Caged Ca2 Research Articles

Modes of exocytosis and electrogenesis underlying canine biphasic insulin secretion.

Biphasic insulin secretion in response to glucose consists of a transient first phase followed by a progressive second phase. It is a well described feature of whole perfused pancreases as well as isolated pancreatic islets of Langerhans. Applying to single cell assays of exocytosis (capacitance monitoring and amperometry) to single canine Beta-cells we have examined the time courses of granule exocytosis in response to voltage-clamp depolarizations that mimic two modes of glucose-induced electrical activity, and then compared these to biphasic insulin secretion. Action potentials evoked in short trains at frequencies similar those recorded during first phase insulin secretion trigger phasic exocytosis from a small pool of insulin granules that are likely docked near voltage-activated Ca²⁺ channels. In contrast, prolonged voltage-clamp pulses mimicking plateau depolarizations occur during second phase insulin secretion and trigger tonic or continuous exocytosis. Comparing the latter results with ones obtained using photorelease of caged Ca²⁺ in other insulin-secreting cells, we suggest that tonic exocytosis likely results from granule release from a highly Ca²⁺-sensitive pool of insulin granules, likely located further from Ca²⁺ channels. Both phasic and tonic modes of exocytosis are enhanced by glucose, via its metabolism. Hence, in canine Beta-cells we propose that two distinct modes of exocytosis, tuned to two types of electrical activity, may underlay biphasic insulin secretion.

Modes of exocytosis and electrogenesis underlying canine biphasic insulin secretion

Biphasic insulin secretion in response to glucose consists of a transient first phase followed by a progressive second phase. It is a well described feature of whole perfused pancreases as well as isolated pancreatic islets of Langerhans. Applying to single cell assays of exocytosis (capacitance monitoring and amperometry) to single canine Beta-cells we have examined the time courses of granule exocytosis in response to voltage-clamp depolarizations that mimic two modes of glucose-induced electrical activity, and then compared these to biphasic insulin secretion. Action potentials evoked in short trains at frequencies similar those recorded during first phase insulin secretion trigger phasic exocytosis from a small pool of insulin granules that are likely docked near voltage-activated Ca²⁺ channels. In contrast, prolonged voltage-clamp pulses mimicking plateau depolarizations occur during second phase insulin secretion and trigger tonic or continuous exocytosis. Comparing the latter results with ones obtained using photorelease of caged Ca²⁺ in other insulin-secreting cells, we suggest that tonic exocytosis likely results from granule release from a highly Ca²⁺-sensitive pool of insulin granules, likely located further from Ca²⁺ channels. Both phasic and tonic modes of exocytosis are enhanced by glucose, via its metabolism. Hence, in canine Beta-cells we propose that two distinct modes of exocytosis, tuned to two types of electrical activity, may underlay biphasic insulin secretion.

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.

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP or caged Ca. We use a flash lamp to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration.

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.

Beat-to-beat Ca2+-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm

Whether intracellular Ca2+ regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca2+ regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca2+ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca2+. Prior to introduction of the caged Ca2+ buffer, spontaneous local Ca2+ releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r2=0.9). The buffer markedly prolonged the decay time (T50) and moderately reduced the amplitude of the AP-induced Ca2+ transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca2+ was acutely released from the caged compound by flash photolysis, intracellular Ca2+ dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca2+ dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca2+ regulates normal SANC automaticity on a beat-to-beat basis.

Munc18-1 and Munc18-2 Proteins Modulate β-Cell Ca2+ Sensitivity and Kinetics of Insulin Exocytosis Differently

Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in β-cell stimulus-secretion coupling. We show that pancreatic β-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. Ramp-like photorelease of caged Ca(2+) and high resolution whole-cell patch clamp recordings show that Munc18-1 and Munc18-2 overexpression shift the Ca(2+) sensitivity of the fastest phase of insulin exocytosis differently. In addition, we reveal that Ca(2+) sensitivity of exocytosis in β-cells depends on the phosphorylation status of the Munc18 proteins. Even though Munc18-1 emerges as the key SM-protein determining the Ca(2+) threshold for triggering secretory activity in a stimulated β-cell, Munc18-2 has the ability to increase Ca(2+) sensitivity and thus mediates the release of fusion-competent granules requiring a lower cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i)(.) Hence, Munc18-1 and Munc18-2 display distinct subcellular compartmentalization and can coordinate the insulin exocytotic process differently as a consequence of the actual [Ca(2+)](i).

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, & Totman Medical Research Trust).

Calcium entry through slow-inactivating L-type calcium channels preferentially triggers endocytosis rather than exocytosis in bovine chromaffin cells

Calcium (Ca(2+))-dependent endocytosis has been linked to preferential Ca(2+) entry through the L-type (α(1D), Ca(V)1.3) of voltage-dependent Ca(2+) channels (VDCCs). Considering that the Ca(2+)-dependent exocytotic release of neurotransmitters is mostly triggered by Ca(2+) entry through N-(α(1B), Ca(V)2.2) or PQ-VDCCs (α(1A), Ca(V)2.1) and that exocytosis and endocytosis are coupled, the supposition that the different channel subtypes are specialized to control different cell functions is attractive. Here we have explored this hypothesis in primary cultures of bovine adrenal chromaffin cells where PQ channels account for 50% of Ca(2+) current (I(Ca)), 30% for N channels, and 20% for L channels. We used patch-clamp and fluorescence techniques to measure the exo-endocytotic responses triggered by long depolarizing stimuli, in 1, 2, or 10 mM concentrations of extracellular Ca(2+) ([Ca(2+)](e)). Exo-endocytotic responses were little affected by ω-conotoxin GVIA (N channel blocker), whereas ω-agatoxin IVA (PQ channel blocker) caused 80% blockade of exocytosis as well as endocytosis. In contrast, nifedipine (L channel blocker) only caused 20% inhibition of exocytosis but as much as 90% inhibition of endocytosis. Conversely, FPL67146 (an activator of L VDCCs) notably augmented endocytosis. Photoreleased caged Ca(2+) caused substantially smaller endocytotic responses compared with those produced by K(+) depolarization. Using fluorescence antibodies, no colocalization between L, N, or PQ channels with clathrin was found; a 20-30% colocalization was found between dynamin and all three channel antibodies. This is incompatible with the view that L channels are coupled to the endocytotic machine. Data rather support a mechanism implying the different inactivation rates of L (slow-inactivating) and N/PQ channels (fast-inactivating). Thus a slow but more sustained Ca(2+) entry through L channels could be a requirement to trigger endocytosis efficiently, at least in bovine chromaffin cells.

Determinants of the Site of Ca 2+ Wave Initiation in Smooth Muscle

Many smooth muscle activities including contraction, transcription and apoptosis are regulated by inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c). Activation of surface receptors, such as muscarinic acetylcholine M3 receptors (mAChR3), leads to the production of InsP3 to evoke Ca2+ release via receptors (InsP3R) present on sarcoplasmic reticulum. Ca2+ release usually begins in a single ‘eager’ region and regeneratively propagates along the length of the cell as a Ca2+ wave. The Ca2+ wave repeatedly originates at the same ‘eager’ site. We addressed the mechanisms which determine the Ca2+ wave initiation site. One possibility is that the ‘eager’ site has a higher sensitivity to InsP3 to evoke larger Ca2+ release. This does not appear to be the case because the site where waves initiated was not the site of largest Ca2+ release (as determined by local photolysis of caged InsP3). The expression patterns of mAChR3 and InsP3R may provide an explanation. Although, there was no apparent regional receptor clustering, dual labelling of mAChR3 and InsP3R showed some receptor co-localization. Ca2+ wave initiation site may be determined by regions where the proximity of mAChR3 and InsP3R generate higher local [InsP3] and [Ca2+]c. To explore this possibility the adaptability of the Ca2+ wave initiation site was examined by changing the local Ca2+ buffer capacity using the caged Ca2+ buffer diazo-2. Photolysis of diazo-2, at the site of wave initiation, during agonist application prevented initiation at this location. Yet, after a time lag the Ca2+ wave initiated at a new ‘eager’ site. These results indicate that the Ca2+ wave initiation site may be determined by co-localisation of mAChR3 and InsP3R and that the ‘eager’ site is altered when the [Ca2+]c increase is prevented. Supported by the Wellcome Trust and British Heart Foundation.

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.

CAMP increases the sensitivity of exocytosis to Ca 2+ primarily through protein kinase A in mouse pancreatic beta cells

Cyclic AMP regulates the late step of Ca 2+-dependent exocytosis in many secretory cells through two major mechanisms: a protein kinase A-dependent and a cAMP-GEF/Epac-dependent pathway. We designed a protocol to characterize the role of these two cAMP-dependent pathways on the Ca 2+ sensitivity and kinetics of regulated exocytosis in mouse pancreatic beta cells, using a whole-cell patch-clamp based capacitance measurements. A train of depolarizing pulses or slow photo-release of caged Ca 2+ were stimuli for the exocytotic activity. In controls, due to exocytosis after slow photo-release, the C m change had typically two phases. We observed that the Ca 2+-dependency of the rate of the first C m change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9 ± 0.2 μM. The intracellular depletion of cAMP did not change amp1, while rate1 and amp2 were strongly reduced. This manipulation pushed the Ca 2+-dependency of the exocytotic burst to significantly lower [Ca 2+] i. To address the question of which of the cAMP-dependent mechanisms regulates the observed shifts in Ca 2+ dependency we included regulators of PKA and Epac2 activity in the pipette solution. PKA activation with 100 μM 6-Phe-cAMP or inhibition with 500 μM Rp-cAMPs in beta cells significantly shifted the EC 50 in the opposite directions. Specific activation of Epac2 did not change Ca 2+ sensitivity. Our findings suggest that cAMP modulates Ca 2+-dependent exocytosis in mouse beta cells mainly through a PKA-dependent mechanism by sensitizing the insulin releasing machinery to [Ca 2+] i; Epac2 may contribute to enhance the rates of secretory vesicle fusion.

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.

Calcium Regulates Vesicle Replenishment at the Cone Ribbon Synapse

Cones release glutamate-filled vesicles continuously in darkness, and changing illumination modulates this release. Because sustained release in darkness is governed by vesicle replenishment rates, we analyzed how cone membrane potential regulates replenishment. Synaptic release from cones was measured by recording postsynaptic currents in Ambystoma tigrinum horizontal or OFF bipolar cells evoked by depolarization of simultaneously voltage-clamped cones. We measured replenishment after attaining a steady state between vesicle release and replenishment using trains of test pulses. Increasing Ca(2+) currents (I(Ca)) by changing the test step from -30 to -10 mV increased replenishment. Lengthening -30 mV test pulses to match the Ca(2+) influx during 25 ms test pulses to -10 mV produced similar replenishment rates. Reducing Ca(2+) driving force by using test steps to +30 mV slowed replenishment. Using UV flashes to reverse inhibition of I(Ca) by nifedipine accelerated replenishment. Increasing [Ca(2+)](i) by flash photolysis of caged Ca(2+) also accelerated replenishment. Replenishment, but not the initial burst of release, was enhanced by using an intracellular Ca(2+) buffer of 0.5 mm EGTA rather than 5 mm EGTA, and diminished by 1 mm BAPTA. This suggests that although release and replenishment exhibited similar Ca(2+) dependencies, release sites are <200 nm from Ca(2+) channels but replenishment sites are >200 nm away. Membrane potential thus regulates replenishment by controlling Ca(2+) influx, principally by effects on replenishment mechanisms but also by altering releasable pool size. This in turn provides a mechanism for converting changes in light intensity into changes in sustained release at the cone ribbon synapse.

Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16b-transfected HEK 293T cells

Ca(2+)-activated Cl(-) channels play relevant roles in several physiological processes, including olfactory transduction, but their molecular identity is still unclear. Recent evidence suggests that members of the transmembrane 16 (TMEM16, also named anoctamin) family form Ca(2+)-activated Cl(-) channels in several cell types. In vertebrate olfactory transduction, TMEM16b/anoctamin2 has been proposed as the major molecular component of Ca(2+)-activated Cl(-) channels. However, a comparison of the functional properties in the whole-cell configuration between the native and the candidate channel has not yet been performed. In this study, we have used the whole-cell voltage-clamp technique to measure functional properties of the native channel in mouse isolated olfactory sensory neurons and compare them with those of mouse TMEM16b/anoctamin2 expressed in HEK 293T cells. We directly activated channels by rapid and reproducible intracellular Ca(2+) concentration jumps obtained from photorelease of caged Ca(2+) and determined extracellular blocking properties and anion selectivity of the channels. We found that the Cl(-) channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and DIDS applied at the extracellular side of the membrane caused a similar inhibition of the two currents. Anion selectivity measured exchanging external ions and revealed that, in both types of currents, the reversal potential for some anions was time dependent. Furthermore, we confirmed by immunohistochemistry that TMEM16b/anoctamin2 largely co-localized with adenylyl cyclase III at the surface of the olfactory epithelium. Therefore, we conclude that the measured electrophysiological properties in the whole-cell configuration are largely similar, and further indicate that TMEM16b/anoctamin2 is likely to be a major subunit of the native olfactory Ca(2+)-activated Cl(-) current.

Functional coupling of α2-isoform Na+/K+-ATPase and Ca2+ extrusion through the Na+/Ca2+-exchanger in cardiomyocytes

Intracellular Na+ concentration ([Na+]i) is an important regulator of Ca2+ homeostasis in cardiomyocytes through its influence on the Na+/Ca2+-exchanger (NCX). [Na+]i is regulated by the Na+/K+-ATPase (NKA) of which two catalytic isoforms are expressed in cardiomyocytes, α1 and α2. Here we report that the NKA α2-isoform can modulate NCX mediated Ca2+ transport by establishing a shared microdomain of Na+ where [Na+]i can be different from that in the bulk cytosol. Ca2+ transients and the corresponding NCX currents were induced by [Ca2+]i jumps via UV-flash photolysis of caged Ca2+ (DM-nitrophen), while Ca2+ release and uptake by the sarcoplasmic reticulum was blocked pharmacologically. We determined that specific inhibition of 55% of the α2-isoform was obtained with 0.3μM ouabain, whereas 5μM ouabain blocked ≈95% of α2-isoform and in addition, ≈8% of the α1-isoform. Specific inhibition of the α2-isoform caused slowing of Ca2+ extrusion by the NCX. However, this NCX inhibition could not be explained by an increase in global [Na+]i as assessed by the Na+ indicator SBFI, but rather by Na+ microdomains built up by the NKA α2-isoform in the “fuzzy space”. The results suggest that regulation of NKA α2-isoform activity is crucial for the regulation of Ca2+ homeostasis in cardiomyocytes.

Agonist‐evoked Ca2+ wave progression requires Ca2+ and IP3

Smooth muscle responds to IP(3)-generating agonists by producing Ca(2+) waves. Here, the mechanism of wave progression has been investigated in voltage-clamped single smooth muscle cells using localized photolysis of caged IP(3) and the caged Ca(2+) buffer diazo-2. Waves, evoked by the IP(3)-generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) over a single though substantial length (approximately 30 microm) of the cell. During regenerative propagation, the wave-front was about 1/3 the length (approximately 9 microm) of the initiation site. The wave-front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP(3) may explain the amplitude changes. Ca(2+) was required for IP(3)-mediated wave progression to occur. Increasing the Ca(2+) buffer capacity in a small (2 microm) region immediately in front of a CCh-evoked Ca(2+) wave halted progression at the site. However, the wave front does not progress by Ca(2+)-dependent positive feedback alone. In support, colliding [Ca(2+)](c) increases from locally released IP(3) did not annihilate but approximately doubled in amplitude. This result suggests that local IP(3)-evoked [Ca(2+)](c) increases diffused passively. Failure of local increases in IP(3) to evoke waves appears to arise from the restricted nature of the IP(3) increase. When IP(3) was elevated throughout the cell, a localized increase in Ca(2+) now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP(3) and Ca(2+) are required for active propagation of the wave front to occur.

Quantitative Analysis of Synaptic Release at the Photoreceptor Synapse

Exocytosis from the rod photoreceptor is stimulated by submicromolar Ca2+ and exhibits an unusually shallow dependence on presynaptic Ca2+. To provide a quantitative description of the photoreceptor Ca2+ sensor for exocytosis, we tested a family of conventional and allosteric computational models describing the final Ca2+-binding steps leading to exocytosis. Simulations were fit to two measures of release, evoked by flash-photolysis of caged Ca2+: exocytotic capacitance changes from individual rods and postsynaptic currents of second-order neurons. The best simulations supported the occupancy of only two Ca2+ binding sites on the rod Ca2+ sensor rather than the typical four or five. For most models, the on-rates for Ca2+ binding and maximal fusion rate were comparable to those of other neurons. However, the off-rates for Ca2+ unbinding were unexpectedly slow. In addition to contributing to the high-affinity of the photoreceptor Ca2+ sensor, slow Ca2+ unbinding may support the fusion of vesicles located at a distance from Ca2+ channels. In addition, partial sensor occupancy due to slow unbinding may contribute to the linearization of the first synapse in vision.

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.

Agonist-Evoked Calcium Wave Progression Requires Calcium and Ip3 in Smooth Muscle

Smooth muscle responds to IP3-generating agonists by producing Ca2+ waves to spread information within and between cells. The mechanism of wave progression has been investigated in voltage-clamped single smooth muscle cells. Agonist-evoked waves initiated as a uniform rise in [Ca2+]c over a substantial length (∼30 μm) of the cell. During regenerative propagation, the wave-front was approximately 1/3 the length (∼9 μm) of the [Ca2+]c change at the initiation site. The wave-front progressed at a constant velocity though amplitude varied through the cell. Differences in sensitivity to IP3 may explain the variation in amplitude; local release of IP3 evoked [Ca2+]c increases of varying amplitude in different regions of the cell. The wave-front does not progress by Ca2+-dependent positive feedback alone. In support, colliding [Ca2+]c increases evoked in response to IP3, released locally in two parts of the cell, did not annihilate but approximately doubledin amplitude. This result suggests that the [Ca2+]c increase, generated by local release of IP3, had not regeneratively propagated but diffused passively from the release site. Notwithstanding, Ca2+ was required for IP3-mediated wave progression to occur. Increasing the Ca2+ buffer capacity in a small (2 μm) restricted region of the cell immediately in front of a carbachol-evoked Ca2+ wave, by photolyzing the caged Ca2+ buffer diazo-2, halted progression at the site of photolysis. Failure of local increases in IP3 to evoke waves appears to arise from the restricted nature of the IP3 increase to small areas within the cell. When IP3 was elevated throughout a localized increase in Ca2+ propagated as a wave. Together, these results suggest that waves initiate over a relatively large length of the cell and both IP3 and Ca2+ are required for active propagation of the wave-front to occur.