.3 Brains were exposed by partial removal of the head capsule. During this procedure, the underlying neural sheath is also removed in order to expose the outer surfaces of the brain. The flies are mounted upside down in an acrylic block, which is then mounted inside a diamond shaped slice chamber (Harvard Apparatus). The slice chamber containing the prepared fly was then transferred to an inverted microscope for recording. Recordings were undertaken in a fly ringers buffer containing, 130 mM NaCl, 5 mM KCl, 2 mM MgCl2+-induced bioluminescence activity has been previously described 2.Detection of CaNicotine was prepared as a stock solution (100 mM) and diluted further in fly ringers (see composition above) to a concentration of 100 µM. KCl was prepared in fly ringers to a concentration of 70 mM and the concentration of NaCl was adjusted accordingly. Thapsigargin was dissolved in 100% DMSO and diluted to a final concentration of 2 µM . TTX was stored as a 1 mM stock solution and diluted to 1 µM in fly ringers. Verapamil was dissolved in fly ringers to a concentration of 10 µM. All drugs were purchased from Sigma.Data was analyzed in Excel and statistical significance was determined using a one-tailed student t test, with unequal variance.Figure S1Schematic drawing of the setup. Setup used to monitor and quantify the bioluminescence light emitted by the Ca2+activated GFP-aequorin. An inverted microscope is equipped with a CCD camera, allowing a fluorescent image of GFP expression in the whole brain to be recorded. Then, by switching off an automatised mirror, this allows the light emitted to directly reach the photon counting camera. Each photon emitted are detected and the X,Y coordinates as well as its time (t) are recorded. After the recording session, the data are analysed by performing various desired and appropriated integration time to visualize the neuronal activity. Remarks that because the photon detector (IPD) is placed below the preparation (inverted microscope), this limits our approach to pharmacological manipulations (similar to a perfusion bath) or to spontaneous recording events. An upright built system will allow physiological and even behavioural manipulations, like odor presentation, locomotor activity recording or else (works in progress).(1.02 MB TIF)Click here for additional data file.Movie S1In-vivo bioluminescence imaging of K+-depolarised Ca2+ uptake in the MBs. Ca2+ uptake in the mushroom bodies (MBs) was directly visualised after K+-depolarisation of the fly brain. Each frame represents 60 s of light accumulation and is shifted by 10 s (6 frames/s). The movie is seen 60 times faster. The light emission is coded in pseudocolors (0-5 photons/pixel) .(0.49 MB MOV)Click here for additional data file.Movie S2In-vivo bioluminescence imaging of K+-depolarised Ca2+ uptake in the ellipsoid-body. Ca2+ uptake in the ellipsoid-body and cell bodies  was directly visualised after K+-depolarisation of the fly brain (70 mM KCl). Each frame represents 60 s of light accumulation and is shifted by 10 s (6 frames/s). The movie is seen 60 times faster. The light emission is coded in pseudocolors (0-5 photons/pixel) .(0.68 MB MOV)Click here for additional data file.Movie S3Spontaneous activity in the ellipsoid-body, a sub-structure of the central complex. Each frame represents 120 s of light accumulation and is shifted by 30 s (6 frames/s). The movie is seen 180 times faster. The light emission is coded in pseudocolors (0-5 photons/pixel) .(0.81 MB MOV)Click here for additional data file.Movie S42+-signalling in the MBs after application of nicotine. Nicotine (100 µM) induces transient increase in [Ca2+]i in all parts of the MBs (OK107) and a delayed secondary Ca2+ response in the lobes 4 to 15 min after the primary response. Each frame represents 60 s of light accumulation and is shifted by 10 s (6 frames/s). The movie is seen 60 times faster. The light emission is coded in pseudocolors (0-5 photons/pixel) .Ca(1.02 MB MOV)Click here for additional data file.Movie S5A2+-response in the MB lobes. The nicotine induced delayed secondary response in the MB lobes occurs sequentially, first, in the α'/β' lobes, second, in γ lobes, and finally in α/β lobes. Each frame represents 60 s of light accumulation and is shifted by 10 s (12 frames/s). The movie is seen 120 times faster. The light emission (photons/pixel) is coded in pseudocolors (2-10 photons/pixel) .Sequential activation of the delayed Ca(1.36 MB MOV)Click here for additional data file.Movie S5B2+-response in the MB lobes. The nicotine induced delayed secondary response in the MB lobes occurs sequentially, first, in the α'/β' lobes, second, in γ lobes, and finally in α/β lobes. Each frame represents 90 s of light accumulation and is shifted by 5 s (12 frames/s). Resolution is 256×256. The light emission (photons/pixel) is coded in pseudocolors (1-6 photons/pixel) .Sequential activation of the delayed Ca(2.78 MB MOV)Click here for additional data file.Movie S62+-response in the MB lobes. Application of thapsigargin, 15 min before and during the nicotine application (100 µM) blocks the secondary response in the MB lobes, suggesting that this delayed response requires the intra-cellular calcium store in the endoplasmic reticulum. Each frame represents 60 s of light accumulation and is shifted by 10 s (6 frames/s). The movie is seen 60 times faster. The light emission (photons/pixel) is coded in pseudocolors (0-5 photons/pixel) .Thapsigargin blocks the nicotine induced delayed Ca(0.55 MB MOV)Click here for additional data file.Movie S72+-response in MB lobes is reduced in dunce mutant flies. The nicotine  induced delayed secondary response in MB lobes is reduced and in some cases, absent in 1dunce mutant. As a positive control, KCl was applied  30 min after nicotine application to verify that the Kenyon cells of the MBs are still physiologically functional. This induced a strong response simultaneously in all parts of the MBs, including the complex calyx/cell-bodies as well as in the medial and vertical lobes. Each frame represents 60 s of light accumulation and is shifted by 10 s (6 frames/s). The movie is seen 60 times faster. The light emission (photons/pixel) is coded in pseudocolors (1-8 photons/pixel) .The nicotine induced delayed Ca(0.86 MB MOV)Click here for additional data file.


Hyphae of the human pathogenic fungus Candida albicans reorient their growth axis in response to underlying surface topography (thigmotropism) 2+ dependent. Tropisms were attenuated in media containing low Ca2+, or calcium-channel blockers, and in mutants where calcium channels or elements of the calcium signaling pathway were deleted. Therefore galvanotropism and thigmotropism may both be mediated by localized Ca2+ influx at sites of polarized growth via Ca2+ channels that are activated by appropriate environmental signals.Eukaryotic cells from fungal hyphae to neurites that grow by polarized extension must coordinate cell growth and cell orientation to enable them to exhibit growth tropisms and to respond to relevant environmental cues. Such cells generally maintain a tip-high Ca C. albicans, but high-affinity and low-affinity calcium-uptake systems (HACS and LACS) have been described and partially characterized in S. cerevisiaeC albicans, homologs of components of these systems—the voltage-gated Cch1p channel Calcium channels have not been identified previously in CaCCH1 encodes a putative 2254 amino acid protein with 38.4% identity to its S. cerevisiae homolog. The 24 predicted transmembrane (TM) regions in CaCch1p are arranged in four repeated units (I to IV) of six TM domains, as they are in mammalian calcium channels where they tetramerize to form the core α1-subunit of L-type Ca2+ channels C. albicans Cch1p and the human voltage-gated calcium channel CaV1.2 are 62.9% similar and 37.7% identical over a 20 amino acid region in the four Ca2+ selective, pore-forming P domains. In the voltage-sensitive S4 domains, 13 of the 23 basic residues in CaV1.2 are identically positioned in CaCch1p. The CaMID1 gene sequence had 36.9% and 34.4% identity to ScMID1 and Schizosaccharomyces pombe yam8(+), respectively. The 559 amino acid protein it encodes contains four putative TM regions (H1-4), potential N-glycosylation sites, a helix-loop-helix domain and 10 conserved cysteines that, in S. cerevisiae, form the C1/C2 domains that are essential for activity and localization S. cerevisiae, the C1/C2 regions in C. albicans are located between H3 and the C-terminal H4. CaFig1p shares 48.5% identity with ScFig1p, a putative homolog of mammalian PMP-22/EMP/MP20/Claudins, which are involved in the trafficking and assembly of membrane-associated proteins N-glycosylation sites in the first of its four TM domains. The role of ScFig1p in S. cerevisiae is not well-defined, but it localizes predominantly to the plasma membrane MRP1 maltose-regulatable promoter (CCH1 or MID1) or by reintegration of the gene at a high-expression locus (FIG1 or MID1) . Also, both HACS and LACS mutants were affected in the thigmotropic response (see below). Therefore, both HACS and LACS are expressed in C. albicans during the normal in vitro and in vivo growth of this fungus.In 2+-depleted solid minimal medium (>14 days), wild-type C. albicans colonies produced aberrant lobed margins that could be alleviated by the addition of 10 mM Ca2+ to the medium. Emerging colonies of the Cacch1Δ and Camid1Δ mutants produced lobed colonies at 2 days , but the extension rates of hyphae were not affected by the mutations (data not shown). Consistent with the putative roles of CaMid1p and Cch1p as Ca2+ channels, Ca2+ accumulation in the Camid1Δ and Cacch1Δ mutants was significantly reduced after 2 hr culture in Ca2+-depleted medium supplemented with 45Ca2+  have been observed in migratory and tip-growing cells d medium A and 1B d medium A. Therefreversed C.Figure CaCCH1 resulted in a significant reduction in the cathodal orientation of germ-tube emergence (p = <0.001)  B. In con2+] deprivation and CaCCH1 deletion primarily affected the site of germ-tube emergence. In low [Ca2+], in the Cacch1Δ mutant and in the presence of L-type Ca2+-channel blockers, cathodal germ-tube emergence was approximately half that of the control strain, but after 6 hr exposure to an electric field, this effect was almost lost   compared to the control strain  A and 3C.l strain . Thigmot was 90° C, suggesC. albicans, calcineurin acts on the transcription factor, Crz1p CaCRZ1 affected both cathodal germ-tube emergence  was calculated with p = Σ (−sin θ/n) × 100, for n measurements. A minimum of 100 cells was measured in each of three independent experiments for each treatment. Tracings of hyphal growth patterns were generated with Adobe Photoshop.Yeast cells were adhered to poly-L-lysine-coated microscope slides and placed on the flat bed of a Biorad midi-sub cell electrophoresis tank Yeast cells were adhered to poly-L-lysine-coated quartz slides featuring ridges of 0.79 μm ± 40 nm and a pitch of 25 μm . This ridge height caused maximal hypha reorientation in a preliminary trial of five heights (data not shown). Slides were placed in 20 ml prewarmed 20% (v/v) newborn-calf serum and 2% (w/v) glucose at 37°C for 6 hr for inducing hyphae. The number of hyphae reorienting on contact with a ridge was expressed as a percentage of the total observed interactions. A minimum of 100 interactions was observed per strain in each experiment, and results were reported as the mean value from three independent experiments ± SD.