Drosophila photoreceptors Ca2+-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, we studied the distribution and physiological properties of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca2+ channel cacophony is also present there. Measurements in the lamina with the Ca2+ fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca2+ increments mediated by cacophony. Additional Ca2+ influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca2+ also contributes to exocytosis, since this process was reduced after Ca2+-store depletion. Therefore, several Ca2+ pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves cacophony; this is complemented by internal Ca2+ release and the activation of TRP and TRPL coupled to Ca2+ depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons.In  Drosophila occurs in retinal microvillar arrangements running along the photoreceptor soma, termed rhabdomere. The axon of this non-spiking neuron releases histamine in a tonic manner q-protein, activates phospholipase C (PLCβ4). This enzyme, encoded by norpA2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). This signaling cascade has been widely implicated in the activation of TRP and TRPL 23 receptor does not 2+ depletion of the endoplasmic reticulum (ER). Here we confirmed the presence of TRP in the lamina 2+ influx via a store-operated channel (SOC) mechanism 2+ channel cacophony, the only fly homologue of vertebrate N-, P/Q- and R-type 2+ signalling.Light transduction in 2+ in non-spiking neurons.These results contribute to understand tonic neurotransmitter release in ribbon-type synapses and presynaptic enhancement by intracellular CaDrosophila brain slices suitable for functional and immunohistochemical studies in the visual system  to create binary images  and TRPL (trpl) single mutants the level of exocytosis induced by depolarization was comparable to wt flies  was added to the external solution in the presence of FM4-64. This treatment induced vigorous vesicle release, being the number of labeled boutons in trpl;trp flies similar to wt in the same conditions , which is strongly expressed in the lamina (26). The Drosophila serca gen has about 70% identity with the mammalian serca1, with which it shares identical binding sites for its inhibitor thapsigargin (Thg) 2+ released from the ER contributes to synaptic activity in the lamina. As an independent test for the involvement of ER Ca2+ in exocytosis, we used the thermosensitive mutant (TSserca) wt flies were loaded with FM4-64 at 41°C for 2 minutes, the number of fluorescently labeled boutons , as compared to norpA not exposed to high-K+  in the photoreceptors axons. We utilized a well-established experimental protocol to test the participation of SOCs, known as the “Ca2+-depletion protocol” 2+-free (0-Ca2+) Ringer supplemented with Thg (10 µM) to deplete the ER of Ca2+, a condition that should open SOCs, if present. If a subsequent restitution of regular Ringer induces a transient Ca2+ influx, it is taken as an indication of SOC activity. In GMR-Gal4/UAS-G-CaMP flies, this protocol induced a significant increase in Ca2+-dependent GCaMP fluorescence in the lamina . This result is consistent with the existence of a SOC mechanism in photoreceptors terminals in the lamina. Considering the difficulty to express G-CaMP in trpl;trp flies due to genetic constraints  exposure should actually increase cytosolic Ca2+, however after 10 min in this solution, internal Ca2+ should be back to original level It might be thought that CaAltogether, these results suggest that TRP and TRPL activation in the photoreceptor axons in the lamina is mediated by a store-operated mechanism.2+ increase associated with TRP/TRPL SOC activity was able to evoke exocytosis in the varicosities. Slices from wt flies were exposed to Thg in 0-Ca2+ external solution for 10 min and then returned to normal Ringer supplemented with FM4-64. If the depletion protocol triggers exocytosis, the dye will be subsequently incorporated into axonal varicosities. As shown in 2+ Ringer could be by itself responsible for the exocytosis observed upon return to regular external solution, we repeated the experiment in the absence of Thg. In this case, the number of loaded boutons  demonstrated that the double mutant has the potential for vesicle release  2+ pathway with the ionophore induced exocytosis in this mutant, an observation that opposes to a generalized degeneration of synaptic machinery. This evidence shows that TRP and TRPL are involved in exocytosis. Only one of these channels was sufficient for sustaining exocytosis.The observations that TRP and TRPL are also in the photoreceptors axons and are considerably CaFM4-64 is presumably incorporated by all lamina neurons and therefore not only photoreceptor boutons should be labeled. However, we expect that the dramatic changes in release observed include photoreceptor terminals, which represent the most numerous synaptic contacts in the lamina Altogether, our results support the participation of TRP, TRPL and cacophony in synaptic transmission in photoreceptor terminals.norpA mutant, suggesting a role of PLC in neurotransmitter release. An obvious possibility is that it mediates TRP/TRPL activation. In principle, PLC may act by either DAG or IP3. PUFAs can activate the light-dependent channels when added to intact ommatidia What is PLC doing in photoreceptors synaptic terminals? Depolarization-induced exocytosis was markedly reduced in Drosophila photoreceptors, a level of PLC activity has been observed both in vitro and in vivoq-protein 2+ has been reported in Drosophila head membranes 2+ influx through cacophony  during depolarization-induced vesicle exocytosis, representing a feed-forward mechanism in this graded synapse. Alternatively, PLC activation may be a consequence of a direct activation of Gq by depolarization, as reported in other insects How is PLC activated? In TSserca to the non-permissive temperature considerably decreased bouton labeling compared to permissive temperature, and we showed that this decrease cannot be explained exclusively by a temperature effect. These results strongly implicate ER Ca2+ release in photoreceptors exocytosis.Calcium reservoirs appear to be involved in exocytosis, since inhibition of SERCA with Thg deeply affected vesicle release. Moreover, exposure of 2+ signals in the lamina after Ca2+ depletion implicated TRP/TRPL, as it was absent in trpl;trp animals. This supports the function of TRP/TRPL as SOCs in the synaptic terminals, allowing Ca2+ influx. This mechanism drives exocytosis, as indicated by the Ca2+-depletion protocol, where bouton labeling was significant. Interestingly, TRP and TRPL function as SOCs in heterologous expression systems The robust CaDrosophila TRP, TRPC1, 2, 4 and 6, are proposed to function as SOCs in different cell types 2+ influx related to neurotransmission in rods and cones Drosophila genome has one gene encoding STIMh 2+ sensor protein that forms functional SOCs in association with TRPC1 Mammalian homologues of 2+-depletion experiments TRPL/TRP opening could be induced by a PLC-dependent mechanism mediated by phospholipase activation by a cytoplasmic Ca2+ increase due to altered reticular release/uptake balance during Thg treatment. In these experiments PLC contribution to exocytosis was possibly by-passed. In normal conditions, this enzyme may elicit Ca2+ elevation in the synaptic terminals by DAG-mediated activation of TRP/TRPL and/or by inducing Ca2+ release.We showed that it is improbable that in the Ca2+2+ increments induce correspondent changes in release, something that would be implausible if a threshold were involved, as in non-graded synapses.Photoreceptors synaptic transmission must accurately follow the fast photoresponses generated in the rhabdomere. As graded synapses support rapid changes in neurotransmitter release, they should undergo fast variations in internal free Ca2+-induced Ca2+ release (CICR), but we lack direct evidence for this. CICR regulates exocytosis in rods allowing high rates of neurotransmitter release Drosophila photoreceptors use all available Ca2+ pathways  to satisfy the synaptic demands required by their extremely fast photoresponses 3R might reinforce transmitter release, but showed no direct evidence for it. This possibility is supported by our results implicating PLC. Moreover, our observation that Ca2+ from the ER contributes to depolarization-induced exocytosis strengthens the possibility of internal release via IP3R and/or RyR.Besides cacophony contribution to exocytosis, the presence of the ryanodine receptor (RyR) in the lamina 2+ plays an essential role in neurotransmission. In tonic synapses, this mechanism may be crucial to sustain synaptic transmission for extensive periods of time.Bouton labeling experiments were conducted under prolonged depolarization, implying that vesicle exocytosis was at steady-state. Thg experiments under such conditions show that released Ca2+ enters through cacophony inducing vesicle release, perhaps enhanced by CICR. Additionally, PLC activated by an unknown mechanism which may be Ca2+ itself or depolarization, generates IP3, triggering Ca2+ release through IP3Rs. ER Ca2+ depletion in turn opens TRP/TRPL by a SOC mechanism, incrementing the Ca2+ supply. These channels may also be opened by lipid and pH changes resulting of PLC activity 2+ increment guarantees efficient, rapid and sustained neurotransmitter release. After depolarization, resting Ca2+ levels would be restored by extrusion by the Na+/Ca2+ exchanger We propose the following model for the synaptic events at the axon terminals : the rec3-induced Ca2+ depletion and TRP/TRPL to presynaptic Ca2+ for vesicle release can vary widely. Also, the activation of TRP/TRPL may rely on ER depletion and/or lipids associated to PLC activity. It may be thought that the Ca2+ influx through cacophony should be sufficient to account for exocytosis, making Ca2+ release redundant and rather unnecessary. However, in this graded synapse the level of cacophony activation will follow the graded depolarization. The amplitude attained by the receptor potential are most likely within a small voltage range above the threshold for cacophony activation  Oregon Red, 343w;trp, 302w;trpl, 302;trpCM, w;trpl302;trp343w;cn,trpl and w;norpA. We confirmed the integrity of the retinae of these flies with the pseudopupil test TS2cac and TS/Cyodserca were kindly provided by R.W. Ordway and S. Sanyal, respectively. For Ca2+ fluorescence experiments, UAS-G-CaMP flies were crossed to GMR-Gal4/Cyo and the progeny of 2–4 days post-eclosion was utilized. Flies were reared at 18°C under dim light conditions.The following 2 and kept in ice for sectioning with a vibratome  under continuous illumination. For each experiment, around 10 flies were stuck to the stainless steel tray with 1 µL of cyanoacrylate ester glue. Flies were immersed in chilled extracellular solution containing (in mM): 120 NaCl, 5 KCl, 8 MgSO4, 1 CaCl2, 25 L-proline, 1.25 NaH2PO4 and 25 NaHCO3, 2.5 sucrose, 10 HEPES. This solution was bubbled with a mixture of 95% O2-5% CO2, which maintained the pH at 7.15. The whole body was sectioned in transversal slices (200 µm thick), yielding one slice per individual. These slices were suitable for both immunofluorescence and functional analysis. In the latter case, slices were kept in iced Ringer in the dark, for at most 2 hours, before imaging. The experiments were carried out at room temperature (approximately 20°C). We considered depolarization-induced FM4-64 or Rhod-2 AM loading into axonal varicosities as a criterion for slice functionality. In mutants where depolarization-induced loading was disrupted, we performed internal positive controls using pharmacological tools to show that the terminals can effectively be loaded. All functional experiments were performed in chilled medium, except for those with the temperature-sensitive mutants at non-permissive temperatures and experiments in wt at 37/41°C to evaluate the effect of temperature.Adult male and female flies were anesthetized in COtrpl;trp. The α-TRPL polyclonal antibody AB5912 was obtained from Chemicon and the pan α-N-type Ca2+ α-1B polyclonal antibody (L-17), from Santa Cruz q-Protein and Rh1 were kindly provided by Charles Zuker (Columbia University). Specificity of the latter four antibodies was previously tested The α-TRP monoclonal antibody MAb83F6 developed by Seymour Benzer was purchased from the Developmental Studies Hybridoma Bank (DSHB) under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City. A second α-TRP monoclonal antibody was kindly provided by Craig Montell (Johns Hopkins University); both antibodies were tested for specificity in the double mutant 2 and fixed for 48 h in 4% formaldehyde in phosphate buffer saline (PBS) at 4°C. 200 µm thick horizontal head sections were cut with a vibratome in chilled PBS under illumination, as described above. The sections were incubated for 2 h at room temperature (20–22°C) in blocking solution containing: 50% goat serum, 10% bovine seroalbumin (BSA) in PBST . Primary antibodies were incubated overnight at the appropriate dilution in blocking solution at 4°C . Slices were washed 4×20 min in PBS and incubated with secondary antibody (1∶200) for 1 h at room temperature. Either goat α-rabbit AlexaFluor594 or goat α-mouse AlexaFluor546 conjugated IgG antibodies were used. Sections were then washed (4×20 min) in PBS and stored in glycerol at 4°C. For imaging, slices were enclosed between two coverglasses and immersed in mounted media  and 20% glycerol.Flies were anesthetized in COFor all quantifications we utilized as statistical tests one or two-way ANOVA with Dunnett’s and Bonferroni post-test, p<0.05. Error bars represent ± S.E.M.Drosophila slices from GMR-Gal4/UAS-G-CaMP flies were covered with low melting point Agarose  to prevent movement. They were constantly perfused with normal extracellular solution  and a glass micropipette positioned on the lamina was used for local application of high-K+ solution . Ca2+ signals were recorded by the fluorescent protein G-CaMP, ectopically expressed in the photoreceptors of GMR-Gal4/UAS-G-CaMP flies. This approach was not used in trpl;trp mutants because in GMR-Gal4 flies the gal4 construct localizes in the same chromosome as trpl. This could be addressed by making GMR-Gal4,trpl;trp recombinants and UAS-GCaMP;trpl;trp segregant. Since this is a rather demanding and time consuming approach, we circumvented it by measuring Ca2+ in 302; trp343trpl mutants and wt flies with the fluorophore Rhod-2-AM. The slices were incubated for 30 minutes in the dye  and then washed for 10 minutes. In the SOC protocol, after Rhod-2-AM loading, Ringer solution was replaced by 0-Ca2+ Ringer supplemented with 10 µM Thg for 10 min; after this, normal Ringer was restored. Confocal z-stacks images were acquired before, during and after stimulation. See SI for details on image capture and analysis.wt and mutant flies. Loading procedure was as follows: in darkness, slices were stimulated in ice with high K+ extracellular solution (90 mM) in the presence of FM4-64 for 10 minutes and then washed for 5 minutes in normal extracellular solution. In store-depletion experiments, FM4-64 was applied after 8 min of thapsigargin  pre-treatment and washed out with Ringer. Basal dye labeling was evaluated without stimulation, in the presence of FM4-64 for 10 minutes. Along the paper the notation “control” refers to basal labeling of FM4-64 or other dyes. Only slices with morphological preservation of the lamina were used. See below and SI for details on image acquisition and analysis.Vesicle exocytosis in the lamina was evaluated by the membrane fluorophore FM4-64 imaging in brain slices. Synaptic terminals were labeled with FM4-64 (10 mM) in an activity-dependent manner www.scian.cl) based on interactive data language . These procedures were used for ROI segmentation, quantification, and to determine colocalization coefficients (see below).Acquisition of the different types of fluorescence images obtained in this study is described in detail in SI. Raw confocal image stacks were deconvolved by Huygens Scripting software  using an algorithm based on the Classic Maximum Likelihood Estimator. Image-processing routines were developed in SCIAN laboratory . The selected ROIs were automatically quantified by IDL. Results are expressed as mean number of fluorescently labeled boutons  from several projection images, each from a different fly. One- and two-way ANOVA with Dunnett’s and Bonferroni post-test statistical analysis was performed using GraphPad Prism 4 for Windows .ROI(random), see SI). M1ROI(random) was subtracted from the Manders colocalization coefficient calculated without any displacement M1ROI(d = 0) and referred to as effective colocalization M1ROI(effective). Expressed as a percentage, Eff-Colocalization (%)  =  M1ROI(effective) * 100.The confined displacement algorithm (CDA) was performed according to Ramírez et al, Figure S1Segmentation of ROIs for colocalization analysis by the confined displacement algorithm (CDA). (A) Representative 2-channel confocal image with α-TRP  and mCD8-GFP . (B) Confined region defines the photoreceptors axons (from Ch2) after segmentation (left). Merge of the segmented signals for Ch1 (red) and Ch2 (green) within the confined ROI (grey) (right).(TIF)Click here for additional data file.Figure S2Bouton detection and quantification in confocal images. (A) Representative image of lamina boutons stained with FM4-64 in wt flies. (B) Binary image showing the ROIs segmented by gradient filtering of the boutons shown in A. (C) Histogram showing the areas of the objects detected in B. The black columns correspond to the area of the objects selected by size filtering. The object areas shown in the gray columns were not considered in the analyses.(TIF)Click here for additional data file.Figure S3Activity-dependent exocytosis in the lamina. (A) Boutons previously loaded with FM4-64 in the lamina are shown, before (a) and after (b) a second exposure to 90 mM K+. Fluorescence decay was detected in the boutons pointed out by arrowheads. (Ac) ROIs used for the quantification. (B) Mean fluorescence decay measured 3 min after exposure to 90 mM K+ in the ROIs shown in (Ac). Error bars: mean ± SEM.(TIF)Click here for additional data file.Text S1Detailed protocols for image capture and analysis.(DOCX)Click here for additional data file.


The complexity of the signaling network that underlies astrocyte-synapse interactions may seem discouraging when tackled from a theoretical perspective. Computational modeling is challenged by the fact that many details remain hitherto unknown and conventional approaches to describe synaptic function are unsuitable to explain experimental observations when astrocytic signaling is taken into account. Supported by experimental evidence is the possibility that astrocytes perform genuine information processing by means of their calcium signaling and are players in the physiological setting of the basal tone of synaptic transmission. Here we consider the plausibility of this scenario from a theoretical perspective, focusing on the modulation of synaptic release probability by the astrocyte and its implications on synaptic plasticity. The analysis of the signaling pathways underlying such modulation refines our notion of tripartite synapse and has profound implications on our understanding of brain function. The simultaneous recognition that astrocytes sense neighboring neuronal activity and release neuroactive agents (or “gliotransmitters”) has been instrumental in the uncovering of the many roles played by these cells in the control of genesis, function and plasticity of synapses Haydon, . These f3)-mediated Ca2+ signaling. This was observed at both glutamatergic, cholinergic, noradrenergic, and GABAergic synapses in the hippocampus, in the thalamus and in the cortex   and ATP which can be converted into adenosine (Adn) in the extracellular milieu    from the synapse to the astrocyte whereby synaptically-released glutamate (or other synaptic agents) promotes Ca2+ signaling in the astrocyte and the other two (B and C) from the astrocyte to synaptic terminals, whereby glutamate or ATP released from the astrocyte affects synaptic function  can be carried out by opening the feedback loop (for example by inhibiting elements of A or B) and analyzing the signaling components separately. From a theoretical point of view, this approach is put forth by at least three steps of analysis that are: (1) characterization of Ca2+ dynamics in the astrocyte as a function of different synaptic inputs ; (2) characterization of how gliotransmitter release from the astrocyte depends on different astrocytic intracellular Ca2+ dynamics; and (3) characterization of the effect on synaptic release of Ca2+-dependent gliotransmitter release from the astrocyte (i.e. pathway B). These three aspects are discussed below from a modeling perspective, focusing on their possible roles in synaptic information processing.In principle the two pathways could coexist at the same synapse where they are expected to display different dynamics and respond to different preferred input stimuli. Therefore their coexistence at the same synapse could give rise to complex effects that are hard to quantify when considered altogether. Accordingly, a common approach in experiments is to characterize their effects on synaptic function by separate manipulations of pathways C Figure  using diC Figure . For exa2+ elevations in the astrocyte are not simple on–off signals  from the ER is considered the primary mechanism responsible for intracellular Ca2+ dynamics in astrocytes  mediated by endoplasmic-reticulum Ca2+-ATPase (SERCA) pumps which contributes to the maintenance of higher Ca2+ concentrations in the ER stores than in the cytosol; (2) a passive Ca2+ leak (JL) from the ER to the cytosol that is driven by the Ca2+ gradient between the ER and the cytosol; and (3) an efflux (JNL) from the ER to the cytosol through IP3 receptor (IP3R) channels, which depends both on IP3 and Ca2+ concentrations in the cytosol in a nonlinear fashion  and by inositol polyphosphate 5-phosphatase (IP-5P)  Figure  (Zhang e) Figure . However3 can also be controlled by gap junction mediated diffusion of IP3 from other regions of the same astrocyte or from neighboring cells , is encoded by the modulation of Ca2+ oscillations and pulsations either in their amplitude (AM), their frequency (FM), or both (AFM). While available experimental data suggest a preferential FM mode of encoding . With low Ca2+, the activity of IP3-3K is reduced and the resulting IP3 degradation slows down. The contribution to IP3 production by Ca2+-dependent PLCδ is reduced as well, so that intracellular IP3 mostly depends on the frequency of synaptic release. Rapid successions of synaptic release events produce crisp increases of IP3  while, between two remote release events, IP3 tends to relax to resting levels. As a result, IP3 dynamics overall evolves as the integral of synaptic activity. If IP3 reaches the CICR-triggering threshold, intracellular Ca2+ increases fast and so does IP3-3K activity. Then, IP3 is rapidly degraded and resting IP3 levels are restored, thus resetting the integral of synaptic activity to initial values.Experimental evidence suggests that Ca3 degradation (a high Ca2+—high IP3-3K-activity phase and a low Ca2+—low IP3-3K-activity phase), that endows IP3 signal with high amplitude variability. On the other hand, such AM features still allow fast variations, thus rich spectral content , in response to changes in frequency of synaptic release. This enticing possibility could endow the IP3 signal with the necessary properties to function as optimal interface between synaptic stimuli and intracellular Ca2+ signals. Since neural information is carried by the timing of spikes rather than by their amplitude  and degradation (IP3 degr.) (dashed black lines) underlying simulated IP3 and Ca2+ signals  in response to two events of synaptic glutamate release (bottom panel). While Ca2+-dependent IP3 production by PLCβ (cyan trace) and PLCδ (pink trace) could modulate the threshold frequency of synaptic stimuli that triggers Ca2+ signaling in the astrocyte, existence of different regimes of IP3 degradation within a single Ca2+ oscillation cycle could be responsible for different cutoff frequencies of synaptic release beyond which Ca2+-mediated astrocyte processing of synaptic stimuli ceases. In particular, the cutoff frequency during low IP3-3K activity could be mainly set by the rate of IP3 degradation by IP-5P (orange trace). When Ca2+ is high instead, IP3 degradation by IP3-3K (purple trace) could also become very strong, thus sensibly reducing the cutoff frequency. That is, the cutoff frequency is dependent on the context of underlying Ca2+ signaling which, in turn, depends on the history of activation of the astrocyte by synaptic stimuli.The tight dynamical coupling between IP3 signaling could also be responsible for spatial heterogeneity of the frequency response of the astrocyte. Although the subcellular localization of IP3 production and degradation enzymes in astrocytes remains to be elucidated, studies in brain tissue suggest that PLCβ and IP-5P could localize mainly in proximity to the plasma membrane, whereas PLCδ and IP3-3K are preferentially in the cytoplasm  diffusion  and AFM-encoding (green traces) astrocytes. The synaptic stimulus is restricted to the first cell of the chain (i.e. cell number 1). Only FM-encoding cells guarantee regenerative propagation of Ca2+ signals, whereas AFM cells do not, acting like propagation barriers along the chain. Unlike in AFM cells, Ca2+-dependent IP3 production in FM cells guarantees IP3 diffusion to the next cell in the chain to levels that are beyond the threshold of CICR thus promoting regenerative propagation , though generally before the recovery of basal Ca2+ levels , triggering gliotransmitter release every time. Conversely, AM or AFM oscillations may not be large enough to reach such threshold, resulting in some oscillations that fail to trigger gliotransmitter release. In this fashion, while FM Ca2+ oscillations trigger gliotransmitter exocytosis at their own frequency, the amplitude of AM and AFM oscillations could selectively discriminate which Ca2+ pulse triggers exocytosis, eventually dictating the frequency of “measured” glutamate or ATP release events  likely depend on the type of targeted presynaptic receptors and are not fully understood , then synaptic release is increasing for incoming spikes, i.e. facilitation occurs, and the corresponding PPR is larger than unity. Conversely, when the second peak is less than the first peak (magenta traces), this marks a decrease of neurotransmitter release from the presynaptic terminal which reflects depression, and the corresponding value of PPR is less than one. It should be noted that, by varying the interval between two pulses, the same synapse can be either depressed or facilitated. For simplicity we omit the interpulse interval as a variable in this description.Short-term facilitation and depression can be assessed by measuring the paired-pulse ratio (PPR), i.e. the ratio between the amplitudes of successive postsynaptic currents (PSCs) recorded in response to a pair of action potentials in rapid succession as illustrated in Figure right). Thus, any modulation of synaptic release probability by gliotransmitters that changes the PPR from below unity to values above it or vice versa, could switch the mode of synaptic transmission from depressing to facilitating or vice versa. This scenario was theoretically addressed in De Pittà et al. . Each column in Figure 2+ pulses (top row), yielding to different frequencies of gliotransmitter release when Ca2+ crosses the threshold for exocytosis (dashed red line) , the raster plot of a facilitating synapse (middle row) shows predominant paired-pulse facilitation, i.e. mostly green dots. However, in presence of release of glutamate from the astrocyte (+Glu), as explained above, the stimulatory effect of this gliotransmitter on synaptic release changes paired-pulse plasticity which is marked by the appearance of magenta bands in the raster plots (middle and right columns). These bands locate time intervals where paired-pulse depression becomes prominent (i.e. predominance of magenta dots) on the overall paired-pulse facilitation background (green dots). Notably these bands are almost in coincidence with glutamate release from the astrocyte (top row) and their number increases with the glutamate release frequency. Therefore, for the same time window, the same originally-facilitating synapse gets increasingly depressing as the rate of glutamate release from the astrocyte increases. The opposite is observed for an otherwise depressing synapse . In this case, in presence of release of ATP and its derivative adenosine (+ATP/Adn) from the astrocyte, due to the inhibitory effect of astrocyte-derived purines on synaptic release considered in this example, green bands appear in the raster plots which mark the onset of periods of predominant paired-pulse facilitation. The number of these bands grows for higher rates of ATP release from the astrocyte so that the same originally-depressing synapse behaves more akin of a facilitating one as the rate of ATP release from the astrocyte increases.In absence of astrocytic gliotransmitter (blue line) could exist above which the astrocyte can switch the nature of synaptic transmission, turning depressing synapses into facilitating or vice versa, facilitating synapses into depressing , intracellular Ca2+ levels in astrocytes spontaneously fluctuate in a highly stochastic fashion  of synaptic transmission   model for variation of the threshold for LTP, which predicts that the relationship between synaptic activity and persistent changes in synaptic strength can vary according to the number of NMDARs available during synaptic activation  (Fellin et al., 2+ ions (Cull-Candy et al., Glutamate released from astrocyte could also target extrasynaptically-located NR2+-dependent gliotransmission may not be limited to the very synapses that trigger Ca2+ activity in the astrocyte but it could also affect farther synaptic domains in a multimodal fashion (Kozlov et al., Modulation of synaptic transmission by CaThe latter idea could also bring to a possible role of astrocyte signaling in coherent function of neural networks underlying potential behavioral states (Engel et al., A large body of evidence has accumulated over the last years, suggesting an active role of astrocytes in many brain functions. Collectively these data have fuelled the concept that synapses could be tripartite rather than bipartite, since in addition to the pre- and post-synaptic terminals, the astrocyte could be an active element in synaptic transmission (Araque et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


At the fruit specific level, ABA increased fruit tissue water-soluble apoplastic Ca2+ concentrationABA prevented blossom-end rot development in tomato fruit by whole plant and fruit specific mechanisms. At the whole plant level, ABA increased fruit Ca 2+ partitioning and allocation in plants and fruit remain poorly understood. The objectives of this study were to determine Ca2+ partitioning and allocation in tomato plants and fruit in response to whole-plant and fruit-specific abscisic acid (ABA) treatments, as well as to analyse the effect of changes in Ca2+ partitioning and allocation on fruit susceptibility to the Ca2+ deficiency disorder blossom-end rot (BER) under water stress conditions. Tomato plants of the cultivar Ace 55 (Vf) were grown in a greenhouse and exposed to low Ca2+ conditions during fruit growth and development. Starting 1 day after pollination (DAP), the following treatments were initiated: (i) whole plants were sprayed weekly with deionized water (control) or (ii) with 500mg l−1 ABA; or fruit on each plant were dipped weekly (iii) in deionized water (control) or (iv) in 500mg l−1 ABA. At 15 DAP, BER was completely prevented by whole-plant or fruit-specific ABA treatments, whereas plants or fruit treated with water had 16–19% BER incidence. At 30 DAP, BER was prevented by the whole-plant ABA treatment, whereas fruit dipped in ABA had a 16% and water-treated plants or fruit had a 36–40% incidence of BER. The results showed that spraying the whole plant with ABA increases xylem sap flow and Ca2+ movement into the fruit, resulting in higher fruit tissue and water-soluble apoplastic Ca2+ concentrations that prevent BER development. Although fruit-specific ABA treatment had no effect on xylem sap flow rates or Ca2+ movement into the fruit, it increased fruit tissue water-soluble apoplastic Ca2+ concentrations and reduced fruit susceptibility to BER to a lesser extent.The mechanisms regulating Ca The plants were grown in an organic substrate of equal portions of peat, sand, and redwood compost, with 2.6g kg−1 dolomite lime. Plants were irrigated once a day at about 08:00h with a nutrient solution containing N (102mg l–1), P (26mg l–1), K (124mg l–1), Ca (90mg l–1), Mg (24mg l–1), S (16mg l–1), Fe (1.6mg l–1), Mn (0.27mg l–1), Cu (0.16mg l–1), Zn (0.12mg l–1), B (0.26mg l–1), and Mo (0.016mg l–1). At full bloom, fully open flowers in the first cluster were tagged and manually pollinated. After pollination, plants were irrigated once a day at 08:00h with 450ml of the same nutrient solution, but without Ca2+, for the remaining period of the study to induce BER development in the fruit. The volume of nutrient solution added daily to the plants after pollination was determined from the volume of total plant water loss per day determined as described below (~450ml plant–1 d–1). This approach was used to induce plant hydration followed by plant dehydration in a 24h cycle. Wilting was typically observed in the control plants prior to irrigation in the morning, but plants recovered turgor upon irrigation. The water stress condition was used to enhance fruit susceptibility to BER.Tomato plants [−1 ABA , or 4–6 fruit on separate plants from those sprayed were dipped weekly in 300ml of deionized water (control) or 500mg l−1 ABA . All solutions contained 0.5ml l−1 of Silwet L-77  as a surfactant. The dipping treatments were applied by completely immersing the fruit in each solution for a period of 10 s. All treatments were applied once a week until 30 DAP. Each treatment was composed of four single plant replications at each evaluation time. After evaluation, plants were eliminated from the experiment. On each plant, 6–8 flowers on the first cluster were tagged, resulting in 6–8 tagged fruit per replication. No thinning treatment was applied to non-tagged flowers or fruit. Plants and fruit were analysed at 15 and 30 DAP as described below. Fruit sampling was accomplished at the end of the irrigation cycle, before 08:00h.Beginning 1 day after pollination (DAP), each plant was sprayed weekly with 200ml of deionized water (control) or 500mg lThe relative humidity and air temperature inside the greenhouse were monitored daily every 15min. The results presented are the hourly averages from 15 to 30 DAP. VPD was calculated based on the hourly measured relative humidity and air temperature as described by The percentage BER was determined by dividing the number of tagged fruit with BER symptoms by the total number of tagged fruit per plant and multiplying this value by 100.Analysis of electrolyte leakage was carried out according to the method described by The SWP was determined from 13:00h to 15:00h by bagging one leaf directly connected to the main stem in a reflective envelope for ~20min. Each bagged leaf was then cut from the plant and placed inside a pressure chamber  for SWP measurement .This was analysed in two opposite, fully expanded leaves located on the base, middle, and top regions of each tomato plant. The base, middle, and top regions were defined by visually dividing the height of the plant into three equal sections. The stomatal conductance measurements were accomplished from 13:00h to 15:00h using a steady-state porometer .The total water loss was determined by irrigating the plants early in the morning. After draining, the pots were bagged and weighed. Twenty-four hours later, the pots were weighed again and the volume of water lost was calculated as the difference between the first and the last weight per plant.These were accomplished using the heat ratio method (HRM) with an external sap flow sensor . After h2+, to each plant pot, and collecting the drained leachate. After collecting the soil leachate, plants were decapitated 15cm above the soil level and the pots containing the roots plus stem stump were used to extract the stem xylem sap. Two fruit and two middle leaves were harvested at the end of the irrigation cycle from each replicate plant with the entire peduncle and pedicel attached and kept in a sealed plastic bag for xylem sap extraction. Xylem sap extraction was accomplished by placing the plant pot, fruit, or leaf blade inside a pressure chamber (PMS Instrument Company), while the cut end of the stem, peduncle, or pedicel was exposed to the outside of the chamber through a seal. After sealing, the pressure inside the chamber was increased up to 0.8MPa with N2. The initial xylem sap moving out of the stem, peduncle, or pedicel cut end was blotted dry to reduce Ca2+ contamination from the cut. The following 100 μl was collected over a period of 5min and used to determine the Ca2+ concentration in the xylem sap . A standard Ca2+ calibration curve (R2=0.98) was used to determine the Ca2+ concentration in the samples.The Ca2+ concentration in leaf and fruit tissues was determined in freeze-dried leaf blades, as well as pericarp tissues manually cut from the peduncle and blossom regions of the fruit. Freeze-dried samples were subjected to microwave acid digestion and analysed by inductively coupled plasma atomic emission spectrometry . One tagged fruit on each plant replication was used for fruit growth rate analysis. All sensor signals were logged  at 10min intervals and averaged every hour.The fruit weight was determined by dividing the total weight of all tagged fruit on the plant by the total number of tagged fruit on each plant replication. The final result represents the average of all replications in each treatment. The average fruit water uptake for growth (WUG) was determined by subtracting the fruit water content quantified at 15 DAP from the fruit water content quantified at 30 DAP and dividing that value by 15 d. The results are presented as volume of sap uptake per fruit per day.Analysis of the PSU was based on the assumption that the total fruit water uptake from 15 to 30 DAP was supplied only by xylem and phloem. Therefore, PSU was determined as the sum of WUG plus transpiration (Ts) minus XSU. In this case,–1 d–1, for the same tomato fruit developmental stage (–1 d–1. Phloem sap solutes concentration (PSC) was calculated as the fruit dry weight (DW) accumulated from 15 to 30 DAP plus the carbon (C) weight loss due to fruit respiration (CWR) divided by the PSU from 15 to 30 DAP  or presented as means ±standard deviation (SD).Analysis of variance (ANOVA) was performed for each variable using the Statistical Analysis System (SAS) software package. The mean values were compared by Tukey’s test . The average xylem sap Ca2+ concentrations were 0.71±0.06mM and 0.86±0.02mM in top leaves, 0.77±0.03mM and 0.87±0.02mM in middle leaves, and 0.64±0.03mM and 0.81±0.02mM in basal leaves at 15 and 30 DAP, respectively. The Ca2+ concentration in the peduncle xylem sap was higher in fruit from ABA-sprayed plants at 15 and 30 DAP, compared with all other treatments , and statistically lower in plants sprayed with ABA (average=27.4±0.42mg g DW–1) than all other treatments (average=32.8±0.53mg g DW–1) at 30 DAP. The Ca2+ concentration in fruit tissue collected at the peduncle and blossom ends of the fruit was higher in ABA-sprayed plants at 15 and 30 DAP . In that case, ABA treatment may have also reduced the hydraulic resistance within the fruit, favouring xylemic water movement in the fruit towards the blossom-end tissue, provided a hydrostatic gradient responsible for xylem sap flow was present in the fruit  of the Ca2+ accumulation quantified by the difference in total fruit Ca2+ content observed at 30 DAP minus the total fruit Ca2+ content observed at 15 DAP. Considering that fruit water uptake is via the xylem and phloem, the results suggest that the phloem may have also contributed to fruit Ca2+ uptake under the experimental conditions described in this study. The results also show a greater difference between the quantified and estimated Ca2+ accumulation in the fruit of ABA-sprayed plants (6.7 μg Ca2+ fruit–1 d–1) than in the fruit of other treatments (1.2–1.5 μg Ca2+ fruit–1 d–1), suggesting that spraying plants with ABA also enhanced fruit phloem Ca2+ uptake. Considering that spraying tomato plants with ABA decreased fruit phloem sap uptake, it is possible that this treatment increased Ca2+ concentration in the phloem sap to increase fruit Ca2+ uptake to compensate for the reduction of phloem sap uptake. These results agree with previous studies suggesting that phloem can also have an important contribution to fruit Ca2+ uptake depending on the phloem sap Ca2+ concentration and phloem sap flow rate into the fruit (The results show that ABA spray treatment increased fruit xylem sap uptake4.72-fold, fruit xylem sap Ca2+ can be taken up by the cells, bind to active exchange sites within the cell walls, or remain soluble in the xylem vessels to be translocated towards the blossom-end tissues of the fruit (2+ content at the fruit peduncle end resulted in higher fruit Ca2+ translocation to and Ca2+ accumulation in the blossom-end tissues in response to whole-plant ABA treatment.In the xylem vessels, after reaching the peduncle end of the fruit, Cahe fruit . Accordi2+ content at the fruit peduncle end, but resulted in higher Ca2+ accumulation and higher Ca2+ in the apoplast in the blossom-end tissue at 15 DAP, suggesting that ABA also triggered a fruit-specific mechanism that favoured Ca2+ translocation from the peduncle end towards the blossom-end region of the fruit. This latter effect was not observed at 30 DAP. According to the present data, spraying the whole plant with ABA or dipping the fruit in ABA maintained a higher number of functional xylem vessels that reduced the resistance to xylemic water and Ca2+ movement into the blossom-end tissue, which could help to explain the observed higher Ca2+ content in the distal end of the fruit. In ABA-dipped fruit, the increase in Ca2+ concentration in the blossom-end tissue was only observed at 15 DAP, possibly due to the reduction in any ABA effect on maintaining a higher number of functional xylem vessels at late stages of fruit growth and development. It is possible that ABA could also increase the number of functional xylem vessels connecting the fruit to the plant, which should be determined in future studies. In addition, higher cuticular wax content in epidermal cells at 30 DAP compared with fruit at 15 DAP (Dipping the fruit in ABA did not affect xylem sap or tissue Ca2+ flow to the leaves, which maintained higher stem water potential. Under such conditions, whole-plant ABA treatment favoured xylemic water and Ca2+ movement into the rapidly expanding fruit, resulting in higher Ca2+ content reaching the fruit peduncle end. However, the data suggest that xylem sap uptake could not fully explain fruit Ca2+ accumulation due to the difference between the observed total fruit Ca2+ accumulation and the estimated fruit Ca2+ accumulation based on the Ca2+ concentration in the xylem sap and xylem sap flow rate into the fruit. These results suggest that phloem could have acted as a source of Ca2+ to the fruit under the experimental conditions described in this study. More detailed studies should include direct measurements of fruit transpiration rates to better characterize the role of phloem in fruit Ca2+ uptake. In addition, a better understanding of phloem contributions to fruit Ca2+ uptake can be accomplished by developing efficient methods to extract and quantify Ca2+ in the phloem sap moving into the fruit.At the whole-plant level, ABA treatment triggered stomatal closure, decreasing xylemic water and Ca2+ movement in the fruit and thus allowing Ca2+ to be translocated towards the blossom-end tissue. Such Ca2+ accumulation in the blossom-end tissue was enhanced in response to whole-plant ABA treatment, compared with fruit-specific ABA treatment, possibly due to the additional effects on increasing xylem sap flow rate into the fruit, and Ca2+ concentration in the xylem sap taken up by the fruit.At the fruit-specific level, ABA maintained a higher number of functional xylem vessels at early stages of growth and development, reducing the resistance to Ca2+ in the fruit, and for that reason is the most susceptible tissue in the fruit to Ca2+ deficiency disorders (2+ deficiency symptoms are triggered by a depletion of the apoplastic pool of Ca2+ required to bind to phospholipids and proteins on the plasma membrane (2+, the plasma membrane can become leaky, leading to cell plasmolysis and eventually death (2+ accumulation in the blossom-end tissue in response to ABA treatment resulted in higher water-soluble apoplastic Ca2+ concentration, lower membrane leakage, and reduced fruit susceptibility to BER development. In this context, new tomato cultivars with higher ABA biosynthesis could be selected not only for water saving purposes, but also for reduced fruit susceptibility to BER. However, it is possible that fruit water content will be higher in high ABA genotypes, which may reduced the post-harvest life of fresh fruit.Blossom-end tissue has the lowest concentration of Caisorders . Studiesmembrane . Under c


However, if and how STIM1 and SOCE function in nerve growth cones during axon guidance remains to be elucidated.Stromal interaction molecule 1 (STIM1), a CaXenopus spinal growth cones to regulate Ca2+ signaling and guidance responses. We found that STIM1 works together with TRPC1 to mediate SOCE within growth cones and filopodia. In particular, STIM1/TRPC1-dependent SOCE was found to mediate oscillatory filopodial Ca2+ transients in the growth cone. Disruption of STIM1 function abolished filopodial Ca2+ transients and impaired Ca2+-dependent attractive responses of Xenopus growth cones to netrin-1. Finally, interference with STIM1 function was found to disrupt midline axon guidance of commissural interneurons in the developing Xenopus spinal cord in vivo.We report that STIM1 and transient receptor potential channel 1 (TRPC1)-dependent SOCE operates in 2+ signals that mediate guidance responses of nerve growth cones.Our data demonstrate that STIM1/TRPC1-dependent SOCE plays an essential role in generating spatiotemporal Ca We found that store Ca2+ depletion and re-addition of external Ca2+ experiment led to Ca2+ entries in about 25% filopodia  was found to potentiate both the incidence and frequency of filopodial Cas Figure : Movie 7N Figure E and F. 1 Figure E and F. 2+ in filopodia. We thus analyzed the sites of initial filopodial Ca2+ entry in Xenopus growth cones by kymography analysis. We found that, although the initial sites of Ca2+ entry distributed throughout the length of a filopodium, a large portion of the Ca2+ entry sites (42-59%) were found at the filopodial tip  were examined, no difference was seen on the location of Ca2+ entry sites in filopodia  without laminin coating exhibited robust chemoattractive turning within 30 minutes , PLC-γ and IP3 production, which further triggers store-depletion, STIM1 activation, and Ca2+ influx through TRPC1 for replenishing ER Ca2+. This notion is further supported by the findings that both netrin-1 and BDNF activate PLC-γ and Phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in neurite elongation  was identified by BLAST searches of the GenBank database using human STIM1 cDNA sequence. The coding region of XSTIM1 gene was isolated by RT-PCR, sequenced and cloned into the pCS2 vector . The following constructs of XSTIM1 and its mutants were generated by site-directed mutagenesis (Strategene) or by PCR based on previous studies of mammalian STIM1 mutants i) along the length of the ROI (y-axis) over time (x-axis).For filopodial Cailopodia , in seruth cones  and mimiXenopus brain explants including spinal cords dissected from the embryos at stage 26-28 using lysis buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-Cl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 0.2 mM Na-orthovanadate, and protease inhibitor cocktail. They were incubated with the appropriate antibody for 3-4 hours at 4°C, followed by incubation with protein A/G agarose beads (Pierce) for overnight at 4°C. Mouse anti-c-Myc monoclonal antibody (Roche Applied Science) and rabbit anti-GFP polyclonal antibody (Abcam) were used for immunoprecipitation and immunoblotting.For co-immunoprecipitation, protein lysates were prepared from the Embryos at stage 25-26 were fixed and processed for immunocytochemistry as previously described ,23,62. MThe authors declare that they have no competing interests.SS designed and performed all the experiments, and wrote the manuscript. JQZ oversaw the project, designed some of the experiments, and revised the manuscript with SS. GLM provided guidance to the project and contributed to the writing of the manuscript. All authors read and approved the final manuscript.Xenopus laevis, Xenopus tropicalis and human. Identical amino acid residues are highlighted in dark. Dashes indicate gaps inserted for maximal alignment score. The red box indicates the amino acid  that was mutated in the dominant negative form of XSTIM1.Alignment of STIM1 amino acid sequences of Click here for fileXenopus embryonic neural tissues.Association of XSTIM1 with TRPC1. Shown are sample westernblots for co-immunoprecipitation of human TRPC1 (myc-hTRPC1-WT) and wild-type (YFP-XSTIM1-WT) or constitutively active STIM1 (YFP-XSTIM1-CA) expressed in Click here for fileMovie 1. Filopodial Ca2+ entries are generated by SOCE. A pseudocolored Lck-GCaMP3 fluorescent Ca2+ images of Xenopus neuronal growth cones were captured at 200 milliseconds intervals over 7 min period of the store-depletion and re-addition of extracellular Ca2+ as shown in Figure Click here for fileMovie 2. SOCE-induced filopodial Ca2+ entries are abolished by inhibition of XSTIM1 with XSTIM1-DN overexpression. A pseudocolored Lck-GCaMP3 fluorescent Ca2+ images of Xenopus neuronal growth cones expressing XSTIM1-DN were captured at 200 milliseconds intervals over 7 min period of the store-depletion and re-addition of extracellular Ca2+.Click here for fileMovie 3. SOCE-induced filopodial Ca2+ entries are attenuated by inhibition of XTRPC1 with XTRPC1-MO overexpression. A pseudocolored Lck-GCaMP3 fluorescent Ca2+ images of Xenopus neuronal growth cones expressing XTRPC1-MO were captured at 200 milliseconds intervals over 7 min period of the store-depletion and re-addition of extracellular Ca2+.Click here for fileMovie 4. Oscillatory spontaneous filopodial Ca2+ entries. A pseudocolored Lck-GCaMP3 fluorescent Ca2+ images of Xenopus neuronal growth cones were captured at 200 milliseconds intervals over 7 min period in Modified Ringer solutions (1 mM extracellular Ca2+) as shown in Figure Click here for fileMovie 5. Spontaneous filopodial Ca2+ entries are abolished by inhibition of XSTIM1 with XSTIM1-DN overexpression. A pseudocolored Lck-GCaMP3 fluorescent Ca2+ images of Xenopus neuronal growth cones expressing XSTIM1-DN were captured at 200 milliseconds intervals over 7 min period in Modified Ringer solutions.Click here for fileMovie 6. Spontaneous filopodial Ca2+ entries are abolished by inhibition of XTRPC1 with XTRPC1-MO overexpression. A pseudocolored Lck-GCaMP3 fluorescent Ca2+ images of Xenopus neuronal growth cones expressing XTRPC1-MO were captured at 200 milliseconds intervals over 7 min period in Modified Ringer solutions.Click here for fileMovie 7. Netrin-1-potentiated filopodial Ca2+ entries. A pseudocolored Lck-GCaMP3 fluorescent Ca2+ images of Xenopus neuronal growth cone were captured at 200 milliseconds intervals in Modified Ringer solution (1 mM extracellular Ca2+) with Sp-cAMP (25 μM) before and after bath application of netrin-1 (10 ng/ml) as shown in Figure Click here for fileMovie 8. Dynamic translocation of STIM1 into neuronal filopodia in response to store-depletion. Time-lapse fluorescent images of Xenopus neuronal growth cone expressing YFP-XSTIM1 before (1 mM Ca2+) and after store Ca2+ depletion (0 mM Ca2+/CPA). Images were captured at 2 seconds intervals.Click here for file


In chiral and non-chiral electrophoretic resolution of basic drugs, adsorption of analytes to negatively charged capillary wall could lead to poor repeatability of migration time and peak area. In addition, chiral resolutions of basic drugs are commonly performed in low pH buffers. Therefore, longer analysis time due to suppression of electroosmotic flow (EOF) is another dilemma. In this work the improvement effect of polybrene (PB), a cationic polymer, on chiral separation of a model basic drug, amlodipine (AML), was investigated. PB both as a semi-permanent coating agent and as an additive in the running buffer was utilized. Better results were obtained with PB as a buffer additive. Compare to untreated bare silica without using PB in running buffer, addition of 0.0005% PB buffer decreased analysis time downed to 3 folds; efficiency improved up to 5 folds; limit of detection (LOD) and limit of quantification (LOQ) downed to 8 folds and within-day migration time and peak area repeatabilities, in terms of relative standard deviations (RSD) downed to 5 and 20 folds, respectively. About 40% of drugs in use are chiral , 2 and iCE has matured to a powerful technique, especially for analytical enantioseparations. This is primarily due to high efficiency and high flexibility with regard to analytes and to the separation conditions, as well as the low consumption of chemicals and solvents. According to a recent discussion forum on the application of CE in the pharmaceutical industry, the major application of the technique is chiral separations , 5.However, CE has some limitations, especially for chiral and non-chiral resolution of basic drugs. Basic analytes are positively charged and electrostatically attracted to the negatively charged capillary walls. This irreproducible adsorption can lead to migration time and peak area variations , and cona > 5) at pH 2.5 , on the improvement of chiral resolution of a model basic drug, amlodipine (AML), was studied. AML  is a potα-cyclodextrin; α-CD , β-cyclodextrin; β-CD , γ-cyclodextrin; γ-CD , and hydroxypropyl β-cyclodextrin; HP-βCD  were used as received. Concentration of 10 mM of each cyclodextrin was dissolved in 50 mM sodium phosphate buffer. A solution of 1 mM mesityl oxide (Aldrich) dissolved in water, was used as the neutral EOF marker. Racemic AML and internal standard, racemic verapamil (VER) were provided by Minoo Co. . S-AML was a kind gift from Cipla . Racemic AML, S-AML and VER solutions were prepared in water and used throughout the experiments. All solutions were prepared in Nanopure 18 MΩ ultrapure water . Sodium phosphate buffer pH 2.5 was prepared from sodium phosphate monobasic monohydrate , and adjusting the pH with orthophosphoric acid . PB with molecular weight of ~4000-6000 was used as received from Aldrich . CE system3DCE capillary electrophoresis system  equipped with a diode array detector. CE Chemstation software  was used for control and data acquisition. The data acquisition rate was 6 Hz, and the rise time was 0.2 s. DAD was set at 200 or 254 nm. Untreated fused-silica capillaries  with a total length of 45.5 cm (37.0 cm to the detector), an inner diameter of 50 µm, and an outer diameter of 360 µm were used. The capillary was thermostated at 25°C. Samples injection was performed hydrodynamically by applying 50 mbar of pressure for 3s.Experiments were performed on a HPEach new capillary was pretreated by rinsing at high pressure (950 mbar) with 0.1 M NaOH for 10 min and with water for 10 min. A Corning model 445-pH meter  was employed to adjust the pH of the running buffer. Separations were performed at 20 kV. Depending on the conditions, normal or reverse polarities were applied.EOF measurementsEOF) was calculated using Equation 1.The neutral marker, mesityl oxide (1 mM in water) was injected into the capillary using hydrodynamic injection (50 mbar) for 3s. The electroosmotic mobility (µμEOF=Ld×LtMT ×V (Equation 1)t and Ld are the total length of the capillary (45.5cm) and the capillary length to the detector (37.0 cm), respectively, MT is the migration time of the neutral marker (in seconds), and V is the applied voltage (in volts).Where LCapillary coatingAll of the rinsing procedures performed at 950 mbar. New capillaries were pretreated by rinsing at high pressure (950 mbar) with 0.1 M NaOH for 10 min and with water for 10 min, sequentially. After that, capillaries were rinsed for 3 min with 1% w/v PB in water. Then, capillaries were rinsed for another 10 min with water followed by running buffer for 3 min.Coating stabilityβCD with applying -20 kV voltage were conducted. Between runs different washing cycles were applied. Along with, EOF was measured by using mesityl oxide.The stability of the PB coatings was indirectly evaluated by monitoring the EOF as a function of time. The stability studies were performed in two ways. In the first method, or hydrodynamic rinsing, the coated capillary was flushed with different solutions at a constant pressure (950 mbar) for various times. The EOF mobility was then determined as described above. In the second method, or successive injections , a serieSample and internal standard preparationStandards of 100 ppm of racemic-AML and S-AML were prepared in nanopure water. VER was selected as internal standard since it migrates close to the AML enantiomers.All samples solutions were spiked with a fixed concentration (20 ppm) of VER.a 8.6) with the primary amine group (AML is a basic drug (pKne group . AML, line group  and has Different strategies have been employed for coating of capillaries by PB. Yeh and coworkers  employedResolution of AML in bare silicaet al. (βCD, adjusted to pH 2.5 was employed as background electrolyte. Initially, chiral resolution of AML in bare silica was performed. On the basis of a report published by Owens et al.  a sodiumThe LOD and the LOQ were estimated as three and ten times the signal-to-noise ratios, respectively. The LOD and LOQ of AML enantiomers in uncoated capillaries were 25 and 80 ppm, respectively.et al. using HP-βCD as a neutral selector (As shown in selector .Resolution of AML in semi-permanent coated capillaries Capillaries were coated with PB solution under the conditions described previously. PB strongly adsorbed onto the inner capillary surface due to electrostatic attraction between this cationic polymer and the anionic silica . Due to For enantioresolution, coating should be also stable when cyclodextrins are added to running buffer. The second study of coating stability was performed by 30 successive separations as detailed in experimental section. Results in In the coated capillaries, due to opposite direction of EOF and electrophoretic mobility of AML enantiomers, the more tightly interacted S-enantiomer reached faster to the detector . TherefoOne of the advantages of strong reversed EOF in the coated capillary is the ability of resolution of AML counter-ion, besylate, with the drug peak in a single run. Furthermore, compare to untreated bare silica, LOD downed from 25 ppm to 3 ppm and LOQ downed from 80 ppm to 10 ppm. Another advantage of strong reversed EOF is decreasing analysis time up to 3 folds.PB as a buffer additiveTo improving peak tailing, PB was used as a buffer additive. It is known that even low concentrations of PB could reverse the electroosmotic flow . As starBoth using PB as buffer additive and semi-permanent coating agent improve detection limit, efficiency and repeatability of AML chiral resolution. In addition, PB increases speed of the analysis by generating strong reversed EOF. Also, due to strong reverse EOF, simultaneous resolution of AML and besylate was achievable. Although results demonstrate high stability of PB semi-permanent coating but peak tailing for both enantiomers of AML was observed. Therefore, PB as buffer additive selected as a superior approach for improvement of chiral analysis of AML.


Caenorhabditis elegans. This worm has only three pairs of olfactory receptor neurons. Intracellular Ca2+ decreases in one pair of olfactory neurons in C. elegans, the AWC neurons, following application of an attractive odor and there is a transient increase in intracellular Ca2+ following removal of odor. The magnitude of this increase is positively correlated with the duration of odor stimulation. Additionally, this Ca2+ transient is induced by a cGMP second messenger system. We identified likely candidates for the signal transduction molecules functioning in this system based on available gene expression and physiological data from AWCs. Our model incorporated a G-protein-coupled odor receptor, a G-protein, a guanylate cyclase as the G-protein effector, and a single phosphodiesterase. Additionally, a cyclic-nucleotide-gated ion channel and a voltage-gated ion channel that mediated calcium influx were incorporated into the model. We posited that, upon odor stimulation, guanylate cyclase was suppressed by the G-protein and that, upon cessation of the stimulus, the G-protein–induced suppression ceased and cGMP synthesis was restored. A key element of our model was a Ca2+-dependent negative feedback loop that ensured that the calcium increases were transient. Two guanylate cyclase-activating proteins acted on the effector guanylate cyclase to negatively regulate cGMP signaling and the resulting calcium influx. Our model was able to closely replicate in silico three important features of the calcium dynamics of AWCs. Specifically, in our simulations, [Ca2+] increased rapidly and reached its peak within 10 s after the odor stimulus was removed, peak [Ca2+] increased with longer odor exposure, and [Ca2+] decreased during a second stimulus that closely followed an initial stimulus. However, application of random background signal (‘noise’) showed that certain components of the pathway were particularly sensitive to this noise.We developed a mathematical model of a hypothetical neuronal signal transduction pathway to better understand olfactory perception in  Caenorhabditis elegans nematode worms exhibit well-characterized responses to defined external stimuli, and their nervous system is also well characterized. Therefore, this species is suitable for mathematical modeling of nerve and nervous system function. The nervous system of C. elegans comprises only 302 neurons; consequently, the role of a single neuron in the worm may be more important than that of any single neuron in the larger nervous systems of higher animals. Accurate physiological and signal-transduction-gene expression data from a neuron are essential for developing models of signal transduction in that neuron. Electrophysiological studies 2+]) after the cessation of odor stimulus C. elegans, cGMP is more likely to be the second messenger because CNG channels in silico the [Ca2+] changes observed in AWC physiological experiments. We propose that, upon odor stimulation, guanylate cyclase is suppressed by a G-protein and that, upon cessation of the stimulus, the suppression ceases and cGMP synthesis is restored. Increases in cGMP concentration induce calcium ions influx via cGMP-sensitive CNG channels. Also the pathway includes feedback regulation of guanylate cyclase activity.Here, we constructed a model of the odor transduction pathway in AWC neurons that can replicate 2+], we searched the literature and the C. elegans database (http://www.wormbase.org) to identify molecules that are reportedly expressed in C. elegans neurons. The physiological responses of AWC neurons to olfactory stimuli have been studied primarily using calcium imaging, and the fluorescent protein G-CaMP 2+] increases transiently after removal of odor stimulation, but the signal transduction mechanism remains unclear. We proposed a hypothetical scheme that could replicate this phenomenon in silico; we introduced likely candidate molecules into this scheme to represent components of this unknown mechanism.  2+] changes in AWCs resemble the well-characterized [Ca2+] responses that occur in the photoreceptor cell of vertebrate retina. In the vertebrate retina, transducin, a G-protein, binds and activates phosphodiesterase 6 (PDE6), the G-protein effector; these events result in cGMP dissociation, which in turn reduces the cation influx through cGMP-gated channels C. elegans, there is no report of expression of the transducin or PDE6, and our BLAST searches did not recover C. elegans homologs of transducin or PDE6. Moreover, a recently published article pde1–pde5) do not produce abnormal chemotaxis toward benzaldehyde, an attractant sensed by AWC neurons. The remaining PDE, PDE6, is described by www.wormbase.org as an isoform of mammalian cAMP-specific PDE8A. Therefore, PDE in C. elegans apparently differs from PDE6 in the vertebrate retina, and we proposed that in C. elegans guanylate cyclase (GCY), an enzyme that synthesized cGMP from GTP, was the Gα effector. We posited that, upon odor stimulation, GCY was suppressed by Gα and, upon cessation of the stimulus, the suppression was removed and cGMP synthesis was restored.The effector of the Gα subunit has not been identified; therefore, we selected a likely candidate, guanylate cyclase (GCY), as the hypothetical effector. The  after removal of odor stimulus, a mechanism for transient increase in GCY activity was included in the model. The level of GCY activity should be positively correlated with length of odor stimulation. A guanylate cyclase activating protein (GCAP) was included in the feedback loop incorporated into our model. C. elegans NCS-1 has high homology to vertebrate GCAP; GCAP activate GCY at low calcium concentrations in vertebrate retina 2+]. GCAP potentiates GCY activity, but the effects of GCAP are suppressed when calcium ions bind GCAP; therefore, Ca2+-dependent negative feedback loops are formed. We posited that Gα-GCY-GCAP complexes accumulated during odor stimulation. We also hypothesized that, when the stimulus was removed, Gα dissociated from the complex, and GCY-GCAP produced cGMP; if these suppositions are true, the peak response would be dependent on the stimulus period.In order to realize a transient increase in [Ca2+ buffer. CaM trapped free calcium ions and reduced [Ca2+]. Excess calcium ions were transported outside the neuron via an extrusion apparatus, possibly a sodium-calcium exchanger (CaX). We posited that CaX was regulated by a calmodulin-Ca2+ complex.We theorized that calmodulin (CaM) acted as a Ca2+] must decrease following odor stimulation and increase within 1 s and reach its peak within 10 s after the odor stimulus is removed; then, [Ca2+] gradually returns to baseline at approximately 40 s. 2) The peak [Ca2+] must increase as the duration of odor stimulus increases between 1–5 min. 3) When the second stimulus is added in the period of high [Ca2+] after removal of the first stimulus, the [Ca2+] must decrease during the second stimulus. The increase in [Ca2*] after stimulus removal may trigger backing or turning behavior when the worm moves in the down-gradient direction of a concentration gradient of attractive odor. G-CaMP-based estimates of [Ca2+] depend on changes in fluorescence intensity; they do not measure [Ca2+] directly. Quantitative relationships between G-CaMP fluorescence and [Ca2+] have been assessed using a cell-free system 2+] and fluorescence vary widely and are dependent on experimental conditions. We adopted the EC50 value of G-CaMP in the cell-free system 2+], G-CaMP fluorescence changes, and membrane potential changes  concerning the above-mentioned sensitive parameters. CaM::Ca4 and cGMP showed transient [Ca2+] increases in the presence of noise, while GTP was insensitive to noise , was sufficient to reproduce the experimental data. To date, there is no report that Gα suppresses GCY, and GCY may be suppressed either directly or indirectly by Gα; however, for simplicity we posited direct suppression. We examined also whether a model with one GCAP and two PDEs could produce the [Ca2+] changes shown in 2+] by the second odor stimulus. Therefore, two GCAP and one PDE were used in the model.Genes 2+] that had magnitudes that were dependent on the duration of the stimulus. We chose GCAP as the feedback factor for cGMP synthesis. However, GCAP could be replaced by another protein while maintaining the basic feedback scheme; for example, the type II calmodulin-dependent protein kinase UNC-43 modulates the function of other proteins in a [Ca2+]-dependent manner, although the feedback loop has to be modified. Moreover, accumulation of the Gα-GCY-GCAP complex during odor stimulation was included in the model. Experimental studies are necessary to verify the model.A negative feedback pathway was essential for transient increases in [Ca2+] activity in AWCs is observed experimentally, and is suppressed by a neuropeptide-mediated feedback from AIA interneurons 2+] fluctuations were only observed when external input was applied. It is possible that the fluctuations observed in imaging experiments may result from synaptic inputs or from external disturbances of the intracellular signaling pathway. The parameters, which satisfied the condition that noise activity shows transient large [Ca2+] increases and only small decreases CNG, nCNG; K−1,GTPsupp; K+1,CaM+Ca, K+1,CaM::Ca+Ca, K+1,CaM::Ca2+Ca, K+1,CaM::Ca3+Ca; K+1,PDE+CaM::Ca4; K+1,PDEactive and EfCaX. These parameters, and therefore the decrease in GTP supply, the increase in concentration of active PDE to bind cGMP, or the calcium extrusion mechanism, might concern the spontaneous activity in AWC neurons. For example, EC50CNG is affected by calmodulin 2+] activity. EC50CNG and nCNG were hyper-sensitive to noise  reported that AWC releases both glutamate and the neuropeptide NLP-1. These transmitters are sensed by AIA interneurons that, when odor is present, release the insulin-like neuropeptide INS-1, which suppresses AWC calcium transients. Although the INS-1 receptors in AWC and their downstream molecules are not yet identified, the whole model of [Ca2+] activity should be constructed in the future based on experimental studies.Spontaneous [Cato noise  and migh2+] that occur in AWC neurons upon odor stimulation. We have also used this model to analyze concentration changes in molecules and to identify possible causes of [Ca2+] fluctuation.We have presented a hypothetical model that can explain the changes in [Ca2+] in AWCs following odor stimulation 2+ influx and efflux were described by Hill equations or modified Hill equations formulated by the authors. Relationship between G-CaMP fluorescence and [Ca2+] (http://www.copasi.org) to perform these simulations (COPASI-file S1).Activation of second messenger proteins and their enzyme reactions were modeled using differential equations in which the mass-action law  is supposed for all processes, if not stated explicitly. The differential equations are presented in d [Ca2+]  was based [Ca2+]  and the The resting membrane potential in AWCs is approximately −28 mV The pulse train noise with the following features was made. The pulse height was A(1+R). A was fixed to the parameter value when the noise was added to the parameter value, whereas A was variable  whenever the noise was added to the concentration of any substance. R was the uniform noise with the range . Pulse width had a uniform random value between 1 and 5 s. The interval between the end of a pulse and the start of the succeeding pulse was a uniform random value between 0.1 and 10 s.COPASI-file S1COPASY-file including the information that is necessary to perform simulation.(CPS)Click here for additional data file.Figure S1Current-voltage relationship for voltage-dependent currents without odor stimulation in AWC neurons. Deduced from electrophysiological data published by Nickell et al. (2002).(PDF)Click here for additional data file.Figure S2active , D. PDEactive::cGMP.Changes in concentrations of intracellular pathway components. These changes are related to PDE and catalytic reactions caused by the addition or removal of odor stimulation. A. PDE, B. PDE::cGMP, C. PDE(PDF)Click here for additional data file.Figure S31, C. CaM::Ca2, D. CaM::Ca3, E. CaM::Ca4. CaM::Cak (k = 1–4) calmodulin binding with k calcium ions.Changes in concentrations of intracellular pathway components. These were related to a combination of calcium buffer and calcium ions caused by addition or removal of odor stimulation. A. Free calmodulin (CaM)-like calcium buffer, B. CaM::Ca(PDF)Click here for additional data file.Figure S42+], G-CaMP fluorescence and membrane potential. The bars indicate periods of stimulation. A, B, C. Responses to 1-min stimulus. D, E, F. After removal of the first 1-min stimulus, a second 20-s stimulus was applied 10 s later. G, H, I. After removal of the first 1-min stimulus, a second was applied 30 s later. J, K, L. Responses to 5-min stimulus. M, N, O. After removal of the first 5-min stimulus, a second 20-s stimulus was applied 10 s later. P, Q, R. After removal of the first 5-min stimulus, a second was applied 30 s later.Changes in [Ca(PDF)Click here for additional data file.Table S1Values of parameters in equations. This table includes the values used for figures in the result section, the lower and upper limits used in the genetic algorithm, and the reference numbers in References in the text.(PDF)Click here for additional data file.Text S1List of equations.(PDF)Click here for additional data file.


Ca2+ is an essential secondary intracellular signaling molecule that regulates fundamental cellular functions in various cell types, including neurons. We present a systematic review of the studies reporting changes in intracellular Ca2+ levels in response to activation of the immunoglobulin superfamily CAMs, cadherins and integrins in neurons. We also analyze current experimental evidence on the Ca2+ sources and channels involved in intracellular Ca2+ increases mediated by CAMs of these families, and systematically review the role of the voltage-dependent Ca2+ channels (VDCCs) in neurite outgrowth induced by activation of these CAMs. Molecular mechanisms linking CAMs to VDCCs and intracellular Ca2+ stores in neurons are discussed.Cell adhesion molecules (CAMs) play indispensable roles in the developing and mature brain by regulating neuronal migration and differentiation, neurite outgrowth, axonal fasciculation, synapse formation and synaptic plasticity. CAM-mediated changes in neuronal behavior depend on a number of intracellular signaling cascades including changes in various second messengers, among which CAM-dependent changes in intracellular Ca Cell adhesion molecules (CAMs) expressed on the neuronal cell surface play crucial roles in neuronal migration, axonal fasciculation, and neurite outgrowth during brain development. They also play an important role in regulation of synaptic plasticity in adult brain and axonal regeneration in injured nervous system -6.2+ levels have been documented to occur in neurons in response to activation of virtually all families of CAMs, including the three main families comprising the immunoglobulin superfamily (IgSF), cadherins and integrins.Functions of CAMs are induced in response to their binding to ligands presented either in the soluble form or on membranes of other cells or on artificial surfaces, a process which is often called CAM activation. CAM activation induces a number of intracellular signaling cascades, which are essential for CAM-mediated functions . A2+ serves as a secondary signaling messenger with Ca2+ channels at the neuronal cell surface and internal Ca2+ stores regulating intracellular Ca2+ concentrations in neurons and other cells. Intracellular Ca2+ has critical roles in all aspects of neuronal development including neurite elongation and neuronal growth cone motility in developing neurons [Intracellular Ca neurons -17.2+ levels in response to activation of IgSF CAMs, cadherins and integrins in neurons. We also analyze the experimental evidence supporting the involvement of the cell surface Ca2+ channels and intracellular Ca2+ stores in intracellular Ca2+ changes induced by CAMs of these families, and review the data showing the effects of Ca2+ channel inhibitors on CAM-induced neurite outgrowth.Intracellular signaling cascades activated by CAMs in response to ligand binding induce a number of physiologically important responses in neurons, among which changes in neurite outgrowth are probably the best characterized for CAMs of different families ,7,11,18.2+ levels were first documented in response to activation of the neural cell adhesion molecule (NCAM) and L1, both members of the IgSF  [2+ levels in PC12 cells and in small cerebellar neurons as measured using fluorimetry and a Ca2+ indicator Quin-2AM [2+ levels in dorsal root ganglion neurons but not in small cerebellar neurons loaded with Fura-2AM [2+ levels.CAM-induced increases in intracellular Ca neurons . Similarter (AM) ,23. The Quin-2AM ,25. In aFura-2AM , suggest2+ levels in PC12 cells [2+ influx in response to L1 activation has also been observed using voltage patch clamp recordings in cortical neurons [2+ levels in Fura-2AM loaded growth cones of dorsal root ganglion neurons [2+ levels in neurons incubated with a monoclonal antibody against an epitope within fibronectin type III repeats of L1, but not in neurons incubated with the monoclonal antibodies against epitopes within immunoglobulin domains [2+ levels. Similar to L1, exposure to immunopurified NgCAM or anti-NgCAM antibodies induced an increase in intracellular Ca2+ levels in neurons from the brains of songbird zebra finch loaded with a Ca2+ indicator Fluo-3AM and analyzed by confocal microscopy [2+ in signaling induced by another member of this family, CHL1, has been recently suggested [2+ levels is still missing.CAMs of the L1 family, including L1, close homolog of L1 (CHL1), neurofascin and Neuron-glia cell adhesion molecule (NgCAM), also mediate homophilic adhesion and play a prominent role in the developing central nervous system (CNS) ,27. Fluo12 cells , small c12 cells ,25,28, c12 cells ,30, and 12 cells . Ca2+ in neurons . Interescroscopy . While tuggested , the dir2+ levels. Np mediates cell-to-cell adhesion via homophilic interactions and is expressed as two isoforms named according to the molecular weight Np55 and Np65. Application of recombinant ectodomains of Np55 and Np65 or a mimicking peptide of Np65 induced an increase in intracellular Ca2+ levels in synapses of cultured hippocampal neurons loaded with a Ca2+ indicator Fluo-4AM [2+ in hippocampal neurons and neurons from visual cortex loaded with Fluo-3AM [In addition to NCAM and L1 family members, activation of the immunoglobulin superfamily cell adhesion molecules neuroplastin (Np) and limbic system-associated membrane protein (LAMP) has also been shown to induce increases in intracellular CaFluo-4AM ,35. SoluFluo-3AM .2+ channels indicate that changes in intracellular Ca2+ levels in response to activation of IgSF CAMs can be mediated by several classes of VDCCs. Inhibitors of L- and T-type VDCCs reduced the increase in intracellular Ca2+ levels observed in response to NCAM activation in cultured hippocampal neurons [2+ response when compared to nifedipine, an inhibitor of L-type VDCCs, in cultured hippocampal neurons [2+ measurements showed that inhibitors of nonselective cation channels also reduced NCAM-dependent Ca2+ influx in Fura-2AM loaded neurons, suggesting that these channels are also activated in response to NCAM ligands [2+[2+ influx in mouse dorsal root ganglion neurons and small cerebellar neurons [2+ influx [2+ increases in hippocampal neurons were fully inhibited by nifedipine but not ω-conotoxin [Experiments with inhibitors of various types of Ca neurons  and PC12 neurons . Pimozid neurons . Fluorom ligands . Nifedipgands [2+,30,37, w neurons . Nifedip+ influx . The LAMonotoxin . Altoget2+ channels, but also depletion of the internal Ca2+ stores by incubation with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase, inhibited an increase in intracellular Ca2+ levels in response to activation of NCAM in cultured hippocampal neurons [2+ influx at the cell surface membrane is accompanied by the release of Ca2+ from the internal stores. Internal Ca2+ stores have also been suggested to contribute to intracellular Ca2+ increases in response to activation of Np55 [Not only inhibitors of cell surface Ca neurons ,38 and i neurons , indicat of Np55 .2+ levels in growth cones and cell bodies of neurons [2+ were also recorded in growth cones of chick retinal ganglion cells grown on N-cadherin and loaded with a membrane targeted Ca2+ indicator, FFP-18AM [2+ were found to be not affected by substrate coated recombinant N-cadherin suggesting that N-cadherin influences predominantly submembrane Ca2+ levels [2+ currents in ciliary ganglion neurons indicating that homophilic interactions of N-cadherin are sufficient to activate a cellular mechanism that regulates Ca2+ influx [Optical recordings of chick ciliary ganglion neurons loaded with Fura-2AM showed that application of soluble fragments of N-cadherin purified from brain resulted in an increase in intracellular Ca neurons . IncreasFFP-18AM . Interes+ levels . Whole c+ influx .2+ channels suggest that VDCCs play an important role in cadherin-induced increases in intracellular Ca2+ levels. In particular, co-application of nifedipine and ω-conotoxin partially reduced the increase in intracellular Ca2+ levels in response to N-cadherin activation in retinal ganglion cells [2+ influx in neurons. It should be noted, however, that inhibitors of N- and L-type VDCCs ω-conotoxin and diltiazem had no significant effect to N-cadherin-induced Ca2+ response in ciliary ganglion neurons [Similarly to members of IgSF, experiments with inhibitors of various types of Caon cells , suggest neurons .2+ levels have been shown to occur in response to activation of atypical cadherins Celsr2 and Celsr3, which are highly expressed in hippocampal and cortical neurons [2+ levels increased in response to recombinant cadherin repeats of Celsr3 and Celsr2 with a more pronounced effect observed in response to activation of Celsr2 when compared to Celsr3 [2+ levels in response to activation of Celsr2 or Celsr3 was inhibited by thapsigargin, indicating that intracellular Ca2+ stores play also a role in Ca2+ increases mediated by cadherin family members [In addition to N-cadherin, increases in intracellular Ca neurons . Opticalo Celsr3 . This in members .2+ levels were reported to occur in response to exposure of neurons to natural ligands of integrins. Optical recordings showed that laminin induced an increase in intracellular Ca2+ levels in growing Fura-2AM loaded chick ciliary ganglion neurons [2+ levels were observed in response to integrin ligand Arginine-Glycine-Aspartic acid (RGD)-sequence containing peptides. Optical recordings of Fluo-4AM loaded cultured Xenopus spinal neurons showed that incubation with soluble RGD peptides elevated intracellular Ca2+ levels in growth cones and increased filopodial Ca2+ transient frequency [2+ levels while larger responses were observed in neurons treated with RGD-containing peptides [2+ currents induced by activation of integrins using multivalent antibodies against integrins were also observed using whole cell patch clamp recordings in neurons acutely dissociated from the medial septum/diagonal band nucleus of the rat [2+ levels in motoneurons isolated from the CNS of the pond snail L. stagnalis[2+ levels, such as via activation the N-methyl-D-aspartate (NMDA) receptors in an integrin-independent manner [2+ to overall increases in intracellular Ca2+ levels in studies using RGD peptides cannot be fully excluded.Modest increases in intracellular Ca neurons , and in  neurons . Much larequency . Similarpeptides ,46. Incr the rat . Both, ostagnalis. It shout manner . Therefo2+ levels were partially blocked by nifedipine and gadolinium III (Gd3+), a broad spectrum VDCC inhibitor, in cortical neurons [2+ increases in somata of chick ciliary ganglion neurons [2+ stores and inhibitors of the ryanodine receptor (RyR) and inositol 1,4,5-triphosphate gated receptor (IP3R), channels through which Ca2+ in intracellular stores is released into the cytosol, also reduced but did not eliminate increases in intracellular Ca2+ levels in response to RGD-containing integrin ligand peptides in cortical neurons [2+ influx via VDCCs and Ca2+ release from internal stores can both contribute to the elevation of intracellular Ca2+ levels in response to integrin activation.Integrin β-dependent increases in intracellular Ca neurons . However neurons . Depleti neurons . Therefo2+ levels have also been reported for other neuronal cell surface molecules involved in neuronal adhesion, notably for amyloid precursor protein (APP) and cellular prion protein (PrP). Optical recordings of B103 rat neuroblastoma cells transfected with APP and loaded with Fluo-4AM showed an increase in intracellular Ca2+ levels in response to incubation with amyloid beta (Aβ), an APP-derived toxic peptide accumulating in brains of Alzheimer’s disease patients. Since no changes in intracellular Ca2+ levels in response to Aβ occured in cells non-transfected with APP, it was proposed that binding of Aβ to APP induced Ca2+ influx in these cells [2+ signaling has been also found in astrocytes from mice missing APP [Changes in intracellular Case cells . Dysregusing APP .2+ levels have been observed in synaptosomes incubated with recombinant PrP, while function blocking antibodies against PrP inhibited depolarization induced Ca2+ influx via synaptosomal VDCCs, indicating that PrP also plays a role in regulation of intracellular Ca2+ levels [2+-influx has been shown to occur in response to such ligands of PrP as laminin and stress-inducible protein 1 in dorsal root ganglion neurons loaded with Fluo-3AM [2+ influx has been observed using Fura-2AM and a Ca2+ indicator Calcium Green-5N in cerebellar granule cells and hippocampal CA1 neurons from PrP deficient mice, respectively [2+ were affected by PrP deficiency [An increase in intracellular Ca+ levels . PrP depFluo-3AM . Reducedectively ,55. Both2+ currents have been also recorded in mice deficient in α-neurexin [2+ levels in neurons. Whether binding of α-neurexins to neuroligins stimulates Ca2+ influx into neurons remains to be investigated.Reduced Caneurexin , indicatVDCCs have been shown to play a multitude of roles in the developing and adult brain being involved in a number of signaling pathways. The role of different types of VDCCs in various brain functions is beyond the scope of this review and we refer the reader to several recent excellent reviews on this subject -63. Belo2+ channels on CAM-induced neurite outgrowth is summarized in Table 2+ influx via different Ca2+ channels is necessary to raise the overall levels of intracellular Ca2+ above the threshold required for NCAM-dependent neurite outgrowth. Similarly, L1-dependent neurite outgrowth was blocked by inhibitors of L-type  and N-type (ω-conotoxin) VDCCs in rat cerebellar neurons and PC12 cells [2+ influx via distinct Ca2+ channels at the cell surface is required to induce a complete set of molecular changes and responses required for IgSF CAM-dependent neurite outgrowth. This scenario is consistent with the observations showing that VDCCs can activate several independent signaling pathways in growth cones of growing neurites [Analysis of studies investigating effects of various inhibitors of Ca12 cells ,67, and 12 cells . Neurite12 cells . Inhibit12 cells ,67,68. I12 cells . Altogetneurites .2+ channels are involved in cadherin-dependent neurite outgrowth in these cells. Surprisingly, inhibitors of L- and N-type VDCCs diltiazem and ω-conotoxin had no effect on integrin-mediated laminin-induced neurite outgrowth in ciliary ganglion neurons [Similarly to xxxxxxxxxxxIgSF CAMs, N-cadherin-mediated neurite outgrowth from PC12 cells has been shown to be inhibited by inhibitors of L- and N-type VDCCs in an additive manner . However neurons . Therefo2+ levels in neurons, the mechanisms of this increase remain incompletely understood and probably involve a number of signaling cascades, which link CAMs to the sources of extra- and intracellular Ca2+ by changing the permeability of the respective channels.While data accumulated over the last two decades indicate that activation of CAMs induces an increase in intracellular Ca2+ influx via VDCCs in response to L1 activation occurs without changes in membrane voltage [2+ influx via changing VDCCs properties. Probably the best characterized signaling pathway activated by IgSF CAMs to induce changes in intracellular Ca2+ levels involves the fibroblast growth factor receptor (FGFR)  or calcium/calmodulin-dependent kinase II α (CaMKIIα), which are activated by NCAM [2+ influx via VDCCs in response to activation of NCAM  and neuunctions ,102. In unctions , and ligunctions . Whether2+ levels in neurons by acting at VDCCs and possibly other types of Ca2+ channels in the neuronal cell surface plasma membrane and in the intracellular Ca2+ stores. Not only VDCCs, but also other neuronal plasma membrane Ca2+ channels such as transient receptor potential channels, stretch-activated channels, and cyclic nucleotide-gated channels have been reported to play a role in neuronal development [2+ channels in neurons remain however unknown. Current data indicate that CAMs activate Ca2+ channels by inducing intracellular signaling cascades which can either activate or remove inhibition of Ca2+ channels to induce an increase in intracellular Ca2+ levels. It remains to be investigated whether formation of the molecular complexes between CAMs and Ca2+ channels directly influences the activity of the latter.In conclusion, a number of studies indicate that CAMs play an important role in regulation of intracellular Caelopment -107. Dir2+ levels. Better imaging technologies which appeared recently may help to investigate the dynamics of local CAM-dependent Ca2+ changes occurring during cell-to-cell contact formation, and particularly during synapse formation. A combination of such technologies with biochemical analysis and optical imaging of the synapse enriched cytoskeleton components and enzymes may provide valuable information about the mechanisms of the molecular rearrangements accompanying contact maturation. The development of genetically encoded Ca2+ reporters [2+ levels in CAM-enriched membrane microdomains in different neuronal compartments, which is a promising new direction for further research.Most of the previous work was performed using artificial stimulation protocols by applying soluble ligands in the culture medium and monitoring bulk changes in intracellular Caeporters -111 withAA: Arachidonic acid; Aβ: Amyloid beta; AM: Acetoxymethyl ester; APP: Amyloid precursor protein; CAMs: Cell adhesion molecules; CaMKIIα: Calcium/calmodulin-dependent kinase II α; CHL1: Close homolog of L1; CHO: Chinese hamster ovary cells; CNS: Central nervous system; DAG: Diacylglycerol; ER: Endoplasmic reticulum; FGFR: Fibroblast growth factor receptor; HEK 293 cells: Human embryonic kidney 293 cells; IP3: Inositol 1,4,5-triphosphate; IP3R: Inositol 1,4,5-triphosphate gated receptor; LAMP: Limbic system-associated membrane protein; NCAM: Neural cell adhesion molecule; NgCAM: Neuron-glia cell adhesion molecule; NMDA: N-methyl-D-aspartate; Np: Neuroplastin; NSCCs: Nonselective cation channels; PLC: Phospholipase C; PKA: Protein kinase A; PKC: Protein kinase C; PTX: Pertussis toxin; PrP: Prion protein; RGD: Arginine-Glycine-Aspartic acid; RyR: Ryanodine receptor; TrpC5: Transient receptor potential channel 5; VDCCs: Voltage-dependent calcium channels.The authors declare that they have no competing interests.LS, IL and VS have performed literature search and analysis and written the manuscript. All authors read and approved the final manuscript.