Both acute and chronic degenerative diseases of the nervous system reduce the viability and function of neurons through changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Disease involvement of numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles has been documented. Diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants. The present review summarizes the discovery, mechanisms and biological activity of neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed. As such, the control of the intracellular Ca2+ concentration is critical to the functioning and survival of the cell. In both acute and chronic degenerative diseases of the nervous system the viability and function of neurons is reduced as a result of changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Furthermore, numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles have been identified to be involved in many of these pathophysiological processes. Neuroprotective strategies targeting different aspects of the complex control of intracellular Ca2+ signaling are a promising approach for pharmaceutical intervention in degenerative diseases of the nervous system. To this end, diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants, each targeting different aspects of fine-tuned control of the intracellular Ca2+ concentration. The present review summarizes the discovery, mechanisms and biological activity of representative neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Furthermore, disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed.Changes in the intracellular concentration of Ca3) receptors (IP3Rs) and ryanodine receptors (RyRs) are the two major intracellular Ca2+ channels (ICCs) that release Ca2+ from intracellular stores such as the endoplasmic reticulum (ER). Release and sequestering of Ca2+ from these intracellular stores is a major determinant in the shape and resting state of intracellular Ca2+ levels. IP3Rs and RyRs are critical for Ca2+ dependent processes such as cellular growth, development, gene expression and neurotransmission [+2 handling also plays a role in a variety of neurodegenerative diseases including Alzheimer’s disease (AD), Huntington’s disease (HD), Amyotrophic lateral sclerosis (ALS), excitotoxicity induced apoptosis, hypoxia, and neuroinflammation [Inositol 1,4,5-trisphosphate IP receptorsmission ,7,8,9,10ammation ,16,17,183 levels are reduced in the brains of patients with AD. The decrease in IP3 binding sites in brains of AD patients correlates with the increased occurrence of amyloid plaques and neurofibrillary tangles [3R activity is altered in cultured neurons from mouse models of AD [2+ homeostasis [3R activity resulting in enhanced Ca2+ release from ER stores [The role of ICCs in AD pathology has become more apparent in the last several years. For example, phosphatidylinositol and IP tangles ,22,23,24ls of AD ,16,17 aneostasis , with PSR stores ,32.2+ release [2+ release, and susceptibility to Aβ [2+ release contributing to neuronal cell death [In addition, RyR function is directly affected by wild type PS1  and PS2  release  and vuln release . Transgety to Aβ ,26. Thisty to Aβ . Aβ treall death ,18.3R type 1 (IP3R1) associates with the HD Htt-HAP1 complex and increases IP3R1 sensitivity to activation by IP3 [3R leads to elevated Ca2+ release and apoptosis in neuronal cells [3R binding to Htt reduces glutamate induced apoptosis by stabilizing intracellular Ca2+ homeostasis [2+ induced Ca2+ release (CICR) in the progression of this disease [HD is a neurodegenerative disorder characterized by the loss of striatal neurons, neuro-inflammation, and increased oxidative stress ,34. HD in by IP3 . This seal cells . Furthereostasis . Genomic disease .2+. This increase in Ca2+ can be ablated by the use of ryanodine and IP3 receptor blockers, suggesting a role for altered CICR and intracellular Ca2+ signaling in ALS [Amyotrophic lateral sclerosis (ALS) is defined by loss of motor neurons, neuroinflammation, and increased oxidative stress. T cell dysfunction and mutations in the superoxide dismutase 1 (SOD-1) gene have been suggested as possible mechanisms for the progression of this disorder ,39. Abnog in ALS .These studies provide a rationale for determining the roles of ICCs in the progression of neurodegenerative diseases as well as for the development of neuroprotective strategies and potential therapeutic agents with ICCs and their signaling and binding partners as targets.2+ concentration is widely considered the single most important contributor to neurodegeneration and neuronal cell death following ischemic or hypoxic insult [2+ channels (VGCCs) are an important target for the development of neuroprotective strategies, given their central role in depolarization-induced Ca2+ influx [Increase of the intracellular Cac insult . As a co+ influx . 1-subunit, high voltage-activated Ca2+ channels form a complex consisting of an additional β-, α2δ- and possibly γ-subunit. The biophysical properties of VGCCs are largely determined by the specific sub-type and splice variant of the α1-subunit, the specific subunit-composition, phosphorylation status as well as the level of channel inactivation  channels and Cav2  channels, as well as low voltage-activated Cav3 (T-type) channels. In addition to the pore-forming αiew, see ). 2+ load. The pivotal role of Cav2 channels in neurotransmitter release may contribute to an overall insult-induced state of neuronal hyperexcitability, however, their role in contributing to increases in intracellular Ca2+ following insult is questionable, given their localization at the active zone of synapses and their intermediate inactivation kinetics. In contrast, Cav1  channels exhibit slow voltage-dependent inactivation [2+ release channels such as RyR in neurons [The rationale for considering VGCCs as possible neuroprotective targets is based on the hypothesis that VGCCs respond to the persistent ischemia- or hypoxia-induced depolarization (reviewed in ). Based tivation . Further neurons ,45. in vitro systems, dihydropyridine blockers of Cav1 channels, such as nimodipine, israpidine, and nicardipine have shown neuroprotective effects by reducing the intracellular rise in Ca2+ following insult [in vivo models have been highly variable: whereas some studies found neuroprotective effects of dihydropyridines [In g insult ,47. Howeyridines ,55,56,57yridines . The finClinical trials have failed to show improved outcome after stroke with nimodipine treatment, as recently reviewed by Ginsberg . It shouin vivo experiments and disappointing clinical trials, Cav1 channels remain a therapeutically-relevant target, as evidence emerges that other known neuroprotective compounds mediate their beneficial effects, at least in part, via Cav1 channels. Nicotine was shown to reduce glutamate excitotoxicity in mouse cortical cultures by lowering intracellular Ca2+ influx by ~40% through a pathway involving β2 nicotinic acetylcholinergic receptors, calcineurin and Cav1 channels [2+ currents [Despite these seemingly contradictory channels . Similarcurrents . 2+ channel blockers purified from the spider toxin of Phoneutria nigriventer has recently been shown to block ischemia-induced glutamate release, neuronal death, and loss of neurotransmission in hippocampal slice preparations subjected to oxygen deprivation and low glucose insult [A novel broad class of Cae insult ,63. 2+ homeostasis and the diverse effects of ischemic, excitotoxic or hypoxic insult thereupon may require more than a single molecular target for successful neuroprotective therapeutic intervention. Rational drug design efforts have recently provided promising results for neuroprotection: novel tacrine-dihydropyridine hybrids that inhibit both acetylcholinesterase and L-type Ca2+ channel-mediated rises in intracellular Ca2+ have been shown to exert neuroprotective effects in a cellular model for excitotoxic insult [The complex regulation of intracellular Cac insult . in vivo animal studies and clinical trials, reducing the activity of dihydropyridine-sensitive Ca2+ channels must continue to be considered in future rational drug design approaches for neuroprotection.Despite disappointing results from early 2+ influx through NMDA receptors and subsequent activation of a variety of signaling events ultimately leads to the expression of proteins involved in synaptic plasticity . Oth2+ signaling and ICCs in a variety of cellular functions including protection from cellular insults, scaffolding proteins that structurally or functionally couple them to ionotropic glutamate receptors (iGluRs) are of great importance to the maintenance of normal cellular function. Targeting of a variety of excitatory ionotropic neurotransmitter receptors is achieved through scaffolding proteins such as PSD-95, Chapsyn-110, SAP97, PICK-1, gephyrin, GRIP, ABP and GIPC [2+ influx generated from NMDA receptors [2+ signaling. Given the critical function of intracellular Caand GIPC ,83,84,85eceptors ,87,88,893Rs [3Rs, NMDA and AMPA receptor subunits, alteration of the actin cytoskeleton may also be a mechanism by which synaptic Ca2+ signals are regulated.Actin, itself, can bind to and alter the function of IP3Rs  and acti3Rs ,91,92,933Rs . The cyt3Rs . Given tSeveral studies provide evidence that synaptic scaffolding proteins are critical for maintaining proper neuronal function and viability. Treatment of cultured mouse cortical neurons with F-actin destabilizing agents selectively disrupts NMDA receptor but not AMPA receptor clustering in dendritic spines . FurtherUsing recombinant mutant NMDA receptor subunits, it was shown that NR2 subunits are required to effectively recruit NR1-1a splice variant subunits into dendritic spines . This ef1-42 peptide increases endocytosis of NMDA receptor subunits (NR1 and NR2B) [1-42 exhibit a reduced surface NMDA receptor density [versus NR2A NMDA receptor subunit expression [2+ influx to nNOS activation [NMDA receptors are affected in models of AD. Treatment of cortical neurons with Aβnd NR2B) . In addi density . Culture density . PSD-95  density . NMDA repression . PSD95 ttivation . Nitric tivation . Reductitivation .2+ influx though NMDA receptors. In addition, the oligomeric Aβ -mediated reduction in spine density is dependent upon the phosphatase calcineurin and actin destabilization [Aβ dimer and trimer treatment of hippocampal tissue slices leads to a significant decrease in the number and density of dendritic spines in pyramidal neurons . Furtherlization . Overexplization . Furtherlization . Hippocalization .Introduction of a GluR2 derived peptide containing a GRIP- and ABP-interacting PDZ domain into hippocampal slices increased AMPA-mediated excitatory postsynaptic current in pyramidal neurons in addition to reducing LTD  suggesti2+ release (CICR) mechanism mediated by RyR and also by regulation of IP3Rs by elevated cytosolic Ca2+ concentrations [3R and RyR activity is regulated in part by posttranslational modifications, such as phosphorylation, triggered by upstream signaling events including Ca2+ influx into the cell [NMDA receptors and ICCs functionally interact through a calcium-induced Catrations ,109,110.the cell ,124,125.2+ influx will be reduced. Furthermore, iGluRs and ICCs interact through a complex network of scaffolding proteins in the postsynaptic density ,128,129.s, Gαi/o, Gαq, and Gα12 [s (stimulatory) and Gαi/o (inhibitory) modulate the production of cAMP by adenylyl cyclase. cAMP regulates cAMP-dependent protein kinase A (PKA) which affects downstream processes such as cAMP response element binding (CREB) protein activation, neuronal excitability, and gene transcription [3 which then activate protein kinase C (PKC) and calcium release by IP3Rs, respectively. GPCR signaling mechanisms has been ably reviewed in [G-protein coupled receptors (GPCR) are heptahelical plasma membrane proteins, which relay information on extracellular conditions to the cytosol. Despite the large variety of GPCRs, signaling is conveyed through a relatively small number of conserved G-protein signaling pathways. Upon receptor activation the Gα subunit is activated by binding of GTP and dissociation from the inhibitory Gβ/γ dimer . G-proteand Gα12 . Gαs  is expressed in the central nervous system (CNS) and has a strong anti-glutamatergic effect. Two endogenous ligands have been identified for CB1, anandamide  and 2-art manner ,141, butt manner ,143,144 on cells . WIN55,2on cells . CB1 recon cells . Agonismon cells ,150,151 on cells . A PKA-d [et al. . Zhuang  release . Promiscin vitro and certain rodent studies [i/o mechanism or α2 receptor activation of the inward rectifying K+ channel [Glaucoma is characterized by increased intraocular pressure (IOP) and leads to retinal cell death and blindness ,156. Dec studies ,169,170  studies ,172. Ago studies . The α2  studies . The α2  studies , however studies . Brimoni studies . Pretrea studies . The obs channel . A follo channel . Alterna channel  though t channel . Furtherth and 7th transmembrane domains [2+ regulation are profound though mixed [Presenilins (PS) are ubiquitously expressed in most tissues ,185,186  domains ,193,195. domains ,195,196.gh mixed ,204,205.gh mixed , PS signgh mixed  and aspegh mixed ,238 were2+ in the cytosol have been implicated in neurodegenerative disorders like AD [2+ release during ischemia as a causative mechanism [Toxic concentrations of Ca like AD ,207,208. like AD ,211,212. like AD  and PS2  like AD , potentiechanism ,211. et al. [et al. [2+) [2+ (>1 mM) [2+.Rybalchenko et al.  and Hayr [et al.  have rec [et al. ,28. Incr [et al. ,28. Bindal. [2+) ,28. On t (>1 mM) ,28 but t (>1 mM) ,28. Theset al. [et al. [An interaction between PS and RyR had been described in other parts of the literature though a specific functional molecular interaction was novel to Rybalchenko et al.  and Hayr [et al. . Both NT [et al. . The cle [et al. . PC12 ce [et al. . The sam [et al. . PS has  [et al. ,214. In  [et al. . Sorcin  [et al. . The CTF [et al. . Though  [et al. , hypothe [et al. .et al. (2008) described an analogous interaction between PS and a second class of ER calcium release channels, the IP3R [et al. (2008) reported a PS-IP3R interaction that increased receptor open probability and calcium release [3 hydrolysis by phospholipase C-β (PLCβ) in human neuroblastoma SH-SY5Y cells transfected with mutant PS1 containing an exon 9 deletion (PS1ΔE9) [3R Ca2+ signaling in a Xenopus oocyte system [Cheung the IP3R . Cheung  release . This pa(PS1ΔE9) . PS1ΔE9 (PS1ΔE9) . PS1ΔE9 (PS1ΔE9) . Interese system . 2+ release and PS. Much more work is required to fully define the role of PS in ER Ca2+ release but the current data indicate therapies could be developed to blunt excessive Ca2+ release found in neurodegenerative conditions like AD and ischemia.To date, studies indicate a causal relationship between ER Ca2+ signaling in both acute and chronic neurodegenerative disease, progress in the development of neuroprotective approaches targeting the control of the intracellular Ca2+ concentration have been limited, largely due to the lack of knowledge about the spatio-temporal properties of Ca2+ signaling in different subcellular domains. However, in recent years a number of novel imaging technologies have become available that will help the accurate measuring the intracellular Ca2+ concentration in different sub-compartments and domains of the cell. In the following two subsections advances in the design of novel genetically-encoded Ca2+ sensors and progress in new superresolution microscopy will be reviewed and their potential use for the development of novel neuroprotective strategies be discussed. Despite the rapidly evolving knowledge on the role of aberrations in Ca2+ sensors are characterized by both strengths and weaknesses associated with these different approaches to Ca2+ imaging and quantification. In addition, current superresolution microscopy technologies expand the spatial resolution of light microscopy resulting in improved strategies for measuring intracellular Ca2+ signaling.Several new technologies are becoming available for the imaging and quantification of cellular processes by microscopic observation. Some research groups, such as the Tsien laboratory have been working to improve the kinetics, stability, and range of fluorescent sensors ; while o2+ sensor dyes. No ideal strategy exists for intra-cellular Ca2+ imaging resulting in the need for compromises in experimental design.The intensity, quantum efficiency, stability, membrane permeability, toxicity, and kinetic parameters determine the overall experimental usefulness of Ca2+ levels are epitomized by the Fura family of calcium sensitive binding dyes. Ratiometric imaging of the Fura2 dye, developed by the Tsien group in 1985, is considered to be the best method for quantification of Ca2+ concentration in living cells [2+ ions in the intracellular milieu. The Fura2 molecule acts as Ca2+ chelator, on binding to free Ca2+ ions, Fura2 changes its peak absorbance wavelength from ~350 nm to~ 340 nm with a concomitant change, a decrease in absorbance at~380 nm, while its emission maxima remains at ~510 nm. After calibration of Fura2 fluorescent intensities with known Ca2+ concentrations, the intracellular concentrations of Ca2+ can be calculated based on the ratio of changes in fluorescent intensities at 510 nm with excitation at 340 nm and 380 nm in living cells. Fura2 has a Ca2+ binding affinity near physiologic levels and a high selectivity for Ca2+ ions [2+ includes high selectivity, fast kinetics, and the ability to quantify Ca2+ concentrations using ratiometric imaging. Ratiometric imaging techniques reduce quantification errors caused by indicator concentration, excitation intensity, and detector efficiency. However, ratiometric image analysis is subject to errors [The small molecule sensors of intracellular Cang cells ,219. Typa2+ ions ,219. Theo errors , resulti2+ Indicators (GECIs). Sensors using Förster Resonance Energy Transfer (FRET) pairs or Bimolecular Fluorescence Complementation (BiMC) can be used to quantify Ca2+ levels using ratiometric techniques. However, the fusion of Fluorescent Proteins (FP’s) to calcium sensitive proteins is not a trivial task. Fusion of FPs can change the behavior of the sensor, change the protein folding pattern, or cause autophagocytosis of the sensor and in addition consideration of Heisenberg’s uncertainty principle of quantum theory [Recently, Tsien and others have been focused on producing Genetically Encoded Cam theory  is needeAequorea victoria and DsRed from Discosoma sp. to form a wide spectrum of genetically encoded fluorescent protein variants. These new genetically encoded fluorescent proteins can be used for multiple spectral and temporal labeling of target proteins and events. These fluorescent proteins can be fused to calcium chelators containing the C2 domain or the EF hand domain to create genetically encoded Ca2+ sensors, whose construction, coordination, and geometry has been reviewed recently [Tsien’s group has produced directed mutations of Green Fluorescent Protein (GFP) from recently . These cet al. used structure guided engineering to produce GCaMP3, which has a 3 fold increase in fluorescence, a 3 fold increase in dynamic range, and a 1.3 fold higher affinity for calcium than its parental GCaMP2 sensor [2+ analysis in vivo will likely be dominated by improvements of sensor signal to noise ratios and of binding affinities. GECIs have expanded the experimental capabilities including targeting the sensor to specific organelles or cellular sites, the generation of transgenic animals, conditional expression, and photo-activation of the indicators. At the same time, the genetically encoded sensors do not share the fast kinetics of the small molecule sensors. The slow kinetics and the lower signal to noise ratio prevents detection of fast or weak changes in calcium concentration. However, GECIs continue to be improved. Recently, Tian 2 sensor . The futAdvances in light microscopy have led to the breaking of the diffraction limit of resolution. These new methods have been referred to as superresolution microscopy or nanoscopy ,225. UntImpressive sub-diffraction limit resolution of point fluorescence sources has been achieved using these methods ,235,236;2+ concentration and study the effects of novel neuroprotective compounds. The combination of new dye technologies with dramatically improved binding affinities and faster kinetics with robust superresolution techniques will be instrumental in measuring the intracellular CaDisease-mediated changes in the regulation of intracellular calcium signaling are characteristic for acute and chronic degenerative diseases of the nervous system as evidenced by studies at the cellular and system level. While the homeostasis of the intracellular calcium concentration is the ultimate readout, numerous molecular and cellular components contribute to the overall change and at the same time represent qualified targets for the design of neuroprotective strategies. The capacity to both identify such targets as well as to reproducibly measure effects on the regulation of intracellular calcium signaling determine critically the success of neuroprotection.


Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca2+ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation.Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Cai) GTPγS induces Ca2+ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca2+; iii) GDPβS has little effect on the amplitude of the Ca2+ peak, but significantly expedites the initial Ca2+ waves induced by InsP3 photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca2+ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca2+ signaling.By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: 2+ release.Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca Within 1–2 min after addition of the hormone, free Ca2+ is quickly released from internal stores. This Ca2+ release takes place in the cortex of the vegetal hemisphere 2+ release, we found that the Ca2+ release initiated much before any structural changes became evident in the endoplasmic reticulum (ER). This finding prompted a study for the roles of the actin cytoskeleton in the mobilization of Ca2+ in the cortical domain of the oocytes The meiotic maturation of starfish oocytes starts with intracellular Ca2+ release in starfish oocytes is not well known. Nonetheless, it could be inferred that the activation of heterotrimeric G-proteins by 1-MA would lead to the stimulation of PLCβ and thereby to the increased production of InsP3, the Ca2+-inducing second messenger 2+ release could be more complicated than this canonical model. We have found that the Ca2+-releasing mechanism induced by InsP3 was significantly influenced by changes of the actin cytoskeleton 2+ release 3 pathway, e.g. heparin and U73122, caused alterations of the cortical actin cytoskeleton in starfish oocytes added weight to the suggestion that dynamic modulation of the actin cytoskeleton was also a crucial element in the regulation of intracellular Ca2+ signaling The detailed mechanism in which 1-MA induces Ca2+ signaling in the way that is related to the changes of the actin cytoskeleton. Hence, in this contribution, we have investigated the effects of nonhydrolyzable analogues of GTP and GDP on the 1-MA-induced Ca2+ signaling patterns in starfish oocytes. As expected, we found that injection of oocytes with GTPγS mimicked the Ca2+-releasing effect of 1-MA. Conversely, the Ca2+ release was blocked by GDPβS injection. To our surprise, however, the preinjection of oocytes with GDPβS not only modulated the Ca2+ signaling pattern and kinetics, but also produced a significant enhancement of the actin network underneath the plasma membrane. This and other results in our study suggest that the dynamic change of the actin cytoskeleton is a crucial component of the mechanisms controlling intracellular Ca2+ signaling and cortical granule exocytosis.The finding that the 1-MA-induced meiotic maturation is mediated by G-proteins leads to the question whether the nucleotides acting on the G-proteins modulate the CaA. pectinifera) were captured in the Mutzu Bay  during the breeding season (September) and transported to the Stazione Zoologica in Naples, Italy. Animals were maintained in circulating cold seawater (16°C). The gonads containing numerous oocytes were dissected from the central dorsal area and transferred to cold filter-sterilized seawater. Fully grown immature oocytes were isolated as single cells by sieving through gauze several times. Nearly all the oocytes released from the gonads were arrested at the first prophase of meiosis, as judged by the presence of the germinal vesicle (nucleus). Free oocytes were isolated by repeated rinsing and low speed  sedimentation in cold filtered seawater. For meiotic maturation, immature oocytes were stimulated with 1 µM of 1-MA for 1 h in filtered seawater.The Japanese species of starfish (3 (Molecular Probes) by microinjection . To activate the caged InsP3, microinjected oocytes were irradiated with 330 nm UV light for 25 seconds with the use of the computer-controlled shutter system Lambda 10-2 . Cytosolic Ca2+ changes were detected with a cooled CCD camera  mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 20x/0.50 objective. The quantified Ca2+ signal at a given time point was normalized to the baseline fluorescence (F0) following the formula Frel = [F−F0]/F0, where F represents the average fluorescence level of the entire oocyte. Fluorescent Ca2+ images were analyzed with the MetaMorph Imaging System software .Microinjection of oocytes was performed with an air-pressure Transjector (Eppendorf). Typically, the amount of the injected material is estimated 1% of the oocyte volume. Hence, the final concentration of the injected material inside the oocyte is to be 100-fold lower than the concentration in the injection pipette. The fluorescent calcium dye (Oregon Green conjugated with 10 kDa dextran) was purchased from Molecular Probes, and was used in 5 mg/ml pipette concentration with the injection buffer . The same injection buffer was used for delivering caged InsPTo visualize F-actin in living oocytes, the microinjection pipette was loaded with 50 µM Alexa Fluor 568-conjugated phalloidin in DMSO as previously described 3 were prepared in aqueous solution (injection buffer). The incubation conditions for jasplakinolide (JAS), GDPβS, and GTPγS were indicated in the figure legend. Denuded oocytes devoid of the vitelline coat were prepared by 30 minutes' incubation in 0.01% actinase E  at room temperature. Unless specified otherwise, the control cells refer to the oocytes that have been treated with the same vehicles for drug delivery.GDPβS and GTPγS were purchased from Calbiochem, and jasplakinolide (JAS) from Molecular Probes. While JAS was dissolved in DMSO, GTPγS, GDPβS, and caged InsP2+ release. To test if GTPγS can induce the same characteristic Ca2+ release seen in 1-MA-treated oocytes 2+ dye were subjected to microinjection with GTPγS. As expected, GTPγS produced a very similar Ca2+ wave to that of the 1-MA-incubated oocytes. In both cases, the Ca2+ wave started from a single spot in the cortex of the vegetal hemisphere and propagated to the opposite side  than in 1-MA-treated oocytes  (P<0.05). This is likely due to the additional lag time required for the microinjected material to diffuse to the Ca2+ release sites in the oocyte cortex. Although the peak of the Ca2+ signal in GTPγS-injected oocytes displayed considerably lower amplitude  than in 1-MA-incubated oocytes  (P<0.01), the kinetics of the Ca2+ rise to the peak was strikingly similar in both cases (2+ spot to the peak in GTPγS-injected and 1-MA-treated oocytes were 21.4±4.7 and 26.0±5.1 sec (P>0.05), respectively.The 1-MA-induced maturation of starfish oocyte is mediated by heterotrimeric G-proteins ite side . Howeverth cases . The tim2+ response  and late (GVBD) stages of meiotic maturation.Starfish oocytes were then microinjected with GDPβS, a nonhydrolyzable analogue of GDP. As expected, preinjection of oocytes with GDPβS completely blocked 1-MA-induced Caresponse . Furtherresponse . Taken t2+ signaling mechanism of InsP3, starfish oocytes were microinjected with the same dose of GDPβS  that had totally eliminated the 1-MA-induced Ca2+ signaling. At variance with the effect on 1-MA-induced Ca2+ signals, GDPβS failed to block the Ca2+ wave that was generated by uncaged InsP3  in comparison with the control values   , the lated cells . As a reed cells . In paraed cells . While t3 receptors and rearrange the actin cytoskeleton 2+ response. The results showed that the treatment enhanced the cortical actin networks underneath the plasma membrane  and the GDPβS-injected  eggs were not significantly different (P>0.1)  was again much shorter than in the control   . HoweverP<0.001) . Apparenenvelope .2+ signals generated by 1-MA or GTPγS are influenced by the changes of the actin cytoskeleton, we have used jasplakinolide (JAS), a well-established agent provoking actin polymerization. JAS rearranges the actin cytoskeleton by inducing actin polymerization while inhibiting depolymerization of actin filaments in vivo2+ signaling , with a slight ‘compensatory’ enhancement of F-actin bundles in the inner cytoplasm. These denuded oocytes still responded to 1-MA with a substantial Ca2+ release from the internal stores , as was the case with the 1-MA-induced Ca2+ wave propagation 2+ waves, but also inhibited progression of meiotic maturation , arguing against the role of this type of monomeric G-proteins in meiotic maturation.Considering the broad targets of GTPγS and GDPβS inside the cell, it is conceivable that the effect of these nucleotides might have been mediated by downstream effectors other than heterotrimeric G-proteins. However, the specific inhibitors of Rho families of G-proteins such as 2+ signaling in maturing oocytes of starfish. Whereas the 1-MA-evoked Ca2+ release was completely blocked by GDPβS  reduced the amplitude of the InsP3-evoked Ca2+ response  and heparin (an antagonist of InsP3 receptors) blocked GTPγS-evoked Ca2+ transient 3-dependent pathways. In neuronal cell lines, however, InsP3 and GTPγS activated Ca2+ release by distinct mechanisms 2+ release is still an open question. In support of the idea that tight regulation of the actin cytoskeleton should be considered in interpreting these data, we have found that both heparin and U73122 induce hyperpolymerization of cortical actin as well as blocking Ca2+ signaling 2+, several lines of evidence have suggested that the status of actin polymerization can modulate the intracellular Ca2+-releasing mechanisms 2+ release in starfish oocytes may be mediated by a novel mechanism involving F-actin and its associated proteins 2+-releasing mechanism to InsP3 during meiotic maturation was strongly inhibited by latrunculin-A (LAT-A) 3-evoked Ca2+ signaling  or 100 mM GTPγS. The actin changes were monitored by subsequent microinjection of Alexa Fluor 568-phalloidin respectively 3 and 15 min after the GTPγS injection.Hyperpolymerization of cortical actin by GTPγS. Immature oocytes of (0.27 MB PDF)Click here for additional data file.Data S22+ in response to fertilizing sperm. The postmeiotic eggs of A. pectinifera loaded with Ca2+ dyes were fertilized after 15 min incubation in the presence (violet curves) or absence (green curves) of 6 µM of jasplakinolide. The Ca2+ response to the fertilizing sperm is substantially faster in jasplakinolide-treated eggs.Jasplakinolide-treated eggs display quicker release of Ca(0.04 MB PDF)Click here for additional data file.


Human neutrophils express two pattern recognition GPCRs, FPR1 and FPR2, which belong to the family of formyl peptide receptors. The high degree of homology between these two receptors suggests that they share many functional and signal transduction properties, although they exhibit some differences with respect to signaling. The aims of this study were to determine whether FPR2 triggers a unique signal that allows direct influx of extracellular calcium without the emptying of intracellular calcium stores, and whether the gelsolin-derived PIP2+ induced by agonists for FPR1 or FPR2 in human neutrophils occurred also in the presence of a chelator of Ca2+ (EGTA). PBP10 inhibited not only FPR2-induced oxidase activity, but also the transient rise in intracellular Ca2+.The transient rise in intracellular Ca2+ signaling mediated via FPR2 follows the same route as FPR1, which involves initial emptying of the intracellular stores. PBP10 inhibits selectively the signals generated by FPR2, both with respect to NADPH-oxidase activity and the transient rise in intracellular Ca2+ induced by agonist exposure.Ca FPR1 sequence i i i . Emptyin [Ca2+]i ,26.2-binding peptide PBP10 [via FPR1 [2+ influx across the plasma membrane [2+ across the plasma membrane is not preceded by an increase in [Ca2+]i that originates from the release of Ca2+ from the intracellular stores i that involves Ca2+ release from intracellular stores, and this signaling pathway is inhibited by the FPR2-specific inhibitor PBP10.In the present study, we characterize the neutrophil responses to the FPR2-specific agonist WKYMVM. We confirm the difference between FPR1-dependent and FPR2-dependent activation of the neutrophil NADPH-oxidase. However, in apparent discrepancy with previously published results ,29, we s-7 M final concentrations) of these peptides were also very similar, with peak activity observed after approximately 1 minute. In accordance with earlier findings [The NADPH-oxidase activities induced by the FPR1-specific peptide agonist fMLF and the FPR2-specific peptide agonist WKYMVM were of similar magnitude  a second phase that is regulated by the stores [It is well known that binding of a specific ligand to FPR1 results in PLC-dependent cleavage of PIPe stores  but reli2+]i and activation of the NADPH-oxidase. This is clearly shown in desensitized cells in which the cytoskeleton is disrupted by cytochalasin B. These cells produce large quantities of oxygen radicals  are used, and we set out to determine if the insensitivity to PBP10 of the WKYMVM induced response, is due to the concentration used. We found that when the concentration of agonist was reduced . To ass2+]i, which affects different cellular functions. For a resting neutrophil, the free cytosolic calcium concentration is maintained at a very low level (approximately 100 nM) relative to the level in the extracellular fluid (approximately 1 mM). To maintain this concentration gradient, cells are equipped with ion pumps that transport Ca2+ out of the cells as well as to the intracellular calcium storing organelles, which contain high levels of the calcium-binding protein calreticulin. It is generally accepted that the main pathway for the control of rapid changes of [Ca2+]i in neutrophils is that represented by the capacitive model, which starts with the production of inositol-1,4,5-trisphosphate (IP3). The main pathway for IP3 production involves PLC-mediated hydrolysis of membrane PIP2; the generated IP3 releases calcium from intracellular stores by binding to specific receptors on the intracellular storage organelles. The depletion of the intracellular Ca2+ stores regulates the opening of store-operated Ca2+ channels (SOC) in the plasma membrane, thereby providing a rich source of capacitive entry of calcium ions, originating from the extracellular space [2+]i is unique to FPR2. Binding of an agonist to either of the two FPRs results in a signaling cascade, which in turn leads to an increase in intracellular calcium through the influx of ions across the plasma membrane. Whereas FPR1 influences the plasma membrane channels through the emptying of intracellular stores, as described above, FPR2 appears to generate a signal that acts directly on the ion channels in the plasma membrane so as to facilitate the influx of Ca2+ independently of the filling status of the storage organelles i was achieved when the calcium in the extracellular milieu was depleted through the addition of EGTA; these data are in accordance with recent results from experiments in which a much higher concentration (10-7 M) of agonist was used i. The transient rise in [Ca2+]i peaks at agonist concentrations that are much lower than those used in the oxidase assay, and the inhibitory effects of PBP10 are, thus, disclosed only when the concentration of the FPR2 agonist is reduced. No inhibitory effect of PBP10 is seen when fMLF is used as the agonist, irrespective of the concentration of the chemoattractant used to activate the neutrophils.In the present study, we confirm the difference between FPR1-dependent and FPR2-dependent activation of the neutrophil NADPH-oxidase. Moreover, we show that PBP10 inhibits not only the oxidase activity, but also the FPR2-induced increase in [CaThe molecular mechanism underlying the effects of PBP10 on FPR2-mediated induction of cell functions is complex. PBP10 displays clearly higher specificity for FPR2 than for FPR1, although this pattern of sensitivity is not unique to this receptor ,18. The 2+ signaling mediated via FPR2 follows the same route as FPR1-mediated signaling, which involves initial emptying of the intracellular stores. However, we found that the FPR2-mediated oxidase activity and Ca2+ signaling pathway are inhibited by the FPR2-specific inhibitor PBP10, whereas there is no inhibitory effect of PBP10 when the high affinity FPR1 agonist fMLF is used, irrespective of the concentration of the chemoattractant used to activate the neutrophils. These data clearly demonstrate that there is a fundamental difference between the two very closely related receptor members, in that one is PBP10-sensitive (FPR2) and the other (FPR1) is PBP10-insensitive.The neutrophil FPR family members FPR1 and FPR2 share 69% amino acid identity and mediate almost indistinguishable cellular responses. Therefore, these two FPRs use the same basic signaling pathways. Accordingly, we show that Ca-2 M, and stored at -70°C until use. Further dilutions were made in Krebs-Ringer phosphate buffer that was supplemented with glucose (10 mM), Ca2+ (1 mM), and Mg2+ (1.5 mM) (KRG).The hexapeptide WKYMVM was synthesized and purified by Alta Bioscience . The formylated peptide N-formylmethionyl-leucyl-phenylalanine (fMLF) was purchased from Sigma Chemical Co. . The peptides were dissolved in dimethyl sulfoxide to a concentration of 106 per ml were washed with Ca2+-free KRG and centrifuged at 220 × g. The cell pellets were resuspended at a density of 2 × 107 cells/ml in KRG that contained 0.1% BSA, and loaded with 2 μM Fura 2-AM  for 30 minutes at room temperature. The cells were then diluted to twice the original volume with RPMI 1640 culture medium without phenol red  and centrifuged. Finally, the cells were washed once with KRG, and resuspended in the same buffer at a density of 2 × 107 cells/ml. Calcium measurements were carried out in a Perkin Elmer fluorescence spectrophotometer (LC50), with excitation wavelengths of 340 nm and 380 nm, an emission wavelength of 509 nm, and slit widths of 5 nm and 10 nm, respectively. The transient rise in intracellular calcium is presented as the ratio of fluorescence intensities (340 nm: 380 nm) detected. The measuring cuvette contained catalase (2000 U), to counteract inactivation of the chemoattractants by the MPO-H2O2-system [Cells at the density of 1-3 × 102+. A small volume (10 μl) of EGTA-containing buffer was added to the measuring vial, and 20 seconds later the cells were activated by the addition of ionomycin . The lowest concentration of EGTA that inhibited more than 90% of the ionomycin-induced response was used in subsequent studies to ensure that no Ca2+ entered the cells across the plasma membrane.The concentration of EGTA required to achieve a calcium-free environment was determined by titration of the ionomycin-triggered production of oxidants by neutrophils resuspended in KRG that contained 1 mM Ca6 cells/ml) were incubated at 37°C for 5 minutes in an NADPH-oxidase measuring apparatus . The activity of the cells was then using a luminol/isoluminol-enhanced chemiluminescence (CL) system [5 cells/ml), horse radish peroxidase  and isoluminol . The tubes were equilibrated at 37°C for 5 minutes in the absence or presence of inhibitor, after which the stimulus (0.1 ml) was added. The light emission was recorded continuously.Neutrophils (5 × 10) system ,40. The 5 neutrophils/ml, superoxide dismutase , catalase (2000 U), and luminol . The tubes were equilibrated at 37°C for 5 minutes in the luminometer, after which the stimulus (0.1 ml) was added, and light emission was recorded continuously.To determine intracellular NADPH-oxidase activity levels, we used mixtures that contained 5 × 10HF performed the experiments. HF and CD planned the project, designed the experiments, wrote the paper, and approved the final version of the paper.


In search for this mechanism we found that Aβ1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca2+ in neurons and promote mitochondrial Ca2+ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca2+ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca2+ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca2+ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca2+ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD.Dysregulation of intracellular Ca This view is supported by reports on dysregulation of intracellular Ca2+ promoted by Aβ and mutant presenilins 2+ entry into neurons but results are controversial 2+ levels are important for AD since overexpression of calbindin28k, an endogenous Ca2+ buffer, prevents neuron death in AD models 2+ and cell damage is unknown. A rise in mitochondrial Ca2+ concentration ([Ca2+]mit) might contribute to neurotoxicity but monitoring [Ca2+]mit in individual neurons has been challenging. We have addressed the effects of Aβ assembly state on Ca2+ influx and mitochondrial Ca2+ uptake using photon counting imaging of neurons expressing targeted aequorin 2+ concentrations. Accordingly we asked for the role of mitochondrial Ca2+ uptake on neurotoxicity induced by Aβ oligomers. Finally, we tested whether NSAIDs may protect against Aβ toxicity acting on subcellular Ca2+ fluxes. For these studies we have used mainly cerebellar granule cells although some experiments have been also carried out in cortical and hippocampal neurons.Alzheimer's Disease (AD) is a devastating neurodegenerative disorder due to a massive neuron dysfunction and loss related to development of senile plaques that are made of amyloid β peptide (Aβ), a cleavage product of the amyloid precursor protein. Early affected areas in the brain are the cortex and hippocampus. However, neuropathological studies show frequent and varied cerebellar changes in the late stages of the disease 1–42 obtained from a commercial source (Bachem). Since the standard protocol of preparation includes a precipitation step in which some protein sample is lost, we hydrolyzed an aliquot of the final solution of both oligomers and fibrils in order to carry out an amino acid analysis. This procedure allowed us to obtain the real concentration of these compounds in solution. In the second place we characterized the quaternary structure  of both the oligomers and fibrils using non-denaturing SDS-PAGE (pseudo-native gels). We were unable to stain the Aß1–42 peptides using Coomassie blue even when 2 µg of peptide were loaded per lane (data not shown). However, using silver staining we were able to determine the presence of high-molecular mass species in the SDS-PAGE. We rationalized that despite the fact that the samples were not boiled, a significant population of protein-protein interactions might be lost in the presence of SDS. However, were able to clearly identify monomers, dimers and tetramers in our preparation of oligomers (1–42 oligomers in SDS-PAGE gels is well characterized 1–42 (data not shown). In the third place, electron microscopy was used in order to characterize our Aß1–42 fibrils. Negative staining using uranyl acetate undoubtedly showed the presence of large fibrils in solution  that have been shown previously to be neurotoxic and produce large increases in [Ca2+]cyt in several neuron models including GT1 neural cells 1–42 oligomers, Aβ1–42 fibrils and the toxic fragment Aβ25–35 (a surrogate of Aβ) on cytosolic Ca2+ concentration ([Ca2+]cyt) were tested by fluorescence microscopy  after Aβ25–35 (1–42 in its different assembly states (fibrils and oligomers) prepared from synthetic Aβ1–421–42 fibrils are less toxic than Aβ1–42 oligomers but produce the same toxicity when used at larger concentrations 1–42 fibrils are equally toxic than Aβ1–42 oligomers at concentrations ranking 100 nM–1 µM. We found that, at 500 nM, Aβ1–42 fibrils induce no [Ca2+]cyt increase (data not shown). At 2 µM, Aβ1–42 fibrils induce little or no [Ca2+]cyt rise despite that Aβ25–35 increases [Ca2+]cyt in the same neurons and cells responded also to N-methyl D-aspartate (NMDA) and K+ . Most cells showing a Ca2+ response to Aβ1–42 oligomers also respond to NMDA suggesting that responsive cells are neurons rather than glia  and K+ . In contst cells . The inclar Ca2+  and addihan glia . To conf1–42 oligomers on [Ca2+]cyt in cortical and hippocampal neurons. 1–42 oligomers (500 nM) induce very large increases in [Ca2+]cyt in both cortical  and hippocampal  cells. In addition, the same cells that responded to Aβ1–42 oligomers also responded to NMDA suggesting that responsive cells are neurons rather than glia . This probe contains also GFP to select transfected neurons for bioluminescence imaging . + medium (to open voltage-gated Ca2+ channels) release typically 5–10% of photonic emissions (expressed as the % of remaining counts) that, when calibrated, reported increases in [Ca2+]mit of about 100–300 µM. In addition, these [Ca2+]mit rises were limited to a pool of mitochondria close to sites of Ca2+ entry and release 25–35  whereas 62±10% of the neurons responded to Aβ1–42 oligomers with a large increase in [Ca2+]mit . In contrast, Aβ1–42 fibrils failed to induce any increase in [Ca2+]mit  consistently with the lack of effects on [Ca2+]cyt. The rises in [Ca2+]mit induced by Aβ25–35 and Aβ1–42 oligomers in intact neurons were similar to the increase in [Ca2+]mit induced by perfusing permeabilized neurons with intracellular medium  with 10 µM FCCP (data not shown). Aβ25–35 induced a similar large increase in both [Ca2+]cyt and [Ca2+]mit in GT1 neural cells (data not shown). We tested also the effects of Aβ1–42 on [Ca2+]mit in cortical neurons. Aβ1–42 oligomers also induced large [Ca2+]mit increases in a large population  of cortical neurons (2+]mit was 1099±93 µM (mean±SEM). Taken together, these results show that Aβ25–35 and Aβ1–42 oligomers, but not fibrils, induce a large and sustained entry of Ca2+ through the plasma membrane followed by mitochondrial Ca2+ overload in all neuronal cell models tested here. We asked next whether Aβ1–42 oligomers induce apoptosis and cell death and if so, for the possible role of mitochondrial Ca2+ overload in that process.We studied next whether Aβ peptides promote also mitochondrial Case 25–35  and Aβ1–ligomers  releaseddium see  containi neurons . In the 2+ overload may promote opening of the mitochondrial permeability transition pore (mPTP) and the intrinsic pathway to apoptosis 1–42 oligomers promote apoptotic processes in cerebellar granule cells. For assessing mPTP opening we used the calcein/Co2+ method 2+. Opening of mPTP promotes mitochondrial calcein quenching. 1–42 oligomers induce quenching of mitochondrial calcein fluorescence in cells loaded with calcein and CoCl2 and this effect is prevented by cyclosporin A, a mPTP blocker. Aβ1–42 oligomers also promote release of cytochrome c  did not affect TMRM fluorescence excluding the possibility that TMRM is reporting changes in the plasma membrane potential (data not shown). Next, we studied the effects of such an small mitochondrial depolarization on mitochondrial Ca2+ uptake. We found that FCCP, at 100 nM, prevents the increase in [Ca2+]mit induced by Aβ1–42 oligomers . FCCP also inhibited the rise in [Ca2+]mit induced by Aβ25–35 . This effect is not due to inhibition of Ca2+ entry through the plasma membrane since 100 nM FCCP does not prevent at all the increase in [Ca2+]cyt induced by Aβ1–42 oligomers  production and contribute to neuron cell damage 1–42 oligomers on ROS production in cerebellar granule cells. Incubation of cerebellar granule cells with 500 nM Aβ1–42 oligomers for 4 h elicited a clear increase in number of neurons strongly stained by the ROS probe CM-H2DCFDA. This effect was absent in cells incubated previously with 100 nM FCCP added here to prevent the Aβ1–42 induced increase in [Ca2+]mit . Indomethacin, sulindac sulfide and R-flurbiprofen, all tested at 1 µM, inhibited by 77±3, 86±4 and 68±1%, respectively, the Ca2+-induced rise in [Ca2+]mit in permeabilized neurons (data from 188 cells studied in 6 independent experiments). Salicylate and R-flurbiprofen also prevented the [Ca2+]mit rise induced by Aβ1–42 oligomers . These results indicate that NSAIDs inhibit mitochondrial Ca2+ uptake induced by Aβ1–42 oligomers and Aβ25–35 in intact cells and by high Ca2+ in permeabilized cells. In addition, they suggest that this effect is probably secondary to mitochondrial depolarization and therefore, inhibition of the driving force for Ca2+ uptake through the mitochondrial Ca2+ uniporter, rather than inhibition of Ca2+ entry through the plasma membrane. To support further this view we assessed the effects of NSAIDs on the increases in [Ca2+]cyt induced by Aβ1–42 oligomers and Aβ25–35. None of the NSAIDs tested including indomethacin, R-flurbiprofen and sulindac sulfide, all tested at 1 µM, produce any change in resting [Ca2+]cyt or prevent the increase in [Ca2+]cyt induced by Aβ1–42 oligomers  suggesting that the rises in [Ca2+]cyt are not due to membrane permeabilization. It has been shown previously that the increases in [Ca2+]cyt induced by Aβ species can be prevented by the NMDA receptor open channel blocker memantine 2+ induced by Aβ1–42 oligomers promotes mitochondrial Ca2+ overload as shown directly by bioluminescence imaging of neurons expressing a low-affinity aequorin targeted to mitochondria. This probe was originally developed to monitor the high [Ca2+] inside the endoplasmic reticulum that reach the mM range in resting conditions 2+]mit may reach several hundred µM upon stimulation of voltage-gated Ca2+ entry and Ca2+ release from intracellular stores 2+]mit were transient and restricted to a pool of mitochondria close to sites of Ca2+ entry or release 2+]mit may reach values above 200 µM, the sensitivity limit of these probes, stressing that [Ca2+]mit levels actually rise substantially and the requirement of low-affinity probes for actual measurements of [Ca2+]mit, particularly when a mitochondrial Ca2+ overload is to be measured. Using the low-affinity, mitochondria-targeted aequorin, we find that Aβ25–35 and Aβ1–42 oligomers, but not fibrils, induce a massive mitochondrial Ca2+ overload that reaches values close to the mM level. As aequorin is consumed by the high Ca2+ level achieved, the results suggest that, at variance with stimulation with high K+, most mitochondria take up Ca2+ when cells are stimulated by Aβ1–42 oligomers. As mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter requires high [Ca2+]cyt levels, these results suggest that Aβ1–42 oligomers promote a massive entry of Ca2+, large and sustained enough to activate the mitochondrial Ca2+ uniporter of most mitochondria.We show here that Aβ2+ overload may promote mPTP opening and apoptotic cell death 2+ overload contributes to the apoptotic cell death induced by Aβ oligomers. This view is supported by the findings that Aβ1–42 oligomers promote i)a mitochondrial Ca2+ overload in the whole mitochondrial population, ii)mitochondrial calcein quenching in a cyclosporin sensitive manner, iii)release of cytochrome c, iv)apoptosis as determined by TUNEL assay and v)cell death, again in a cyclosporin A-sensitive manner. Furthermore, inhibition of mitochondrial Ca2+ uptake by low concentrations of FCCP inhibit both cytochrome c release and cell death without preventing Aβ1–42 oligomers-induced increases in [Ca2+]cyt or decreasing cell ATP levels. Finally, Aβ1–42 oligomers induce ROS production and this effect is prevented by low concentrations of FCCP. Taken together, our results suggest that the large and sustained entry of Ca2+ induced by Aβ1–42 oligomers, an effect mimicked by larger concentrations of the toxic fragment Aβ25–35, activate the mitochondrial Ca2+ uniporter of most mitochondria leading to a mitochondrial Ca2+ overload. This effect may promote mPTP opening by itself or, cooperate with the excess of ROS production promoted by the own mitochondrial Ca2+ overload. Finally, mPTP opening allows release of pro-apoptotic factors including cytochrome c leading to apoptosis and cell death the chemical structure of NSAIDs resembling mitochondrial uncouplers, ii)the release of Ca2+ induced by salicylate in Ca2+-overloaded cells and iii)direct TMRM fluorescence measurements showing NSAID-induced mitochondrial depolarization. In addition, uncoupling activity is considered a common characteristic of anti-inflammatory agents with an ionizable group 2+ uptake. However, several facts support this view. First the Ca2+ channel associated to the mitochondrial Ca2+ uniporter is inwardly rectifying, making it especially effective for Ca2+ uptake into energized mitochondria 2+ current through the mitochondrial Ca2+ uniporter. Second, the Nernst's equilibrium for Ca2+ across mitochondria predicts that a 50% fall in ΔΨ reduces the free mitochondrial [Ca2+] that can be reached at equilibrium by 1,000 fold 2+ is a divalent cation present at extremely low concentrations (100 nM) in resting mitochondria. Thus, it is thermodynamically possible that even small drops in ΔΨ could influence dramatically the [Ca2+]mit increase achieved during cell stimulation. In support of this view, we have shown recently that small drops in ΔΨ of a few tens of mVs are enough to prevent largely mitochondrial Ca2+ uptake The mechanism of inhibition of mitochondrial Ca2+ overload and, perhaps, to neurotoxicity. For example, in familial AD, loss of function presenilin 1 mutants show exaggerated Ca2+ release from intracellular stores due to either defective Ca2+ leak from the ER or increased activity of Ca2+ release channels at ER . The close coupling between ER and mitochondria 2+ overload in patients carrying these mutations. Further research is required to support this view. If that were the case, the possible PS1 mutation-mediated mitochondrial Ca2+ overload and ensuing neuron damage should be limited by NSAIDs. An even more important factor, at least from the point of view of the potential number of patients affected, could be the reported depletion of endogenous Ca2+ buffers, particularly calbindinD28k, in selected brain regions during aging and sporadic AD 2+ buffer capacity may place mitochondria at risk of Ca2+ overload during normal neural activity, a process that should be ameliorated by NSAIDs. Therefore, our results point to a pivotal role of mitochondrial Ca2+ overload in Aβ1–42 oligomers toxicity and AD. Interestingly, the mitochondrial Ca2+ overloads likely induced by AD related processes such as Aβ oligomers (shown here), excitotoxicity 2+ release from ER 2+ buffers 2+ excess being either intracellular or extracellular.Interestingly, other AD-related factors seemingly independent of Aβ species may contribute as well to mitochondrial CaWistar rats were obtained from the Valladolid University animal facility. Fura2/AM, TMRM, DAPI, CM-H2DCFDA, n coelenterazine, the cytochrome c antibody, Alexa 488 α mouse IgG and Alexa F594 α rabbit IgG were purchased from Invitrogen . DMEM (ref. 41966-029), fetal bovine serum, horse serum, neurobasal medium, B27, penicillin and streptomycin are from Gibco . Papain solution is from Worthington . The kit for TUNEL assay is from Roche Diagnostics . Aβ peptides were purchased from Bachem AG . The mouse anti β-tubulin III is from Covance  and the rabbit anti-GFAP is from Sigma . The mGA plasmid was kindly donated by P. Brulet . Hypothalamic GT1 neural cells were provided by R. Weiner . Other reagents and chemicals were obtained either from Sigma or Merck.et al.et al.et al. 3 cells/dish, and grown in Neurobasal Medium supplemented with B27 and 10% horse serum. Cells were cultured for 7–10 days before experiments. Cerebellar granule cells and cortical neurons were plated on poly-L-lysine coated, 12 mm diameter glass coverslips and cultured in high-glucose, low K+, Dulbeccós modified Eaglés medium  plus 10% fetal bovine serum, 5% horse serum, 100 u ml−1 penicillin and 100 µg ml−1 streptomycin for 2 days. Then the culture medium was replaced by Satós medium −1 penicillin and 100 µg ml−1 streptomycin.Cerebellar granule cells were obtained from 5-day old Wistar rat pups killed by dislocation followed by decapitation as reported previously 1–42 oligomers and fibrils were prepared as reported previously by Klein and Dahlgren et al. 1–42 was initially dissolved to 1 mM in hexafluoroisopropanol and separated into aliquots in sterile microcentrifuge tubes. Hexafluoroisopropanol was removed under vacuum in a speed vac., and the peptide film was stored desiccated at −20°C. For the aggregation protocol, the peptide was first resuspended in dry dimethyl sulfoxide to a concentration of 5 mM and treated differently for preparation of oligomers and fibrils. For preparation of oligomers, Haḿs F-12 was added to bring the peptide to a final concentration of 100 µM and incubated at 4°C for 24 h. The preparation was then centrifuged at 14,000×g for 10 min at 4°C to remove insoluble aggregates (protofibrils and fibrils) and the supernatants containing soluble Aβ1–42 oligomers were transferred to clean tubes and stored at 4°C. For preparation of fibrils, the peptide resuspended in dimethyl sulfoxide at 5 mM concentration was diluted in 10 mM HCl to bring the peptide to a final concentration of 100 µM and incubated at 37°C for 24 h. Actual concentrations of both oligomers and fibrils were measured by amino acid analysis. Aβ25–35 was solved in PBS at a concentration of 1 mM, sonicated 3 times and stored at −20°C. For experiments, Aβ25–35 was solved in medium at a final concentration of 20 µM.Aβ1–42 oligomers and fibrils in glass tubes were extensively vacuum dried in a speed-vac. Hydrolysis was performed using a 5.9 HCl solution containing 0.1% phenol. Norleucine was added to the hydrolytic solution as an internal standard. The tubes were sealed under vacuum and the hydrolysis was performed during 24 h at 110°C. Subsequently, the samples were vacuum dried. Finally, the samples were injected into a Biochrom 30 amino acid analyzer. Both composition and concentration data was obtained from the chromatogram.Samples of both Aß1–42 oligomers or fibrils at a 40 µM concentration were incubated with 1X loading buffer. The samples were loaded in the gel without boiling and the gel was run at constant amperage of 20 mA. The gels were subsequently fixed with a freshly prepared solution of 50% methanol, 12% acetic acid, 0.02% paraformaldehyde for at least 1h. Then, the gel was washed three times with 50% ethanol. The next step consisted on an incubation of the gel for 1 min in 0.2 mg/ml NaS2O3. Subsequently, the gel was washed three times with miliQ water for 20 seconds. Then, the gel was incubated for 20 minutes in 2 mg/ml AgNO3 in 0.025% formaldehyde. After two 20-second washes with miliQ water, color was allowed to develop using a solution consisting of 60 mg/ml Na2CO3, 0.02% formaldehyde, 0.004 mg/ml NaS2O3. After the bands appear, the gel was washed twice with miliQ water and the reaction was stopped using a 50% methanol, 12% acetic acid solution.A standard 17% PAGE-SDS was prepared and 10 µl samples of either Aβ1–42 fibrils (40 µM) was applied to a 200 mesh Formvar-coated copper grid and incubated for 15 minutes at room temperature. The sample was then wicked off with filter paper, washed briefly by placing the grid face down on a droplet of water, and stained by transferring the grid face down on a droplet of 2% uranyl acetate for 2 minutes before wicking off the solution and air drying. Grids were visualized in a JEOL transmission electron microscope.A 10 µl sample of Aß2 1, MgCl2 1, glucose 10 and Hepes 10  and loaded with 4 µM fura2/AM or fura4F/AM for 60 min at room temperature. Then coverslips were placed on the heated stage of an inverted microscope (Nikon Diaphot), perfused continuously with the same prewarmed standard medium containing and epi-illuminated alternately at 340 and 380 nm. Light emitted above 520 nm was recorded with a Magical Image Processor (Applied Imaging). Pixel by pixel ratios of consecutive frames were captured and [Ca2+]cyt was estimated from these ratios as previously reported Coverslips containing cells were incubated in standard medium containing (in mM) NaCl 145, KCl 5, CaCl® device and the VPG-1003 transfection kit . The mGA probe contain a mutated, low affinity aequorin targeted to mitochondria and a GFP sequence to help select transfected neurons °C under a Zeiss Axiovert S100 TV microscope and perfused at 5–10 ml/min with test solutions based on the standard perfusing solution described above prewarmed at 37°C. At the end of each experiment, cells were permeabilized with 0.1 mM digitonin in 10 mM CaCl2 to release all the residual aequorin counts. Bioluminescence images were taken with a Hamamatsu VIM photon counting camera handled with an Argus-20 image processor. Photonic emissions were integrated for 10 s periods. Photons/cell in each image were quantified using the Hamamatsu Aquacosmos software. Total counts per cell ranged between 2·103 and 2·105 and noise was (mean±SD) 1±1 counts per second (c.p.s.) per typical cell area (2000 pixels). Data were first quantified as rates of photoluminescence emission/total c.p.s remaining at each time (% of remaining counts) and divided by the integration period (L/LTOTAL in s−1). Emission values of less than 4 c.p.s were not used for calculations. Calibrations in terms of [Ca2+]mit were performed using as reported previously 2, 1 mM K3PO4, 0,2 mM EGTA, 1 mM ATP, 20 µM ADP, 2 mM succinate, 20 mM HEPES/KOH, pH, 6.8. Then, the cells were incubated with the same medium containing 200 nM Ca2+ (buffered with EGTA) with or without NSAIDs for 5 min. Then, perfusion was switched to “intracellular” medium containing 5 µM Ca2+ (with or without NSAID). Further details have been reported previously Cells were transfected with the mGA plasmid using a Nucleofector II2 1 mM for 30 min at 37°C and subjected to conventional fluorescence imaging or confocal microcopy. Fluorescence traces from individual cells were normalized relative to the value before addition of test solutions and averaged. Background fluorescence corresponding to regions of interest devoid of cells was subtracted. In some experiments, cells were incubated with cyclosporin A 1 µM for 15 min before recordings.mPTP opening was assessed directly by the calcein/cobalt method et al. 1–42 in the absence or in the presence of FCCP 100 nM and R-flurbiprofen 1 µM. ROS formation was assessed using 2 µM CM-H2DCFDA with 40 min of probe loading. Then neurons were superfused for 5 min with prewarmed (37°C) PBS. Fluorescence in cells was immediately visualized using the Zeiss S100 TV inverted microscope, a FITC filter set, an OrcaER digital camera from Hamamatsu and the Hamamatsu Aquacosmos software.ROS formation was evaluated in live neurons using CM-H2DCFDA as reported by De Felice The effects of treatments on ΔΨ were estimated by fluorescence imaging in cells loaded with the ΔΨ sensitive probe TMRM as reported previously Cytochrome c release from mitochondria was tested by immunofluorescence and conventional or confocal microscopy. Cells were treated under the various experimental conditions for 72 h and fixed. Location of cytochrome c was tested by indirect immunofluorescence. In conventional fluorescence, nuclei were identified by DAPI staining. Confocal images were obtained using a BIO-RAD laser scanning system (Radiance 2100) coupled to a Nikon eclipse TE2100U, inverted microscope. For quantification of cytochrome c release by confocal microscopy, the relative abundance (%) of cells showing diffuse vs. punctate immunofluorescence was calculated 4 cells/ml and treated with test solutions for 72 h. Cell death was estimated in the same samples by staining with fluorescein diacetate  and propidium iodide  and assessed by fluorescence microscopy using a Nikon Eclipse 80i microscope coupled to a DM 1200C digital camera using a 20x objective. For determination of apoptotic cells at the single cell level, cells were plated at about 5×104 cells/ml and incubated with test solutions for 72 h. Apoptotic cells were revealed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method by fluorescence imaging and a cell death detection kit following the protocol provided by the manufacturer.Cells were plated in wells at about 5×10−5 to 10−9 M concentration range.Cerebellar granule cells were plated in 4-well plates and cultured with test solutions for 72 h. Then, cells were washed twice with PBS at 37°C and 1 ml of boiling 20 mM Tris, pH, 7.75 and 4 mM EDTA was added. After 2 min, samples were centrifuged for 4 min at 10.000 g. ATP was measured later from the supernatant by the luciferin-luciferase assay using a Cairn photon counting device  and a standard curve prepared using pure ATP over a 10When only two means were compared, Student's t test was used. For more than two groups, statistical significance of the data was assessed by ANOVA and compared using Bonferroni's multiple comparison test. Differences were considered significant at p<0,05.Figure S1Characterization of Aβ oligomers and fibrils.A. SDS-PAGE and silver staining of an oligomeric Aβ1–42 sample. Arrows point to bands reflecting the presence of monomers, dimers and tetramers. Representative of 3 experiments. B, SDS-PAGE and silver staining of a fibrillar Aβ1–42 sample. Arrows point to bands reflecting the presence of monomers, dimers, tetramers. Some larger oligomerization species also are apparent in the gel. In addition, a certain amount of large molecular weight fibrils incapable of entering the separating gel is also pointed on top. Representative of 3 experiments. C. Electron microscopy was used in order to characterize Aß1–42 fibrils. Negative staining using uranyl acetate undoubtedly showed the presence of large fibrils in solution. Most fibrils were similar in width and with a length that usually varied between 200 and 800 nm. Bar represents 200 nm. Representative of 6 experiments. Aβ1–42 oligomers and fibrils were also characterized by amino acid analysis for testing actual concentration values and composition (data not shown).(9.36 MB TIF)Click here for additional data file.Figure S225-35 induces apoptosis and cell death in cerebellar granule cells.AβA. Cerebellar granule cells were cultured for 72 h in vehicle (control) or Aβ25–35 (20 µM) and apoptosis was tested by TUNEL assay. Pictures show nuclei (blue) and apoptotic cells (purple). Bars show % of apoptotic cells . Scale bar represents 10 µm. B. Cerebellar granule cells were cultured for 72 h with vehicle (control) or Aβ25–35 (20 µM) and cell death was assessed by staining with FDA  and PI . Bars show % of dead cells . Scale bar represents 10 µm.(6.71 MB TIF)Click here for additional data file.Figure S32+ rise and cytochrome c release induced by Aβ25–35.FCCP prevents the mitochondrial (but not the cytosolic) CaA. Cerebellar granule cells expressing mGA were subjected to bioluminescence for [Ca2+]mit measurements. FCCP 100 nM inhibits the increase in [Ca2+]mit induced by Aβ25–35 . After washout of FCCP, Aβ25–35 was able to increase [Ca2+]mit. B. Cerebellar granule cells were loaded with fura2/AM and subjected to fluorescence imaging for [Ca2+]cyt measurements. FCCP 100 nM failed to inhibit the increase in [Ca2+]cyt induced by Aβ25–35 (20 µM) . C. Immunofluorescence against cytochrome c was assessed by confocal microscopy in cerebellar granule cells treated with vehicle (Control), Aβ25–35 20 µM (Aβ) and 20 µM Aβ25–35 + FCCP 100 nM (Aβ+FCCP) for 72 h. Aβ25–35 promotes diffusion of cytochrome c that normally shows a punctate staining reflecting mitochondrial location. 100 nM FCCP prevented diffusion of cytochrome C. Scale bar represents 10 µm. D. Bars show % of cells showing diffuse staining for cytochrome c (reflecting cytochrome c release). Aβ25–35 (20 µM) increases the percent of cells showing diffuse staining an this effect was inhibited by FCCP 100 nM. ap<0,05 vs. control; bp<0,05 vs. Aβ treated cells. Bars are mean±SEM of 3 independent experiments.(2.27 MB TIF)Click here for additional data file.Figure S4Aβ oligomers induce ROS formation that is prevented by FCCP and R-flurbiprofen. Cerebellar granule cells were incubated for 4 h with vehicle, Aβ1–42 oligomers (500 nM) and oligomers plus FCCP 100 nM or R-flurbiprofen. Then, ROS production was imaged using the ROS-sensitive probe CM-H2DCFDA. Aβ1–42 oligomers induced an increase in fluorescence compared to the vehicle that was prevented by FCCP and R-flurbiprofen. The Pictures are representative of 5–9 microscopic fields in at least 3 independent experiments for each condition. Addition of FCCP or R-flurbiprofen alone produced similar results than vehicle (data not shown).(2.83 MB TIF)Click here for additional data file.Figure S525–35.NSAIDs prevent cytochrome c release and cell death induced by AβA. Cerebellar granule cells were treated with Aβ25–35 (20 µM) for 72 h with or without 1 µM R-flurbiprofen or 100 µM salicylate and fixed for analysis of cytochrome c location using confocal microscopy. Control cells showed a punctate distribution of cytochrome c. Scale bar represents 10 µm. Aβ25–35 treated cells show a more diffuse pattern of cytochrome c whereas cells treated with Aβ25–35 and R-flurbiprofen show a punctate pattern similar to control cells. Bars show the relative abundance (%) of cells showing diffuse immunostaining in control cells, cells treated with Aβ25–35 and cells treated with Aβ25–35 plus 1 µM R-flurbiprofen (B) or 100 µM salicylate (C). . D. Effects of ibuprofen (Ibu), indomethacin (Indo) and sulindac sulfide (Sul), all tested at 1 µM, on cell death induced by Aβ25–35 (20 µM) as assessed by dye exclusion studies. E. Effects of R- and S-flurbiprofen (Rf and Sf), both tested at 1 µM, on cell death induced by Aβ25–35. *p<0,05 vs. control; #p<0,05 vs. Aβ. All data are representative of 3 experiments.(2.10 MB TIF)Click here for additional data file.


Numerous functional studies have shown that nitrergic neurotransmission plays a central role in peristalsis and sphincter relaxation throughout the gut and impaired nitrergic neurotransmission has been implicated in clinical disorders of all parts of the gut. However, the role of nitric oxide (NO) as a neurotransmitter continues to be controversial because: 1) the cellular site of production during neurotransmission is not well established; 2) NO may interacts with other inhibitory neurotransmitter candidates, making it difficult to understand its precise role.2+) in the myenteric nerve varicosities during neurotransmission using multiphoton microscopy. Our results in mice gastric muscle strips provide visual proof that NO is produced de novo in the nitrergic nerve varicosities upon nonadrenergic noncholinergic (NANC) nerve stimulation. These studies show that NO is a neurotransmitter rather than a mediator. Changes in NO production in response to various pharmacological treatments correlated well with changes in slow inhibitory junction potential of smooth muscles.Imaging NO can help resolve many of the controversies regarding the role of NO in nitrergic neurotransmission. Imaging of NO and its cellular site of production is now possible. NO forms quantifiable fluorescent compound with diaminofluorescein (DAF) and allows imaging of NO with good specificity and sensitivity in living cells. In this report we describe visualization and regulation of NO and calcium (CaDual imaging and electrophysiologic studies provide visual proof that during nitrergic neurotransmission NO is produced in the nerve terminals. Such studies may help define whether NO production or its signaling pathway is responsible for impaired nitrergic neurotransmission in pathological states. Nitric oxide (NO) has been proposed as a neuromuscular neurotransmitter of nonadrenergic noncholinergic (NANC) inhibitory nerves in the parasympathetic While there is strong physiological evidence that NO is involved in inhibitory neurotransmission 2+NO has a unique chemistry. It reacts with 4,5-diaminofluorescein (DAF-2) to produce quantifiable fluorescent product DAF-2 triazole (DAF-2T). Reaction of NO with DAF-2 is highly specific and sensitive (NO detection limit of 5 nM) 2+ signals at the cellular level by fluorescent imaging; 2) on the effect of pharmacological treatments on these signals; 3) on nitrergic slow inhibitory junction potential (sIJP) in electrophysiological studies; and 4) to compare the effects of the pharmacological treatments on NO in the imaging studies and sIJP in the electrophysiological studies.The purpose of the present studies was to examine the effect of electrical field stimulation of mice gastric muscle strips: 1) on NO and Cain situ in the gut and also provides proof that on NANC nerve stimulation, NO is produced in the myenteric nitrergic nerve varicosities and not in the smooth muscle cells, thereby demonstrating that NO is a neurotransmitter rather than a mediator produced in the smooth muscles. They also document that during neurotransmission NO is produced de novo and not stored as a NO donor in the varicosities and released with other classical neurotransmitters. Changes in NO production in response to various pharmacological treatments correlated well with changes in slow inhibitory junction potential of smooth muscles. Functional studies combined with imaging may help elucidate whether NO production or its upstream or downstream signaling is the underlying mechanisms of impaired nitrergic neurotransmission in the pathological states.These results provide, for the first time, visual identification of nerve varicosities 2+ signals identify all nitrergic and non-nitrergic varicosities. These studies show that multiphoton microscopy can vividly visualize varicosities on axons deep below the surface in intact tissue. Elevated Ca2+ signals were not seen in smooth muscles because EFS was applied under nonadrenergic noncholinergic conditions to block muscle excitation.We first sought to visualize varicosities of myenteric neurons in the mouse gastric smooth muscle. Imaging was focused on the varicosities because they are the sites of release of the neurotransmitters. Multiphoton imaging of circular muscle strips preloaded with the calcium indicator after EFS revealed discrete orange-red fluorescent spots. These images were superimposed on image of smooth muscles obtained in the transmission mode. Note that varicosities were linearly oriented along the longitudinal axis of the underlying muscle fibers . These fTo visualize nitrergic varicosities, we examined muscle strips preloaded with DAF-2 after applying EFS under NANC conditions. Green DAF-2T fluorescence represent NO signals . Panel  shows intensity (height and width) of the NO fluorescent signals from the varicosities. Note that very few NO signals were seen in the strips without EFS. The signals increased in the strips that received EFS and were again absent in the strips that receive EFS after L-NAME treatment. Panel (2c) shows relative quantification of the NO signals. The bar graphs revealed that NO signal was 1.5±0.25 in the basal state (unstimulated strips), 4.0±0.97 after EFS, and 1.0±0.03 in strips pretreated with NOS inhibitor, L-NAME . Basal levels of NO may be generated by the tonic activity of the nitrergic neurons. These observations strongly suggest that the green signals are truly due to NO produced in the nerve varicosities.2+ signals, we loaded the muscle strips with both DAF-2 and calcium orange and applied EFS. These strips were imaged for NO and Ca2+ signals  increased NO and Ca2+ signals. The elevation of NO and Ca2+ signals were abolished by tetrodotoxin. Since tetrodotoxin blocks the fast sodium channel that mediates the action potential that is conducted along the axon and depolarizes the nerve varicosities to cause Ca2+ influx 2+ channels, ω-CTX GVIA, so that no significant increase in Ca2+ or NO signals were seen. However, L-type Ca2+ channel blocker, nifedipine, did not alter the increases in Ca2+ or NO signals. These observations indicate that Ca2+ entry into the varicosities that stimulates NO production occurred via N-type Ca2+ channels. Pretreatment of tissues with calmodulin (CaM) inhibitor W7 did not affect Ca2+ increase, but markedly suppressed NO production by EFS , suggesting that increase in internal Ca2+ stimulates nNOS via a Ca2+-CaM mediated process to produce NO. Similarly, pretreatment with the nNOS inhibitor L-NAME suppressed NO signals without affecting the Ca2+ signals, showing that suppression of nNOS caused suppression of NO generation in the presence of normal increase in Ca2+ upon electrical stimulation.We also examined the effect of various known antagonists of nitrergic neurotransmission on CaIn order to correlate pharmacology of imaging studies with functional neurophysiological studies of smooth muscle membrane potentials, we examined the effects of antagonists on the nitrergic slow inhibitory junction potentials (sIJP). EFS of muscle strips under NANC conditions produced two overlapping IJPs called the fast and the slow IJPs. Apamin treatment blocked fast IJP and revealed the nitrergic slow IJP In conclusion, the unique chemical properties of NO and its indicator dyes and multiphoton microscopy allows imaging of NO during nitrergic neurotransmission that is not possible with many other neurotransmitters. These studies provide visual proof that NO is a true neurotransmitter and not a secondary mediator.2+ imaging studies combined with neurophysiology may also provide an important tool for understanding mechanisms of impaired nitrergic neurotransmission in motor disorders of the gut. Such studies may also help better define the underlying defect in nitrergic neurotransmission in conditions such as diabetic gastroparesis 2+ and NO can also help document whether the suppressed nitrergic neurotransmission is due to abnormalities in calcium kinetics, CaM abnormalities or defects in the enzyme, nNOSα. Such studies may also be helpful in elucidating abnormalities in urinary tract and cerebral blood vessels where nitrergic neurotransmission is a major regulatory mechanism.Imaging of nitrergic neurotransmission may help distinguish between disorders due to impaired NO production such as nNOS deficiency and those due to impaired NO action such as seen in deficiencies of NO sensitive guanylyl cyclase The experimental protocol used was approved by the Animal Care Committee of the VA Boston Healthcare System.2 narcosis was used to euthanize adult male mice (22–38 g). Stomach was removed and 4–6 mm wide strips of smooth muscle layer were prepared after shearing the mucosa. The strips were transferred to a tissue bath with a Sylgard  floor and pinned to the floor with mucosal surface facing up. The chamber was continuously perfused with warm oxygenated (95% O2/5% CO2) Krebs solution at a rate of 3 ml/min. The bath temperature was maintained at 37±0.5°C and entire set-up was protected from light.COThe drugs and chemicals were obtained from Sigma  unless specified otherwise. They were prepared fresh before use. DAF-2DA  and Calcium Orange- acetoxymethyl ester (AM)  and nifedipine were dissolved in dimethyl sulfoxide (DMSO). The final bath concentration of chemicals were: apamin (0.3 µM); atropine (1 µM ); calcium orange-AM (10 µM); ω-conotoxin-GVIA (ωCTX) (0.1 µM); 4,5-diaminofluorescein diacetate (DAF-2DA) (10 µM); (100 µM); guanethidine (5 µM); N-ω-nitro-L-arginine methyl ester (L-NAME) (200 µM); nifedipine (1 µM); tetrodotoxin (TTX) (1 µM); and W7  (100 µM).Gastric muscle strips were mounted in a chamber and perfused with Krebs' solution prior to loading with dyes/drugs. Calcium orange-AM and/or DAF-2A were added 1 hour prior to EFS. The emission spectrum (576 nm) of Calcium orange can be well resolved from that of DAF-2T (515 nm), thus facilitating simultaneous imaging of the two components.Tissues were incubated in fluorescent dyes for one hour prior to EFS and antagonists were applied 20–30 min prior to the dye loading. The EFS was applied under NANC conditions (in the presence of atropine (1 µM) and guanethidine (5 µM)) to block cholinergic and adrenergic responses to elicit nonadrenergic noncholinergic inhibitory responses. The EFS consisted of 3 stimulus trains of 0.5 sec each  applied 30 seconds apart. The tissues were immediately mounted on the slides and imaged promptly.2+ (549 nm) and DAF-2T (495 nm) dyes to consistently generate measurable emissions in orange-red (576 nm) and green (515 nm) regions of the spectrum. The average laser power delivered to the sample was 70–150 mW. Narrow band pass filters were used to separate the emission spectra of the two dyes. A Zeiss Axiovert S100 inverted microscope equipped with a high quality water immersion 40×/1.2 NA, C-apochroma objective was used in the epifluorescence and/or transmission mode to image the nerve varicosities. The 512×512 pixel images were collected in direct detection configuration at a pixel resolution of 0.484 µm with a Kalman-5 collection filter. The nerve varicosities were identified by Z scanning the circular smooth muscle layer and were generally at depths of 150 µm. The images were reconstructed using the BioRad LaserSharp software.Dye-loaded and treated tissues after EFS were imaged with BioRad MRC 1024ES multi-photon imaging system . The imaging system was coupled with a mode-locked titanium∶sapphire laser  operating at 82 MHz repetition frequency, 80 fs pulse duration with a wavelength of 820 nm. Tri/sapphire laser tuned to 820 nm in multi-photon excitation mode at 700 mW was able to excite calcium orange-Ca2+ response, normal Ca2+ response but reduced NO response or normal Ca2+ and NO responses to EFS. We determined the relative changes in calcium orange-Ca2+ and DAF-NO fluorescence, comparing the intensities of the signals in the antagonist treated, EFS stimulated tissues with the unstimulated control tissues. Three to six boundaries were drawn around arbitrary areas along the nerve varicosities identified by XYZ scanning in a field of view at 320× magnification using an image processor . Fluorescence intensity was integrated over all pixels within the boundary of each individual enclosed area and quantified using LaserSharp (BioRad) and MetaMorph . The data are presented as the average of at least three blinded experiments performed on different days.Impaired nitrergic neurotransmission may be associated with reduced CaFor colocalization of NO and nNOS, muscle strips were loaded with the NO indicator, DAF-2DA and EFS applied as described above. The tissues were then fixed in 4% freshly prepared formaldehyde in PBS and were labeled with rabbit anti-nNOS antibody.Intracellular membrane potential recordings under NANC conditions were made using sharp microelectrodes with high input impedance as described in details in our previous publications Data were expressed as means±SEM and appropriate tests done to compare significance of difference in means .


In contrast, vertebrate photoreceptors are nonspiking neurons that maintain sustained depolarization and neurotransmitter release from ribbon synapses in darkness and produce light-dependent graded hyperpolarizing responses. Rods transmit single photon responses with high fidelity, whereas cones are less sensitive and exhibit faster response kinetics. These differences are likely due to variations in presynaptic Ca2+ dynamics. Metabolic coupling and cross-talk between mitochondria, endoplasmic reticulum (ER), plasma membrane Ca2+ ATPase (PMCA), and Na+-Ca2+ exchanger (NCX) coordinately control presynaptic ATP production and Ca2+ dynamics. The goal of our structural and functional studies was to determine the spatiotemporal regulation of ATP and Ca2+ dynamics in rod spherules and cone pedicles.In conventional neurons, Ca2+ imaging experiments of dark- and light-adapted rod and cone terminals in retinal slices were conducted.Central retina tissue from C57BL/6 mice was used. Laser scanning confocal microscopy (LSCM) experiments were conducted on fixed-frozen vertical sections. Primary antibodies were selected for their tissue/cellular specificity and ability to recognize single, multiple or all splice variants of selected isoforms. Electron microscopy (EM) and 3-D electron tomography (ET) studies used our standard procedures on thin- and thick-sectioned retinas, respectively. Calibrated fluo-3-Ca2+] than cone pedicles, whereas during light adaptation pedicles rapidly lowered their [Ca2+] below that observed in spherules.Confocal microscopy showed that mitochondria, ER, PMCA, and NCX1 exhibited distinct retinal lamination patterns and differential distribution in photoreceptor synapses. Antibodies for three distinct mitochondrial compartments differentially labeled retinal areas with high metabolic demand: rod and cone inner segments, previously undescribed cone juxtanuclear mitochondria and the two plexiform layers. Rod spherule membranes uniformly and intensely stained for PMCA, whereas the larger cone pedicles preferentially stained for NCX1 at their active zones and PMCA near their mitochondria. EM and ET revealed that mitochondria in rod spherules and cone pedicles differed markedly in their number, location, size, volume, and total cristae surface area, and cristae junction diameter. Rod spherules had one large ovoid mitochondrion located near its active zone, whereas cone pedicles averaged five medium-sized mitochondria clustered far from their active zones. Most spherules had one ribbon synapse, whereas pedicles contained numerous ribbon synapses. Fluo-3 imaging studies revealed that during darkness rod spherules maintained a lower [Ca2+] in darkness, which increases their sensitivity and signal-to-noise ratio. In contrast, cone pedicles utilize low affinity/high turnover NCX to rapidly lower their high [Ca2+] during light adaptation, which increases their response kinetics. Spatiotemporal fluo-3-Ca2+ imaging results support our immunocytochemical results. The clustering of cone pedicle mitochondria likely provides increased protection from Ca2+ overload and permeability transition. In summary, these novel studies reveal that several integrated cellular and subcellular components interact to regulate ATP and Ca2+ dynamics in rod and cone synaptic terminals. These results should provide a greater understanding of in vivo photoreceptor synaptic terminal exocytosis/endocytosis, Ca2+ overload and therapies for retinal degenerations.These findings indicate that ATP demand and mitochondrial ATP production are greater in cone pedicles than rod spherules. Rod spherules employ high affinity/low turnover PMCA and their mitochondrion to maintain a relatively low [Ca The increased subplasmalemmal Ca2+ concentration in these microdomains plays a key role in transient exocytosis  in rod spherules and cone pedicles differed during darkness and light-adaptation. The fourth goal was to determine and discuss the functional significance of our results in relation to rod and cone synaptic terminal bioenergetics, Ca2+ homeostasis and neurotransmitter release. Here we show that mitochondria, ER, PMCA and NCX each exhibited a distinct retinal lamination pattern. At higher spatial resolution, unique functionally related distributions of mitochondria, ER, PMCA and NCX were observed in both rod spherules and cone pedicles. Rod spherule and cone pedicle mitochondria also had marked functionally related differences in the location, total number, size, volume and total surface area of the inner mitochondrial cristae membranes. Furthermore, rod spherules maintained a lower [Ca2+] than cone pedicles during darkness, whereas cone pedicles lowered their intraterminal [Ca2+] faster and to a greater degree than rod spherules during light adaptation. These results provide a more comprehensive understanding of the spatiotemporal control of Ca2+ concentrations and mitochondrial ATP production in rod and cone ribbon synaptic terminals as they relate to exocytosis and endocytosis. Moreover, they provide important groundwork for further understanding compartmental differences in photoreceptors and offer new insight into strategies that will be necessary to treat visual deficits that result from rod and/or cone photoreceptor degeneration.The overall goal of these studies was to develop a comprehensive structural and functional understanding of rod spherule and cone pedicle ribbon synaptic terminals. The specific aims of these laser scanning confocal microscopy (LSCM), electron microscopy (EM), three-dimensional electron tomography (ET), and CaAll chemicals were purchased as analytical or molecular biology grade from Sigma Chemical Co.  or Fisher Scientific  unless otherwise noted. The pH of all solutions was 7.40 at indicated temperatures.All experimental and animal care procedures complied with the principles of the American Physiological Society, the NIH Guide for the Care and Use of Laboratory Animals and Maintenance  and were approved by the Institutional Animal Care and Use Committee of the University of Houston. Wild-type C57BL/6J mice , from litters bred at our facility, were maintained on a 12:12 light:dark cycle (10-20 lux cage luminance) with food and water available ad libitum.2 in 0.1 M cacodylate buffer (Karnovsky's fixative) for 12 h at 4 °C for conventional EM or ET studies as described , to the Ca2+-containing HEPES buffer. Similar to our previous results  for 15 min at 27 °C. The fluo-3 fluorescence measured in dark-adapted photoreceptor synaptic terminals, from these retinal slices, was not above background (data not shown). Third, we attempted an in vivo calibration of fluo-3 fluorescence in photoreceptor synaptic terminals using the standard ionomycin, Mn2+ and digitonin procedure for NIH 3T3 cells  in the photoreceptor synaptic terminals. An intracellular buffer  that contained calibrated Ca2+ buffers  and 5 μM of the penta-ammonium salt of fluo-3 (Molecular Probes) was pipetted onto dual concave glass slides maintained in the dark at 27 °C. Fluo-3 fluorescence was measured with the Zeiss LSM-410 confocal microscope system  as described below.To confirm that the observed fluorescence signals reflected changes in internal Ca results ,94, thisT3 cells ,99 and ireported ,94. Therreported ,100, in 2+ image recordings began one minute after light onset as the light-adapted decrease in photoreceptor oxygen consumption stabilizes during the first minute of the light stimulation , in dark- and light-adapted rod and cone synaptic terminals, only experiments where relatively adjacent photoreceptor synaptic terminals were imaged are included in this data set. To normalize all the images a standard background subtraction was performed on each image. Then the overall RFI within each dark- and light-adapted rod spherule and cone pedicle was determined based on a grey scale with 256 levels. The light-adapted to dark-adapted fluorescence intensity ratio for each rod spherule and cone pedicle from each retina was calculated, means±SEMs were determined, and the data was statistically analyzed. The mean RFI of dark-adapted rod spherules and cone pedicles as well as light-adapted rod spherules and cone pedicles were determined and compared. To analyze and evaluate this data, the RFI values were normalized by the gain of the confocal system expressed on a linear scale as described  than cone pedicles. In contrast, the estimated free [Ca2+] in light-adapted cone pedicles was 3.2 fold lower (about 0.2 μM) than in light-adapted rod spherules (about 0.7 μM). Thus, during light adaptation the estimated free [Ca2+] decreased 2 fold in rod spherules and almost 30 fold in cone pedicles compared to darkness. These latter results reveal that during light adaptation cone pedicles rapidly and efficiently lowered their intraterminal [Ca2+] compared to rod spherules. This is consistent with our immunocytochemical, EM and ET findings that cone pedicles possess structural and functional mechanisms for the more rapid removal of intraterminal free Ca2+ than rod spherules (vide supra).2+] were larger and closer to the plasma membrane in dark-adapted (depolarized) cone pedicles  and intensely stained mitochondria in the OPL. The weaker VDAC labeling might result from a low concentration of ER in the IS, since VDAC is found in ER closely apposed to mitochondria . However2+ overload or oxidative stress  regulation in dark- and light-adapted rod spherules and cone pedicles, we conducted Ca2+ imaging studies on retinal slices utilizing fluo-3: a high affinity fluorescent Ca2+ dye. These experiments produced three novel and functionally relevant results that validated several of our predictions. First, in dark-adapted retinas the estimated free [Ca2+] in rod spherules was about 2 μM, which was significantly (3.2 fold) lower than that in cones pedicles. These findings are consistent with the interpretation of our detailed immunocytochemical findings. That is, rod spherules predominantly possess high affinity/low turnover PMCA that maintains a low intraterminal [Ca2+] in order to increase the sensitivity and signal-to-noise ratio of rods  in cone pedicles was 0.23 μM, which is significantly (3.2 fold) lower than that in rod spherules. This is consistent with our immunocytochemical, EM and ET findings that cone pedicles possess the structural components and functional mechanisms  to more rapidly remove intraterminal free Ca2+ compared to rod spherules as well as PMCA to lower the free [Ca2+] below the Kd of NCX. Furthermore, it is compatible with the observation that the recovery and kinetics of exocytosis is faster in cones than rods  during dark and light-adaptation. Compared to rod spherule mitochondria, individual cone pedicle mitochondria are smaller and more ovoid, reside more remotely from the active zone, possess less cristae surface and volume, and have a very small CJ diameter. These distinct characteristics play a fundamental role in the ability of rod and cone presynaptic terminals to regulate and produce ATP as well as to buffer Ca2+ microdomains. In addition, the ER, PMCA and NCX all play essential and inter-dependent roles in rod and cone presynaptic terminals. Their differences in cellular distribution and location, amount of protein expression, affinity and capacity for Ca2+ and their turnover rate of Ca2+ significantly contribute to the kinetics of exocytosis in rods and cones. The results from these LSCM, EM, ET, and Ca2+ imaging experiments can help design new electrophysiological, pharmacological, molecular/transgenic and imaging experiments that will enable us to gain a deeper and fuller understanding of the spatiotemporal control of vertebrate rod and cone synaptic terminal Ca2+ concentrations and mitochondrial ATP production as they relate to exocytosis and endocytosis. In addition, our results illustrate the need to identify and determine the spatial distribution, compartment-specific differences and interrelation of Ca2+-handling proteins and mitochondria in other neural tissues so that the structure-function relations in different neurons and chemical synapses can be ascertained. Moreover, additional new retinal experiments will provide important data on the role and sensitivity of rod and cone synaptic terminal mitochondria to Ca2+ overload and cell injury as compared to RIS and CIS mitochondria [In summary, rod spherules and cone pedicles have significantly different morphology, ultrastructure, mitochondrial density and location, patterns of protein expression, and regulatory mechanisms that control their [Cachondria ,40,182.