Photolysis Of Caged Ca2 Research Articles

Intercellular Ca2+ waves propagate by regenerative IP3‐induced IP3 release

The endothelium is a sheet of highly‐specialised cells that line every blood vessel. Endothelial cells detect, integrate and respond to information from numerous signals, passing this information to neighbouring endothelial cells via intercellular communication to coordinate vascular contractility, inflammation and exchange of products between the blood and surrounding tissues. A significant form of intercellular communication occurs by travelling spatial gradients of Ca2+ (‘Ca2+ waves’) that move between cells. While the underlying mechanisms are not understood, Ca2+ and IP3 diffusion through gap junctions are each proposed to underlie Ca2+ wave propagation in the endothelium. Our results show that IP3, but not Ca2+ or gap junctions, is required for Ca2+ wave propagation.To investigate the mechanisms underlying endothelial signal propagation, second order mesenteric arteries were dissected from rats, pinned out in an en face configuration and Ca2+ signals measured using Cal‐520 (5μM). Localised photorelease of caged‐IP3 (5μM) generated rapid rises in endothelial Ca2+ at the site of photo‐activation. The Ca2+ rise subsequently propagated across cells outside the photolysis site. The propagated Ca2+ rise reflects cell‐cell communication since no uncaging occurred outside the photolysis site.To investigate the contribution of Ca2+ to signal propagation, Ca2+ buffering was spatiotemporally controlled using local photoactivation of the membrane permeant Ca2+ buffer, Diazo‐2. Release of the Ca2+ buffer prevented signal propagation within the uncaging region alone; signal propagation still occurred outside the uncaging area. This result suggests that IP3, but not Ca2+, underlies wave propagation. Supportive evidence for this conclusion was provided by photolysis of caged Ca2+(NP‐EGTA AM, 5μM). Photorelease of caged Ca2+ generated a Ca2+ rise that remained localised to the uncaging region and did not propagate outside the photolysis site, even with progressively increased Ca2+ loads. To verify these results, ACh (1μM) or ionomycin (10μM) were applied to restricted populations of cells by pressure ejection from a puffer pipette. A comparable Ca2+ rise was generated in each case, however the ACh generated a substantial Ca2+ wave that propagated upstream of the activated cells while the Ca2+ rise from ionomycin did not. These results suggest that Ca2+ is not responsible for propagating Ca2+ waves in the endothelium. The data also questions the involvement of gap junctions in wave transmission. If gap junctions were present and open, the Ca2+ rise generated by ionomycin would have been expected to diffuse to neighboring cells. This was not observed. Furthermore, the gap junction blockers gap27 and 18αGA, while successful in blocking FRAP, did not inhibit wave propagation. Together, these results suggest that IP3 is essential for Ca2+ wave propagation, but Ca2+ or gap junctions are not.

Angiotensin II Disrupts Neurovascular Coupling by Potentiating Calcium Increases in Astrocytic Endfeet.

BackgroundAngiotensin II (Ang II), a critical mediator of hypertension, impairs neurovascular coupling. Since astrocytes are key regulators of neurovascular coupling, we sought to investigate whether Ang II impairs neurovascular coupling through modulation of astrocytic Ca2+ signaling.Methods and ResultsUsing laser Doppler flowmetry, we found that Ang II attenuates cerebral blood flow elevations induced by whisker stimulation or the metabotropic glutamate receptors agonist, 1S, 3R‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (P<0.01). In acute brain slices, Ang II shifted the vascular response induced by 1S, 3R‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid towards vasoconstriction (P<0.05). The resting and 1S, 3R‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid–induced Ca2+ levels in the astrocytic endfeet were more elevated in the presence of Ang II (P<0.01). Both effects were reversed by the AT1 receptor antagonist, candesartan (P<0.01 for diameter and P<0.05 for calcium levels). Using photolysis of caged Ca2+ in astrocytic endfeet or pre‐incubation of 1,2‐Bis(2‐aminophenoxy)ethane‐N,N,N',N'‐tetra‐acetic acid tetrakis (acetoxymethyl ester), we demonstrated the link between potentiated Ca2+ elevation and impaired vascular response in the presence of Ang II (P<0.001 and P<0.05, respectively). Both intracellular Ca2+ mobilization and Ca2+ influx through transient receptor potential vanilloid 4 mediated Ang II‐induced astrocytic Ca2+ elevation, since blockade of these pathways significantly prevented the intracellular Ca2+ in response to 1S, 3R‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (P<0.05).ConclusionsThese results suggest that Ang II through its AT1 receptor potentiates the astrocytic Ca2+ responses to a level that promotes vasoconstriction over vasodilation, thus altering cerebral blood flow increases in response to neuronal activity.

Long extensions with varicosity-like structures in gonadotrope Lh cells facilitate clustering in medaka pituitary culture

Accumulating evidence indicates that some pituitary cell types are organized in complex networks in both mammals and fish. In this study, we have further investigated the previously described cellular extensions formed by the medaka (Oryzias latipes) luteinizing hormone gonadotropes (Lh cells). Extensions, several cell diameters long, with varicosity-like swellings, were common both in vitro and in vivo. Some extensions approached other Lh cells, while others were in close contact with blood vessels in vivo. Gnrh further stimulated extension development in vitro. Two types of extensions with different characteristics could be distinguished, and were classified as major or minor according to size, origin and cytoskeleton protein dependance. The varicosity-like swellings appeared on the major extensions and were dependent on both microtubules and actin filaments. Immunofluorescence revealed that Lhβ protein was mainly located in these swellings and at the extremity of the extensions. We then investigated whether these extensions contribute to network formation and clustering, by following their development in primary cultures. During the first two days in culture, the Lh cells grew long extensions that with time physically attached to other cells. Successively, tight cell clusters formed as cell somas that were connected via extensions migrated towards each other, while shortening their extensions. Laser photolysis of caged Ca2+ showed that Ca2+ signals originating in the soma propagated from the soma along the major extensions, being particularly visible in each swelling. Moreover, the Ca2+ signal could be transferred between densely clustered cells (sharing soma-soma border), but was not transferred via extensions to the connected cell. In summary, Lh gonadotropes in medaka display a complex cellular structure of hormone-containing extensions that are sensitive to Gnrh, and may be used for clustering and possibly hormone release, but do not seem to contribute to communication between cells themselves.

Calmodulin binds to Drosophila TRP with an unexpected mode

Drosophila TRP is a calcium-permeable cation channel essential for fly visual signal transduction. During phototransduction, Ca2+ mediates both positive and negative feedback regulation on TRP channel activity, possibly via binding to calmodulin (CaM). However, the molecular mechanism underlying Ca2+ modulated CaM/TRP interaction is poorly understood. Here, we discover an unexpected, Ca2+-dependent binding mode between CaM and TRP. The TRP tail contains two CaM binding sites (CBS1 and CBS2) separated by an ∼70-residue linker. CBS1 binds to the CaM N-lobe and CBS2 recognizes the CaM C-lobe. Structural studies reveal the lobe-specific binding of CaM to CBS1&2. Mutations introduced in both CBS1 and CBS2 eliminated CaM binding in full-length TRP, but surprisingly had no effect on the response to light under physiological conditions, suggesting alternative mechanisms governing Ca2+-mediated feedback on the channel activity. Finally, we discover that TRPC4, the closest mammalian paralog of Drosophila TRP, adopts a similar CaM binding mode.

Strong stimulation triggers full fusion exocytosis and very slow endocytosis of the small dense core granules in carotid glomus cells

Chemosensory glomus cells of the carotid bodies release transmitters, including ATP and dopamine mainly via the exocytosis of small dense core granules (SDCGs, vesicular diameter of ∼100 nm). Using carbon-fiber amperometry, we showed previously that with a modest uniform elevation in cytosolic Ca2+ concentration ([Ca2+]i of ∼0.5 µM), SDCGs of rat glomus cells predominantly underwent a “kiss-and-run” mode of exocytosis. Here, we examined whether a larger [Ca2+]i rise influenced the mode of exocytosis. Activation of voltage-gated Ca2+ channels by a train of voltage-clamped depolarizations which elevated [Ca2+]i to ∼1.6 μM increased the cell membrane capacitance by ∼2.5%. At 30 s after such a stimulus, only 5% of the added membrane was retrieved. Flash photolysis of caged-Ca2+ (which elevated [Ca2+]i to ∼16 μM) increased cell membrane capacitance by ∼13%, and only ∼30% of the added membrane was retrieved at 30 s after the UV flash. When exocytosis and endocytosis were monitored using the two-photon excitation and extracellular polar tracer (TEP) imaging of FM1–43 fluorescence in conjunction with photolysis of caged Ca2+, almost uniform exocytosis was detected over the cell’s entire surface and it was followed by slow endocytosis. Immunocytochemistry showed that the cytoplasmic densities of dynamin I, II and clathrin (key proteins that mediate endocytosis) in glomus cells were less than half of those in adrenal chromaffin cells, suggesting that a lower expression of endocytotic machinery may underlie the slow endocytosis in glomus cells. An analysis of the relative change in the signals from two fluorescent dyes that simultaneously monitored the addition of vesicular volume and plasma membrane surface area, suggested that with an intense stimulus, SDCGs of glomus cells underwent full fusion without any significant “compound” exocytosis. Therefore, during a severe hypoxic challenge, glomus granules undergo full fusion for a more complete release of transmitters.

Aberrant Deactivation-Induced Gain of Function in TRPM4 Mutant Is Associated with Human Cardiac Conduction Block

A gain-of-function mutation in the Ca2+-activated transient receptor potential melastatin member 4 (TRPM4A432T) is linked to life-threatening cardiac conduction disturbance, but the underlying mechanism is unclear. For deeper insights, we used photolysis of caged Ca2+, quantitative Ca2+, and electrophysiological measurements. TRPM4A432T's 2-fold larger membrane current was associated with 50% decreased plasma membrane expression. Kinetic analysis unveiled 4-fold slower deactivation that was responsible for the augmented membrane current progressively rising during repetitive human cardiac action potentials. Rational mutagenesis of TRPM4 at position 432 revealed that the bulkiness of the amino acid was key to TRPM4A432T's aberrant gating. Charged amino acids rendered the channel non-functional. The slow deactivation caused by an amino acid substitution at position 432 from alanine to the bulkier threonine represents a key contributor to the gain of function in TRPM4A432T. Thus, our results add a mechanism in the etiology of TRP channel-linked human cardiac channelopathies.

CaMKII-mediated phosphorylation of GluN2B regulates recombinant NMDA receptor currents in a chloride-dependent manner.

Some forms of long-term synaptic plasticity require docking of Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) to residues 1290-1309 within the intracellular C-terminal tail of the N-methyl-d-aspartate (NMDA) receptor GluN2B subunit. The phosphorylation of Ser1303 within this region destabilizes CaMKII binding. Interestingly, Ser1303 is a substrate for CaMKII itself, as well as PKC and DAPK1, but these kinases have been reported to have contradictory effects on the activity of GluN2B-containing NMDA receptors. Here, we re-assessed the effect of CaMKII on NMDA receptor desensitization in heterologous cells, as measured by the ratio of steady-state to peak currents induced during 3s agonist applications. CaMKIIα co-expression or infusion of constitutively active CaMKII limits the extent of desensitization and preserves current amplitude with repeated stimulation of recombinant GluN1A/GluN2B when examined using low intracellular chloride (Cl-) levels, characteristic of neurons beyond the first postnatal week. In contrast, CaMKIIα enhances the acute rate and extent of desensitization when intracellular Cl- concentrations are high. The apparent dependence of CaMKIIα effects on NMDA receptor desensitization on Cl- concentrations is consistent with the presence of a Ca2+-activated Cl- conductance endogenous to HEK 293 cells, which was confirmed by photolysis of caged-Ca2+. However, Ca2+-activated Cl- conductances are unaffected by CaMKIIα expression, indicating that CaMKII affects agonist-induced whole cell currents via modulation of the NMDA receptor. In support of this idea, CaMKIIα modulation of GluN2B-NMDA receptors is abrogated by the phospho-null mutation of Ser1303 in GluN2B to alanine and occluded by phospho-mimetic mutation of Ser1303 to aspartate regardless of intracellular Cl- concentration. Thus, CaMKII-mediated phosphorylation of GluN2B-containing NMDA receptors reduces desensitization at physiological (low) intracellular Cl-, perhaps serving as a feed-forward mechanism to sustain NMDA-mediated Ca2+ entry and continued CaMKII activation during learning and memory.

Collecting data to determine the Ca²⁺-binding properties of DM-nitrophen and proteins.

Conventional techniques used to measure Ca(2+) binding are too slow to determine accurately the fast binding kinetics of most molecules such as Ca(2+)-binding proteins (CBPs). We have developed an ultra-fast in vitro technique for measuring the Ca(2+)-binding properties of CBPs following flash photolysis of caged Ca(2+). Although the details of the setup, the mathematics, and the analysis involved in this technique have been published elsewhere, many of the practical details regarding the actual measurements have, until now, only been described minimally. Here, we present a protocol to gather the data necessary to determine the kinetic properties of a caged-Ca(2+) compound and a CBP.

Photolysis of Caged Ca2+But Not Receptor-Mediated Ca2+Signaling Triggers Astrocytic Glutamate Release

Astrocytes in hippocampal slices can dynamically regulate synaptic transmission in a process mediated by increases in intracellular Ca(2+). However, it is debated whether astrocytic Ca(2+) signals result in release of glutamate. We here compared astrocytic Ca(2+) signaling triggered by agonist exposure versus photolysis side by side. Using transgenic mice in which astrocytes selectively express the MrgA1 receptor, we found that receptor-mediated astrocytic Ca(2+) signaling consistently triggered neuronal hyperpolarization and decreased the frequency of miniature excitatory postsynaptic currents (EPSCs). In contrast, photolysis of caged Ca(2+) (o-nitrophenyl-EGTA) in astrocytes led to neuronal depolarization and increased the frequency of mEPSCs through a metabotropic glutamate receptor-mediated pathway. Analysis of transgenic mice in which astrocytic vesicular release is suppressed (dominant-negative SNARE mice) and pharmacological manipulations suggested that glutamate is primarily released by opening of anion channels rather than exocytosis. Combined, these studies show that photolysis but not by agonists induced astrocytic Ca(2+) signaling triggers glutamate release.

Calcium-induced outgrowth of astrocytic peripheral processes requires actin binding by Profilin-1

Peripheral astrocytic processes (PAPs) are highly motile structures that are strategically positioned in close proximity to synapses. Long-lasting PAP retraction in hypothalamus is known to alter synaptic transmission [1]. The PAP motility is likely to be actin-based because they are known to contain actin-related proteins such as Ezrin [2]. However, the link between dynamic activity-dependent changes in astrocytic morphology and the synaptic function has not been established experimentally, presumably due to lack of appropriate tools. To selectively suppress activity-dependent morphological plasticity of astrocytes, we developed a bicistronic construct that allows simultaneous tracing and manipulating the morphology of PAPs. The construct is designed for co-expression of (i) the mutant actin binding protein Profilin-1 (abdProf-1) with a single amino acid substitution (H119E) that prevents its binding to actin monomers [3] with (ii) the membrane-targeted morphological tracer LckGFP [4]. Cultured cortical astrocytes transfected with this construct showed abdProf-1 overexpression at a 5-fold level compared to the endogenous Profilin-1. The cells also expressed LckGFP at a level sufficient for precise morphological tracing. We found that photolysis of caged Ca2+ induced a pronounced outgrowth of PAPs, which was suppressed by abdProf-1 overexpression in terms of PAP number, growth rate and maximal length. In contrast, the morphological complexity of astrocytes, basal motility of their PAPs and major cytoskeletal structures were not affected by abdProf-1 overexpression. In summary, we identified the actin binding by Profilin-1 as a pivotal mechanism in activity-dependent morphological plasticity of PAPs in cultured astrocytes.

Astrocyte-induced cortical vasodilation is mediated by D -serine and endothelial nitric oxide synthase

Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D-serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca(2+) produced dilation of penetrating arterioles in a manner attenuated by scavenging D-serine with D-amino acid oxidase, deleting the enzyme responsible for D-serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D-serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling.

ANO1 Activation does not require Calmodulin

The Ca2+-activated Cl channel anoctamin-1 (ANO1, also called TMEM16A), a member of the Anoctamin superfamily, plays a variety of important physiological roles including epithelial fluid secretion. ANO1 is activated and gated by intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to ANO1 or whether an additional Ca2+-binding subunit such as calmodulin (CaM) is required. Here, we report that CaM is not a necessary component for ANO1 activation for the following reasons: 1) ANO1 activation by photolysis of caged Ca2+ is very rapid (<1ms), suggesting that Ca2+ binds directly to the channel. 2) Exogenous CaM has no effect on the currents generated by either the ac or abc isoforms of ANO1 in inside-out excised patches. 3) Over-expression of functionally defective CaM has no effect on ANO1 (abc isoform) currents while it eliminates the small conductance Ca-activated K (SK2) current. 4) CaM does not physically interact with ANO1 as determined by co-immunoprecipitation. 5) ANO1 is activated in excised patches by Ba, which is a very poor CaM activator. We also re-examined the effect of the CaM inhibitor trifluoperazine (TFP). TFP blocks ANO1 in a voltage-dependent manner, suggesting that TFP blocks the current by lodging in the ANO1 pore. These results show CaM is not required for ANO1 activation and supports the hypothesis that Ca binds directly to ANO1.

Analysis of neurotransmitter release mechanisms by photolysis of caged Ca2+ in an autaptic neuron culture system

Neurotransmitter release is triggered by membrane depolarization, Ca(2+) influx and Ca(2+) sensing by the release machinery, causing synaptic vesicle (SV) fusion with the plasma membrane. Interlinked is a complex membrane cycle in which vesicles are tethered to the release site, primed, fused and recycled. As many of these processes are Ca(2+) dependent and simultaneously occurring, it is difficult to dissect them experimentally. This problem can be partially circumvented by controlling synaptic Ca(2+) concentrations via UV photolysis of caged Ca(2+). We developed a culture protocol for Ca(2+) uncaging in small synapses on the basis of the generation of small glia cell islands with single neurons on top, which are sufficiently small to be covered with a UV-light flash. Neurons are loaded with the photolabile Ca(2+)-chelator nitrophenyl-EGTA and Ca(2+) indicators, and a UV flash is used to trigger Ca(2+)-uncaging and SV fusion. The protocol takes three weeks to complete and provides unprecedented insights into the mechanisms of transmitter release.

Assessment of glial function in the in vivo retina.

Glial cells, traditionally viewed as passive elements in the CNS, are now known to have many essential functions. Many of these functions have been revealed by work on retinal glial cells. This work has been conducted almost exclusively on ex vivo preparations and it is essential that retinal glial cell functions be characterized in vivo as well. To this end, we describe an in vivo rat preparation to assess the functions of retinal glial cells. The retina of anesthetized, paralyzed rats is viewed with confocal microscopy and laser speckle flowmetry to monitor glial cell responses and retinal blood flow. Retinal glial cells are labeled with the Ca(2+) indicator dye Oregon Green 488 BAPTA-1 and the caged Ca(2+) compound NP-EGTA by injection of the compounds into the vitreous humor. Glial cells are stimulated by photolysis of caged Ca(2+) and the activation state of the cells assessed by monitoring Ca(2+) indicator dye fluorescence. We find that, as in the ex vivo retina, retinal glial cells in vivo generate both spontaneous and evoked intercellular Ca(2+) waves. We also find that stimulation of glial cells leads to the dilation of neighboring retinal arterioles, supporting the hypothesis that glial cells regulate blood flow in the retina. This in vivo preparation holds great promise for assessing glial cell function in the healthy and pathological retina.

An Inhibitory Effect of Extracellular Ca2+ on Ca2+-Dependent Exocytosis

AimNeurotransmitter release is elicited by an elevation of intracellular Ca2+ concentration ([Ca2+]i). The action potential triggers Ca2+ influx through Ca2+ channels which causes local changes of [Ca2+]i for vesicle release. However, any direct role of extracellular Ca2+ (besides Ca2+ influx) on Ca2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.ResultsUsing photolysis of caged Ca2+ and caffeine-induced release of stored Ca2+, we found that extracellular Ca2+ inhibited exocytosis following moderate [Ca2+]i rises (2–3 µM). The IC50 for extracellular Ca2+ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca2+ concentration ([Ca2+]o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca2+]o. The calcimimetics Mg2+, Cd2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca2+-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.Conclusion/SignificanceAs an extension of the classic Ca2+ hypothesis of synaptic release, physiological levels of extracellular Ca2+ play dual roles in evoked exocytosis by providing a source of Ca2+ influx, and by directly regulating quantal size and release probability in neuronal cells.

Fundamental Change in Neurovascular Coupling after Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) following cerebral aneurysm rupture is associated with substantial morbidity and mortality against which existing therapeutic options have limited efficacy. The impact of SAH on neurovascular coupling, reflecting the coordinated communication between neurons, astrocytes and parenchymal arterioles, is unknown. Using a combination of two‐photon and infrared‐differential interference contrast microscopy, arteriolar diameter and astrocyte endfoot Ca2+ were simultaneously measured in brain slices from un‐operated, sham‐operated and SAH model of rats. Increased neuronal activity caused by electrical field stimulation (EFS) elicited the predicted elevation in endfoot Ca2+ and vasodilation in brain slices from control animals (Filosa JA et al, 2006). EFS induced a similar increase in endfoot Ca2+, but in marked contrast caused parenchymal arteriolar constriction in brain slices from SAH model animals. Further, elevating endfoot Ca2+ via two‐photon photolysis of caged Ca2+ in the range of 200–500 nM caused parenchymal arteriolar dilation in brain slices from control animals and constriction in brain slices from SAH animals. These data demonstrate that SAH causes a fundamental switch from vasodilation to constriction in response to moderate increases in astrocyte endfoot Ca2+. (Supported by NIH P01 HL095488, R01 HL078983, R01 HL44455, &amp; Totman Medical Research Trust).

Extracellular Ca 2+ Directly Inhibits Exocytosis in Neurons

Exocytosis of transmitter releasing vesicles is elicited by an elevation of intracellular Ca2+ concentration ([Ca2+]i). Given the existing Ca2+ sensor receptor (CaSR), although [Ca2+]i-induced exocytosis is fully established, however, whether extracellular Ca2+ (concentration [Ca2+]o) directly regulates exocytosis is not rigorously examined yet. Here we report that extracellular Ca2+ inhibited exocytosis following moderate [Ca2+]i rises (2-3 μM), which were triggered by either photolysis of caged Ca2+ or caffeine.

Functional Characterization of Putative Synaptotagmin-Binding Interfaces in SNAP-25

SNARE complexes possess multiple negative surface charges that probably mediate association with regulatory factors during exocytosis. Several distinct groups of negatively charged residues in SNAP-25 have been identified as essential for binding synaptotagmin-1 (syt-1). One group exists at the C-terminal end (D172/D179/D186/D193), while another site extends around Layer 0 (D51/E52/E55). A third potential group may consist of D166/E170, as we previously found that E170 is essential for fast release. To investigate the nature of the binding interface, we introduced alanine-substitutions at the three putative binding sites and characterized the resulting mutant variants after expression in SNAP-25 -/- chromaffin cells. Secretion was induced by photolysis of caged-Ca2+ and assayed by capacitance measurements and amperometric recordings. We found substantial differences in the phenotypes of these mutant variants, which excludes that all sites cooperate in the same mode of syt-1 interaction. Though no variant fully phenocopied the changes seen in syt-1 -/- cells, SNAP-25A D51A/E52A/E55A caused a slowdown of secretion reminiscent of the syt-1 -/- phenotype. SNAP-25A D166A/E170A decreased total release substantially and abolished the fast burst component, while SNAP-25A D172A/D179A/D186A/D193A had little effect on secretion. Pull-down assays demonstrated that mutation of the two groups around Layer 0 indeed restricted syt-1 binding, while mutation of the C-terminal site did not decrease affinity towards syt-1. By electron microscopy we found that all mutations significantly affected docking of vesicles, which depends on both SNAP-25 and syt-1 (De Wit et al., 2009, Cell 138:935-946). This implies that SNAP-25/Syt-1 assisted docking might rely on another mode of interaction between SNAP-25 and syt-1 than fusion triggering. Our data thus suggest that the interaction between SNAREs and syt-1 might involve multiple interaction modes, and that negative charges around Layer 0 most likely function as a syt-1 binding interface during exocytosis triggering.

Phosphatidylinositol 3-Kinase Facilitates Microtubule-dependent Membrane Transport for Neuronal Growth Cone Guidance

The activity of PI3K is necessary for polarized cell motility. To guide extending axons, environmental cues polarize the growth cone via asymmetric generation of Ca(2+) signals and subsequent intracellular mechanical events, including membrane trafficking and cytoskeletal reorganization. However, it remains unclear how PI3K is involved in such events for axon guidance. Here, we demonstrate that PI3K plays a permissive role in growth cone turning by facilitating microtubule (MT)-dependent membrane transport. Using embryonic chick dorsal root ganglion neurons in culture, attractive axon turning was induced by Ca(2+) elevations on one side of the growth cone by photolyzing caged Ca(2+) or caged inositol 1,4,5-trisphosphate. We show that PI3K activity was required downstream of Ca(2+) signals for growth cone turning. Attractive Ca(2+) signals, generated with caged Ca(2+) or caged inositol 1,4,5-trisphosphate, triggered asymmetric transport of membrane vesicles from the center to the periphery of growth cones in a MT-dependent manner. This centrifugal vesicle transport was abolished by PI3K inhibitors, suggesting that PI3K is involved in growth cone attraction at the level of membrane trafficking. Consistent with this observation, immunocytochemistry showed that PI3K inhibitors reduced MTs in the growth cone peripheral domain. Time-lapse imaging of EB1 on the plus-end of MTs revealed that MT advance into the growth cone peripheral domain was dependent on PI3K activity: inhibition of the PI3K signaling pathway attenuated MT advance, whereas exogenous phosphatidylinositol 3,4,5-trisphosphate, the product of PI3K-catalyzed reactions, promoted MT advance. This study demonstrates the importance of PI3K-dependent membrane trafficking in chemotactic cell migration.

Endocannabinoids differentially modulate synaptic plasticity in rat hippocampal CA1 pyramidal neurons.

BackgroundHippocampal CA1 pyramidal neurons receive two excitatory glutamatergic synaptic inputs: their most distal dendritic regions in the stratum lacunosum-moleculare (SLM) are innervated by the perforant path (PP), originating from layer III of the entorhinal cortex, while their more proximal regions of the apical dendrites in the stratum radiatum (SR) are innervated by the Schaffer-collaterals (SC), originating from hippocampal CA3 neurons. Endocannabinoids (eCBs) are naturally occurring mediators capable of modulating both GABAergic and glutamatergic synaptic transmission and plasticity via the CB1 receptor. Previous work on eCB modulation of excitatory synapses in the CA1 region largely focuses on the SC pathway. However, little information is available on whether and how eCBs modulate glutamatergic synaptic transmission and plasticity at PP synapses.Methodology/Principal FindingsBy employing somatic and dendritic patch-clamp recordings, Ca2+ uncaging, and immunostaining, we demonstrate that there are significant differences in low-frequency stimulation (LFS)- or DHPG-, an agonist of group I metabotropic glutamate receptors (mGluRs), induced long-term depression (LTD) of excitatory synaptic transmission between SC and PP synapses in the same pyramidal neurons. These differences are eliminated by pharmacological inhibition with selective CB1 receptor antagonists or genetic deletion of the CB1 receptor, indicating that these differences likely result from differential modulation via a CB1 receptor-dependent mechanism. We also revealed that depolarization-induced suppression of excitation (DSE), a form of short-term synaptic plasticity, and photolysis of caged Ca2+-induced suppression of Excitatory postsynaptic currents (EPSCs) were less at the PP than that at the SC. In addition, application of WIN55212 (WIN) induced a more pronounced inhibition of EPSCs at the SC when compared to that at the PP.Conclusions/SignificanceOur results suggest that CB1 dependent LTD and DSE are differentially expressed at the PP versus SC synapses in the same neurons, which may have an impact on synaptic scaling, integration and plasticity of hippocampal CA1 pyramidal neurons.