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Biomedical subjects

S M Wu

Publications and source records attributed to S M Wu.

At least 37 records · Page 2Linked to original sources
PubMed2001

Apoptotic cell death of cultured salamander photoreceptors induced by cccp: CsA-insensitive mitochondrial permeability transition.

Photoreceptor degeneration is mediated by apoptosis in several animal models, although the underlying mechanisms are yet to be elucidated. We present here an apoptotic model based on a primary cell culture of tiger salamander photoreceptors, in which treatment with carbonyl cyanide m-chlorophenylhydrazone (cccp), a protonophore, induced apoptosis. Cells exposed to cccp showed condensed nuclei and displayed positive TdT-dUTP terminal nick-end labeling (TUNEL). In addition, 10-100 microM cccp rapidly induced a reduction of Delta psi(m) and > or = 30 microM cccp induced a significant leakage of calcein from mitochondria to cytosol and nucleus, indicating a change in mitochondrial inner membrane permeability. Cyclosporin A (CsA), a transition pore blocker, did not prevent the cccp-induced MPT or the cccp-evoked apoptotic cell death, suggesting that cccp-induced apoptotic process was mediated by a CsA-insensitive pathway. This cell model provides an in vitro tool for studying mechanisms of photoreceptor apoptosis in isolated photoreceptors and may provide clues to the etiology of retinal degeneration.

Animals↗
PubMed2000

Expression and characterization of the naturally occurring mutation L394R in human gamma-glutamyl carboxylase.

Patients with mutation L394R in gamma-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IX's propeptide and vitamin KH(2). Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K(i) for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V(max)/K(m) toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K(m) for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K(m) for vitamin KH(2). The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH(2) binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.

Amino Acid Substitution↗
PubMed2000

Morphological and electrophysiological properties of dissociated primate retinal cells.

Although isolated retinal cell preparations have been used widely to study retinal function in lower vertebrates, dissociated cells from primate retina have not been developed for routine physiological experiments. In this study, we demonstrated the feasibility of obtaining viable and identifiable dissociated cells from the primate retina. In addition, we characterized voltage-dependent membrane currents in each type of primate retinal cell with the whole-cell patch-clamp technique. Multiple types of ionic conductance with distinctive current profiles were recorded in various types of primate retinal neurons. Photoreceptors exhibited an inward I(H) activated by membrane hyperpolarization and an outward current activated at depolarized potentials. Two types of potassium currents (transient potassium current, I(K(A)), and delayed rectifier potassium current, I(K(V))) were recorded from bipolar cells. I(K(A)) dominated the current response in putative midget bipolar cells, and I(K(V)) was mainly associated with putative rod bipolar cells. L-type calcium currents (I(Ca)) were observed in primate bipolar cells with axon terminals, but not in axotomized bipolar cells. Large voltage-dependent sodium currents (I(Na)) were only recorded from ganglion cells. Muller cells exhibited I(K(V)) and large potassium inward rectifier current (I(K(IR))), and occasionally a small I(Na). Neurons with electrophysiological signatures of amacrine cells and horizontal cells were also studied even though their morphological features were lost during cell dissociation. By using both morphological and physiological criteria outlined in this report, it is possible to use the dissociated retinal cell preparation as an in vitro system for physiological, biochemical and pharmacological studies of the primate visual system.

Amacrine Cells↗
PubMed2000

The conformation-dependent interaction of alpha 2-macroglobulin with vascular endothelial growth factor. A novel mechanism of alpha 2-macroglobulin/growth factor binding.

alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these factors is poorly understood. We examined in detail the interaction between alpha(2)M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to alpha(2)M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of alpha(2)M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated alpha(2)M and to the exterior of native and proteinase-converted alpha(2)M; and 3) VEGF (molecular mass approximately 40 kDa) can access the interior of receptor-recognized alpha(2)M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of alpha(2)M is internalized and degraded by macrophages via the alpha(2)M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized alpha(2)M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of alpha(2)M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca(2+) levels. We demonstrate that under physiological conditions, alpha(2)M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca(2+).

Binding, Competitive↗
PubMed2000

A topological study of the human gamma-glutamyl carboxylase.

gamma-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K-dependent posttranslational modification of glutamate to gamma-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

Animals↗
PubMed2000

Functional architecture of synapses in the inner retina: segregation of visual signals by stratification of bipolar cell axon terminals.

We correlated the morphology of salamander bipolar cells with characteristics of their light responses, recorded under voltage-clamp conditions. Twelve types of bipolar cells were identified, each displaying a unique morphology and level(s) of axon terminal stratification in the inner plexiform layer (IPL) and exhibiting light responses that differed with respect to polarity, kinetics, the relative strengths of rod and cone inputs, and characteristics of spontaneous EPSCs (sEPSCs) and IPSCs. In addition to the well known segregation of visual information into ON and OFF channels along the depth of the IPL, we found an overlying mapping of spectral information in this same dimension, with cone signals being transmitted predominantly to the central IPL and rod signals being sent predominantly to the margins of the IPL. The kinetics of bipolar cell responses correlated with this segregation of ON and OFF and of rod and cone information in the IPL. At light offset, rod-dominated cells displayed larger slow cationic current tails and smaller rapid overshoot responses than did cone-dominated cells. sEPSCs were generally absent in depolarizing bipolar cells but present in all hyperpolarizing bipolar cells (HBCs) and larger in rod-dominated HBCs than in cone-dominated HBCs. Inhibitory chloride currents, elicited both at light onset and light offset, tended to be larger for cone-dominated cells than for rod-dominated cells. This orderly segregation of visual signals along the depth of the IPL simplifies the integration of visual information in the retina, and it begins a chain of parallel processing in the visual system.

Ambystoma↗
PubMed2000

Luteinizing hormone receptor mutations in disorders of sexual development and cancer.

Human male sexual development is regulated by chorionic gonadotropin (CG) and luteinizing hormone (LH). Aberrant sexual development caused by both activating and inactivating mutations of the human luteinizing hormone receptor (LHR) have been described. All known activating mutations of the LHR are missense mutations caused by single base substitution. The most common activating mutation is the replacement of Asp-578 by Gly due to the substitution of A by G at nucleotide position 1733. All activating mutations are present in exon 11 which encodes the transmembrane domain of the receptor. Constitutive activity of the LHR causes LH releasing hormone-independent precocious puberty in boys and the autosomal dominant disorder familial male-limited precocious puberty (FMPP). Both germline and somatic activating mutations of the LHR have been found in patients with testicular tumors. Activating mutations have no effect on females. The molecular genetics of the inactivating mutations of the LHR are more variable and include single base substitution, partial gene deletion, and insertion. These mutations are not localized and are present in both the extracellular and transmembrane domain of the receptor. Inactivation of the LHR gives rise to the autosomal recessive disorder Leydig cell hypoplasia (LCH) and male hypogonadism or male pseudohermaphroditism. Severity of the clinical phenotype in LCH patients correlates with the amount of residual activity of the mutated receptor. Females are less affected by inactivating mutation of the LHR. Symptoms caused by homozygous inactivating mutation of the LHR include polycystic ovaries and primary amenorrhea.

Disorders of Sex Development↗
PubMed2000

Down-regulation by extracellular ATP of rat hepatocyte organic anion transport is mediated by serine phosphorylation of oatp1.

Recent studies implicate a role in hepatocyte organic anion transport of a plasma membrane protein that has been termed oatp1 (organic anion transport protein 1). Little is known regarding mechanisms by which its transport activity is modulated in vivo. In previous studies (Campbell, C. G., Spray, D. C., and Wolkoff, A. W. (1993) J. Biol. Chem. 268, 15399-15404), we demonstrated that hepatocyte uptake of sulfobromophthalein was down-regulated by extracellular ATP. We have now found that extracellular ATP reduces the V(max) for transport of sulfobromophthalein by rat hepatocytes; K(m) remains unaltered. Reduced transport also results from incubation of hepatocytes with the phosphatase inhibitors okadaic acid and calyculin A. Immunoprecipitation of biotinylated cell surface proteins indicates that oatp1 remains on the cell surface after exposure of cells to ATP or phosphatase inhibitor, suggesting that loss of transport activity is not caused by transporter internalization. Exposure of (32)P-loaded hepatocytes to extracellular ATP results in serine phosphorylation of oatp1 with the appearance of a single major tryptic phosphopeptide; oatp1 from control cells is not phosphorylated. This phosphopeptide comigrates with one of four phosphopeptides resulting from incubation of cells with okadaic acid. These studies indicate that the phosphorylation state of oatp1 must be an important consideration when assessing alterations of its functional expression in pathobiological states.

Adenosine Triphosphate↗
PubMed2000

Family size, miscarriage-proneness, and handedness: tests of hypotheses of the developmental instability theory of handedness.

The handedness theory of Yeo and Gangestad (1993) posits that moderate right-handedness is the modal manifestation of genetic handedness and that ''developmental instability'' produces deviations from modal handedness. It is also suggested (1) that sinistral parents may produce fewer offspring than do dextral parents; and (2) that sinistral mothers may be more prone to miscarriages than are dextral mothers. In line with these hypotheses, Gangestad et al. (1996) reported that a human leukocyte antigen (B8) was related to both left-handedness and to reduced offspring number in their study. They also found that left-handedness was related to the human leukocyte antigen DR3, and Yeo and Gangestad (1998) noted that this antigen has been found by Christiansen et al. (1996) to be associated with an increased risk of spontaneous abortion in women. We assessed the first hypothesis through a study of the family sizes of 2083 families with two right-handed parents and 502 families having one or more left-handed parents; we assessed the second hypothesis from miscarriage history data supplied by 300 dextral and 52 sinistral mothers. Results supported the developmental instability theory with respect to the hypothesis regarding family size, but not with respect to the hypothesis regarding miscarriage-proneness.

Journal Article↗
PubMed2000

Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamases and identification of a novel AmpC enzyme (CMY-8) in Southern Taiwan.

Twenty (8.5%) of 234 nonrepetitive clinical isolates of Klebsiella pneumoniae from southern Taiwan were found to produce extended-spectrum beta-lactamases (ESBLs): 10 strains produced SHV-12, 4 produced SHV-5, 2 produced a non-TEM non-SHV ESBL with a pI of 8.3, 3 produced a novel AmpC beta-lactamase designated CMY-8 with a pI of 8.25, and 1 produced SHV-12 and an unidentified AmpC enzyme with a pI of 8.2. The CMY-8 enzyme confers a resistance phenotype similar to CMY-1 and MOX-1, and sequence comparisons showed high homologies (>95%) of nucleotide and amino acid sequences among these three enzymes. Plasmid and pulse-field gel electrophoresis analyses revealed that all isolates harboring an SHV-derived ESBL were genetically unrelated, indicating that dissemination of resistance plasmids is responsible for the spread of SHV ESBLs among K. pneumoniae in this area. All three isolates carrying CMY-8 had identical genotypic patterns, suggesting the presence of an epidemic strain.

Amino Acid Sequence↗
PubMed2000

I4AA-Sensitive chloride current contributes to the center light responses of bipolar cells in the tiger salamander retina.

Light-evoked currents in depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) were recorded under voltage-clamp conditions in living retinal slices of the larval tiger salamander. Responses to illumination at the center of the DBCs' and HBCs' receptive fields were mediated by two postsynaptic currents: DeltaI(C), a glutamate-gated cation current with a reversal potential near 0 mV, and DeltaI(Cl), a chloride current with a reversal potential near -60 mV. In DBCs DeltaI(C) was suppressed by L-2-amino-4-phosphonobutyric acid (L-AP4), and in HBCs it was suppressed by 6,7-dinitroquinoxaline-2,3-dione (DNQX). In both DBCs and HBCs DeltaI(Cl) was suppressed by imidazole-4-acetic acid (I4AA), a GABA receptor agonist and GABA(C) receptor antagonist. In all DBCs and HBCs examined, 10 microM I4AA eliminated DeltaI(Cl) and the light-evoked current became predominately mediated by DeltaI(C). The addition of 20 microM L-AP4 to the DBCs or 50 microM DNQX to HBCs completely abolished DeltaI(C). Focal application of glutamate at the inner plexiform layer elicited chloride currents in bipolar cells by depolarizing amacrine cells that release GABA at synapses on bipolar cell axon terminals, and such glutamate-induced chloride currents in DBCs and HBCs could be reversibly blocked by 10 microM I4AA. These experiments suggest that the light-evoked, I4AA-sensitive chloride currents (DeltaI(Cl)) in DBCs and HBCs are mediated by narrow field GABAergic amacrine cells that activate GABA(C) receptors on bipolar cell axon terminals. Picrotoxin (200 microM) or (1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid (TPMPA) (2 other GABA(C) receptor antagonists) did not block (but enhanced and broadened) the light-evoked DeltaI(Cl), although they decreased the chloride current induced by puff application of GABA or glutamate. The light response of narrow field amacrine cells were not affected by I4AA, but were substantially enhanced and broadened by picrotoxin. These results suggest that there are at least two types of GABA(C) receptors in bipolar cells: one exhibits stronger I4AA sensitivity than the other, but both can be partially blocked by picrotoxin. The GABA receptors in narrow field amacrine cells are I4AA insensitive and picrotoxin sensitive. The light-evoked DeltaI(Cl) in bipolar cells are mediated by the more strongly I4AA-sensitive GABA(C) receptors. Picrotoxin, although acting as a partial GABA(C) receptor antagonist in bipolar cells, does not suppress DeltaI(Cl) because its presynaptic effects on amacrine cell light responses override its antagonistic postsynaptic actions.

Ambystoma↗
PubMed2000

Effects of betaxolol on light responses and membrane conductance in retinal ganglion cells.

PURPOSE: To examine the physiological effects of betaxolol, a beta1-adrenergic receptor blocker commonly used in the treatment of glaucoma, on retinal ganglion cells and to evaluate its potential to elicit responses consistent with a neuroprotective agent against ganglion cell degeneration. METHODS: Single-unit extracellular recording, electroretinogram (ERG), intracellular and whole-cell patch-clamp recording techniques were made from flatmounted, isolated retina, superfused eyecup, and living retinal slice preparations of the larval tiger salamander. RESULTS: Bath application of 20 microM betaxolol reduced the glutamate-induced increase of spontaneous spike rate in retinal ganglion cell by approximately 30%. The glutamate-induced postsynaptic current recorded under voltage-clamp conditions was reduced by 50 microM betaxolol, and the difference current-voltage (I-V) relation (I(Control)-I(betaxolol)) was N-shaped and AP5-sensitive, characteristic of N-methyl-D-aspartate receptor-mediated current. Application of 50 microM betaxolol reversibly reduced the voltage-gated sodium and calcium currents by approximately one third of their peak amplitudes. The times-to-action of betaxolol on ganglion cells are long (15-35 minutes for 20-50 microM betaxolol), indicative of modulation through slow biochemical cascades. Betaxolol, up to 100 microM, exerted no effects on horizontal cells or the ERG, suggesting that the primary actions of this beta1 blocker are restricted to retinal ganglion cells. CONCLUSIONS: These physiological experiments provide supporting evidence that betaxolol acts in a manner consistent with preventing retinal ganglion cell death induced by elevated extracellular glutamate or by increased spontaneous spike rates under pathologic conditions. The physiological actions of betaxolol lead to reducing neurotoxic effects in ganglion cells, which are the most susceptible retinal neurons to glutamate-induced damages under ischemic and glaucomatous conditions. Therefore, betaxolol has the potential to be a neuroprotective agent against retinal degeneration in patients with disorders mediated by such mechanisms.

Adrenergic beta-Antagonists↗
PubMed1999

Glutamate receptors differ in rod- and cone-dominated off-center bipolar cells.

The axon morphology of off-center bipolar cells in salamander retinal slices was correlated with light responses and dendritic glutamate responses of these cells under voltage clamp. Cells with telodendria ramifying exclusively in the distal one-quarter of the inner plexiform layer were rod dominated, whereas cells with more centrally ramifying telodendria were cone dominated. The glutamate responses of the centrally ramifying cells displayed more apparent desensitization and a smaller underlying single channel conductance (1.2+/-0.3 pS) than did the distally ramifying cells (2.8+/-0.4 pS), although the responses for both classes of cells were strongly enhanced by cyclothiazide. These results suggest that different subtypes of AMPA receptors are present on rod- and cone-dominated cells, and that these may differ with respect to desensitization kinetics.

Ambystoma↗
PubMed1999

Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha 2-macroglobulin.

The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.

Arthritis, Rheumatoid↗
PubMed1999

Trace analysis of haloperidol and its chiral metabolite in plasma by capillary electrophoresis.

Capillary zone electrophoresis was developed for the simultaneous determination of haloperidol (HP) and its chiral metabolites [(+)- and (-)- reduced haloperidol, (+)- and (-)-RHP] in human plasma. The method involved the presence of an internal standard and liquid-liquid extraction from plasma. After concentration, the residue from the organic extract was dissolved in aqueous acid for capillary electrophoretic analysis. The background electrolyte was Tris-phosphate buffer with dimethyl-beta-cyclodextrin and PEG 6000. In spiked plasma the quantitative ranges were 40-400 nM for HP and 50-500 nM for (+)-RHP or (-)-RHP. The intra-day and inter-day relative standard deviations (n = 3) were all < 20% for each substance. The detection limits were found to be 15 ng/ml for HP and 30 ng/ml for both enantiomers of RHP (S/N = 3, injection 20 s). All recoveries were > 70%. We investigated the in vivo metabolism of HP in Chinese schizophrenia patients. The results show that (-)-RHP seems to be the only chiral metabolite from these two HP-dosed patients.

Electrophoresis, Capillary↗
PubMed1999

Modulation of glycine receptors in retinal ganglion cells by zinc.

Effects of zinc, an endogenous neuromodulator in the central nervous system, on glycine receptors (GlyRs) in retinal ganglion cells were investigated by using the whole-cell voltage-clamp technique. Zn2+ at low concentration (<2 microM) potentiated the glycine-induced chloride current and at higher concentration (>10 microM) suppressed it. This biphasic regulatory action of zinc acted selectively on the fast component of the glycine-induced current mediated by the strychnine-sensitive GlyRs, but not on the slow component mediated by the 5,7-dichlorokynurenic acid-sensitive GlyRs. Dose-response studies showed that 1 microM Zn2+ increased the maximum glycine response (I approximately) and shifted the EC50 to the left, suggesting that Zn2+ at low concentrations acts as an allosteric activator of the strychnine-sensitive GlyRs. Zn2+ at a concentration of 100 microM did not alter I approximately and shifted the EC50 to the right, indicating that Zn2+ at high concentrations acts as a competitive inhibitor of the GlyRs. Physiological functions of zinc modulation of GlyRs in retinal ganglion cells are discussed.

Ambystoma↗
PubMed1999

Multiple types of spontaneous excitatory synaptic currents in salamander retinal ganglion cells.

Spontaneous and light-evoked excitatory postsynaptic currents (sEPSCs and leEPSCs) in retinal ganglion cells of the larval tiger salamander were recorded under voltage clamp conditions from living retinal slices. sEPSCs were isolated from the spontaneous inhibitory postsynaptic currents (sIPSCs) by application of 100 M picrotoxin+1 microM strychnine. In addition to the previously reported sEPSCs [K. Matsui, N. Hosoi, M. Tachibana, Excitatory synaptic transmission in the inner retina: pair recordings of bipolar cells and neurons of the ganglion cell layer, J. Neurosci. 18 (1998) 4500-4510; W.R. Taylor, E. Chen, D.R. Copenhagen, Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells, J. Physiol. 486 (1995) 207-221] [which are equivalent to our fast AMPA receptor-mediated sEPSCs (fAMPAsEPSCs)], we found another type of AMPA receptor-mediated sEPSC with slower rise and decay time courses and larger peak amplitudes (sAMPAsEPSCs), and the NMDA receptor-mediated sEPSCs (NMDAsEPSCs) in ON-OFF ganglion cells. The frequency of all three types of sEPSCs is greatly reduced by cobalt (with zero calcium) and increased by hyperosmotic solution, suggesting that these events are mediated by calcium-dependent exocytosis of glutamatergic synaptic vesicles. The amplitude histograms of sEPSCs do not show multiple peaks, suggesting that larger events are not discrete multiples of elementary events, or quanta, of similar neurotransmitter contents, as in the neuromuscular junction [P. Fatt, B. Katz, Spontaneous subthreshold activity at motor nerve endings, J. Physiol. 117 (1952) 109-128]. The average I-V relations of the fAMPAsEPSCs and sAMPAsEPSCs were outward rectified with reversal potentials at -12.2 mV and -10.8 mV, and that of the NMDAsEPSCs was N-shaped with a reversal potential at -5.8 mV. The average conductance increase associated with a single fAMPAsEPSC, a single sAMPAsEPSC, and a single NMDAsEPSC were 163. 26+/-51.02 pS, 233.33+/-163.64 pS, and 37.5+/-50.0 pS at -110 mV; 241.67+/-22.92 pS, 444.90+/-469.94 pS, and 25.93+/-70.37 pS at -60 mV; and 440.48+/-183.33 pS, 1,192.68+/-651.22 pS, and 517.71+/-238. 24 pS at +30 mV, respectively. The average frequency of the three sEPSCs at +30 mV were 15 Hz, 3.7 Hz and 3.6 Hz, respectively. The rise time (time to peak) of fAMPAsEPSCs was 1.5+/-1.05 ms and the decay time could be fitted with a single exponential with an average time constant of 3.4+/-4.1 ms. The rise and decay time course of the sAMPAsEPSCs and NMDAsEPSCs were much slower and sawtooth-shaped, and each 'sawtooth' had time course and amplitude similar to those of individual fAMPAsEPSCs. We propose that each fAMPAsEPSC is mediated by single or synchronized multiples of glutamatergic synaptic vesicles from bipolar cells, and each sAMPAsEPSC or NMDAsEPSC is mediated by larger clusters of synaptic vesicles triggered by spontaneous calcium spikes in bipolar cell axon terminals [J. Burrone, L. Lagnado, Electrical resonance and calcium influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina, J. Physiol. 505 (1997) 571-584; D. Zenisek, G. Matthews, Calcium action potentials in retinal bipolar neurons, Vis. Neurosci. 15 (1998) 69-75].

Action Potentials↗