Avidin-horseradish Peroxidase Research Articles

Immunization with a Nontoxic/Nonfibrillar Amyloid-β Homologous Peptide Reduces Alzheimer's Disease-Associated Pathology in Transgenic Mice

Immunization with a Nontoxic/Nonfibrillar Amyloid-β Homologous Peptide Reduces Alzheimer's Disease-Associated Pathology in Transgenic Mice

Positional importance of Pro53 adjacent to the Arg49-Gly50-Asp51 sequence of rhodostomin in binding to integrin αIIbβ3

Rhodostomin (RHO), a disintegrin isolated from snake venom, has been demonstrated to inhibit platelet aggregation through interaction with integrin αIIbβ3, but there is a lack of direct evidence for RHO–integrin αIIbβ3 binding. In addition, no study on the length of Arg49-Gly50-Asp51 (RGD) loop of RHO influencing on its binding to integrin αIIbβ3 has been reported. In the present study we have developed a highly sensitive dot-blot and glutathione S-transferase–RHO pull-down assays; the latter was coupled with a biotin–avidin–horseradish peroxidase enhanced-chemiluminescence detection system. These were able to demonstrate the direct binding of RHO to integrin αIIbβ3. The pull-down assay further showed that four alanine-insertion mutants upstream of the RGD motif and three insertions downstream of the RGD were able to decrease integrin αIIbβ3 binding activity to only a limited extent. By contrast, two insertions immediately next to RGD and one insertion in front of the Cys57 caused almost complete loss of binding activity to αIIbβ3. The results of the platelet-aggregation-inhibition assay and platelet-adhesion assay for the insertion mutants were consistent with results of the pull-down assay. It is thus concluded that, although an insertion of a single alanine residue in many positions of the RGD loop has only minor effects on RHO binding to integrin αIIbβ3, the specific position of Pro53 residue adjacent to the RGD sequence is important for RHO binding to platelet integrin αIIbβ3.

Detection of thiol modification following generation of reactive nitrogen species: analysis of synaptic vesicle proteins

S-nitrosylation is an important means of regulating the activity of proteins. We have developed a method which allows unbiased identification of thiol modified proteins within a complex mixture following NO generation, by taking advantage of the fact that prior nitrosylation will block subsequent modification of cysteine residues with 1-biotinamido-4-[4′-(maleimidomethyl)-cyclohexane-carboxamido] butane (biotin-BMCC). Thiol modified proteins are reduced in intensity when revealed by blotting and overlay with avidin–horseradish peroxidase. In the case of a purified synaptic vesicle fraction we observe a high degree of enrichment of specific biotinylated proteins relative to homogenate. We find that thiol modification of proteins in the presence of NO donors is widespread, occurring in the majority of proteins that will react with biotin-BMCC. In a further development of this technique we have depleted the biotinylated proteins from solubilised synaptic vesicles using avidin–agarose and analysed the supernatants with a panel of antibodies. This has allowed us to identify SNARE proteins (soluble NSF attachment protein receptors) as potential targets for S-nitrosylation.

A method for detecting abasic sites in living cells: age-dependent changes in base excision repair.

Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) [Kubo, K., Ide, H., Wallace, S. S. & Kow, Y. W. (1992) Biochemistry 31, 3703-3708] is used to measure AP sites in living cells. ARP penetrates the plasma membrane of cells and reacts with AP sites in DNA to form a stable ARP-DNA adduct. The DNA is isolated and treated with avidin-horseradish peroxidase (HRP), forming a DNA-HRP complex at each biotin residue, which is rapidly separated from free avidin-HRP by selective precipitation with a DNA precipitating dye (DAPER). The number of AP sites is estimated by HRP activity toward chromogenic substrate in an ELISA assay. The assay integrates the AP sites formed by the different glycosylases of BER during a 1-h incubation and eliminates artifactual depurination or loss of AP sites during DNA isolation. The assay was applied to living cells and nuclei. The number of AP sites after a 1-h incubation in old IMR90 cells was about two to three times higher than that in young cells, and the number in human leukocytes from old donors was about seven times that in young donors. The repair of AP sites was slower in senescent compared with young IMR90 cells. An age-dependent decline is shown in the activity of the glycosylase that removes methylated bases in IMR90 cells and in human leukocytes. The decline in excision of methylated bases from DNA suggests an age-dependent decline in 3-methyladenine DNA glycosylase, a BER enzyme responsible for removing alkylated bases.

Production of functional human alpha 1-antitrypsin by plant cell culture.

Recombinant human alpha 1-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice alpha-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6-5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins.

Hemoglobin Endocytosis in Leishmania Is Mediated through a 46-kDa Protein Located in the Flagellar Pocket

Four lines of evidence indicate that a specific high affinity binding site on the surface of Leishmania donovani promastigotes mediates rapid internalization and degradation of hemoglobin. 1) Binding and uptake of 125I-hemoglobin by Leishmania followed saturation kinetics and were competed by unlabeled hemoglobin but not by globin or hemin or other heme- or iron-containing proteins. 2) Immunogold labeling studies revealed that, at 4 degreesC, hemoglobin binding was localized in the flagellar pocket of the promastigotes. Indirect immunofluorescence assays showed that, at 37 degreesC, the bound hemoglobin in such cells entered an endocytic compartment within 2 min and dispersed throughout the cell body by 15 min. 3) After incubation with hemoglobin-gold conjugates at 25 degreesC or 37 degreesC, the particles accumulated in discrete intracellular vesicles. 4) A single biotinylated protein of 46 kDa was revealed when solubilized membranes from surface biotinylated intact Leishmania adsorbed by hemoglobin-agarose beads were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting with avidin-horseradish peroxidase. Considered together, these data indicate that this 46-kDa protein on the cell surface of L. donovani promastigotes mediates the binding of hemoglobin and its rapid internalization through a vesicular pathway characteristic of receptor-mediated endocytosis.

Use of PCR in detection of Mycobacterium avium complex (MAC) bacteremia: sensitivity of the assay and effect of treatment for MAC infection on concentrations of human immunodeficiency virus in plasma.

We evaluated the sensitivity and specificity of a PCR-based qualitative test for the rapid diagnosis of Mycobacterium avium-M. intracellulare complex (MAC) bacteremia in patients with AIDS disease. Eleven subjects with newly culture-proven MAC bacteremia had the following tests performed at biweekly intervals during the first 8 weeks of therapy: blood culture, Mycobacterium-specific PCR, and quantitative human immunodeficiency virus (HIV) viral-load testing. Mycobacterium genus-specific biotinylated primers were used to amplify a sequence of approximately 582 nucleotides within the 16S rRNA genes of M. avium and M. intracellulare. Detection of the amplified product was performed with an oligonucleotide probe-coated microwell plate combined with an avidin-horseradish peroxidase-tetramethylbenzidine conjugate-substrate system. While not as sensitive as BACTEC culture, PCR detected 17 of 18 specimens which grew >/=40 organisms/ml (94.4% sensitivity) and 9 of 16 specimens which grew </=40 organisms/ml (56.3% sensitivity). No clear change in HIV viremia occurred in response to successful treatment of patients' MAC bacteremia. Use of the PCR test allowed detection of MAC bacteremia in 1 day, with a sensitivity similar to those of quantitative blood culture techniques, and it may prove useful for rapid screening of suspected cases. HIV viremia was unaffected by 8 weeks of MAC therapy.

Identification of ampullary and isthmic oviductal fluid proteins that associate with the bovine sperm membrane.

The objective of this study was to define the complement of ampullary and isthmic oviductal fluid proteins that associate with the sperm membrane during the non-luteal and luteal stages of the oestrous cycle. Oviductal fluid was obtained from three dairy cows via indwelling cannulae in the ampulla and isthmus of the same oviduct. Daily samples of oviductal fluid were combined by region and stage of the cycle to create pools consisting of non-luteal isthmic, luteal isthmic, non-luteal ampullary, and luteal ampullary oviductal fluid. An aliquot from each oviductal fluid pool was biotinylated by incubation in biotin reagent. Spermatozoa from three fertile bulls were pooled, washed and incubated for 4 h in each of the four pools of biotinylated oviductal fluid. After incubation, sperm membrane proteins were solubilized, and proteins subjected to one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biotinylated oviductal fluid proteins that associated with the sperm membranes were detected by avidin-horseradish peroxidase and diaminobenzidine substrate. Eight oviductal fluid proteins with apparent molecular weights of 97, 75, 66, 55, 48, 34, 28 and 24 kDa were consistently detected to associate with the sperm membrane. Differences in ampullar and isthmic fluid proteins that associated with sperm during the luteal and non-luteal stage of the oestrous cycle were not detected.

In vitro association of six oviductal fluid proteins with the bovine zona pellucida.

Oviductal fluid proteins have been shown to associate with gametes in several species. The objective of the present study was to identify bovine oviductal fluid proteins that associate with the bovine zona pellucida. Oviductal fluid was obtained daily from two dairy cows with normal oestrous cycles via indwelling oviductal cannulae. Fluid was collected from the ampullary and isthmic regions of the same oviduct. Oviductal fluid samples were pooled by oviduct region and according to stage of the oestrous cycle as determined by the concentration of serum progesterone. Samples collected when serum progesterone concentrations were > 1.5 ng ml-1 were combined into luteal pools. Non-luteal pools consisted of oviductal fluid samples collected on days when serum progesterone concentrations were < or = 1.5 ng ml-1. Each oviductal fluid sample was assayed fpr protein concentration, and an aliquot equivalent to 10 mg ml-1 was biotinylated using biotinamidocaproate N-hydroxysuccinimide ester at a concentration of 1 mg ml-1. Cumulus-free, non-viable bovine oocytes were incubated in the biotinylated oviductal fluid samples for 3.5 h. Oocytes were washed, the zonae mechanically ruptured, solubilized, and subjected to one-dimensional SDS-PAGE. Separated proteins were transferred to nitrocellulose and probed with avidin-horseradish peroxidase. Five biotinylated oviductal fluid proteins were found to associate with the zona pellucida in all treatments. These proteins had apparent molecular masses of 80, 74, 60, 45, and 30 kDa. An additional protein, of molecular mass 95 kDa, was found associated with zonae from oocytes incubated in non-luteal fluid of both regions, but not from oocytes incubated in luteal fluid. This protein was shown to be bovine oestrus-associated protein by using a monospecific polyclonal antibody.

Purification of Hemocyanin from White Shrimp (Penaeus vannamei Boone) by Immobilized Metal Affinity Chromatography

Hemocyanin (Hc) was isolated from white shrimp (Penaeus vannamei Boone) plasma by density gradient ultracentrifugation and immobilized metal affinity chromatography. Hc was contained in the subnatant high density fraction and was adsorbed by an iminodiacetic acid (Ni-IDA) column that bound Hc and apohemocyanin. Hc molecular weight was estimated by pore limiting electrophoresis as 400 kDa. It is composed of two subunits of approximately 75 and 82 kDa. This 400-kDa protein contained copper and was detected by antibodies raised against the purified subunits. Isoelectric points of the affinity purified native protein were 4.8 and 4.9. Presence of covalently bound carbohydrates in both subunits was detected by biotinylated lectins and avidin-horseradish peroxidase.

COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR

The COBAS AMPLICOR system automates amplification and detection of target nucleic acids, making diagnostic PCR routine for a variety of infectious diseases. The system contains a single thermal cycler with two independently regulated heating/cooling blocks, an incubator, a magnetic particle washer, a pipettor, and a photometer. Amplified products are captured on oligonucleotide-coated paramagnetic microparticles and detected with use of an avidin-horseradish peroxidase (HRP) conjugate. Concentrated solutions of amplicon or HRP were pipetted without detectable carryover. Amplified DNA was detected with an intraassay CV of < 4.5%; the combined intraassay CV for amplification and detection was < 15%. No cross-reactivity was observed when three different target nucleic acids were amplified in a single reaction and detected with three target-specific capture probes. The initial COBAS AMPLICOR menu includes qualitative tests for diagnosing infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and hepatitis C virus. All tests include an optional Internal Control to provide assurance that specimens are successfully amplified and detected.

A new reliable chemiluminescence immunoassay (CLIA) for prostaglandin E 2 using enhanced luminol as substrate

A sensitive and reliable chemiluminescence immunoassay suitable for the quantitative determination of prostaglandin E 2 (PGE 2) has been developed using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE 2 antibody (mouse), free antigen and solid phase bound antigen. The MTP was first coated with a bovine serum albumin (BSA)-PGE 2 conjugate. Then, after preincubating, the anti-PGE 2 antibody (Ab) and the analyte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE 2 conjugate. The monoclonal antibody captured on the MTP was determined using biotinylated antimouse-Ab and a complex of avidin and biotin-labelled horseradish peroxidase (HRP). Substrate for HRP was the cyclic diacyl hydrazide compound luminol, enhanced by p-iodophenol. Photons emitted during the reaction were measured using a photomultiplier tube. The assay has been validated with assay buffer and human plasma over a concentration range of 10–50,000 pglml. The lower limit of quantification is 100 pglml (2 pglwell) and 150 pglml (3 pglwell) for buffer and plasma, respectively. The intea-day coefficients of variation (CV) for the range of 100–50,000 pglml are 3.2–8.9% (buffer) and 4.2–17.7% (plasma) and inter-day CV are 2.9–19.8% (buffer) and 3.6–21.2% (plasma). The method can be used for quantification of PGE2 in biological fluids like plasma and suction blister fluid.

Lipoic acid (thioctic acid) analogs, tryptophan analogs, and urea do not interfere with the assay of biotin and biotin metabolites by high-performance liquid chromatography/avidin-binding assay

Lipoic acid, urea, and tryptophan show structural similarities to the vitamin biotin. If these compounds can successfully compete with biotin or a biotinylated protein for binding to avidin, their presence in serum and urine would cause artifacts in avidin-binding assays for biotin. We assessed the ability of lipoic acid and certain analogs, urea, and l-tryptophan and tryptophan derivatives to interfere with the measurement of 16 biotin analogs by a high-performance liquid chromatography (HPLC)/avidin-binding assay. In this assay, compounds are separated by reversed-phase HPLC, followed by assay of each fraction based on binding to avidin-horseradish peroxidase. At physiologic concentrations, neither lipoic acid analogs ( d-lipoate, l-lipoate, d,l-lipoamide, bisnorlipoate, β-hydroxybisnorlipoate, and tetranorlipoate) nor tryptophan derivatives exhibited detectable avidin-binding. Minor avidin-binding was seen for urea and l-tryptophan; binding ratios relative to biotin were 1 × 10 −9 and 3 × 10 −6, respectively. Urea did not co-elute on HPLC with any of the 16 biotin analogs, but l-tryptophan did co-elute with bisnorbiotin methyl ketone. However, because the relative avidin affinity of l-tryptophan is five orders of magnitude smaller than that of bisnorbiotin methyl ketone, we conclude that none of the tested compounds are likely to interfere with the measurement of biotin or its metabolites by an HPLC/avidin-binding assay.

Detection of enteroviruses by polymerase chain reaction in urine samples of patients with aseptic meningitis.

Detection of enteroviruses by polymerase chain reaction in urine samples of patients with aseptic meningitis.

Trypanosome-binding proteins of the tsetse flies Glossina palpalis gambiensis and G. morsitans morsitans

In this paper we describe a new, selective approach to identify protein ligand-receptor interactions between an arthropod vector and the parasite it transmits. Biotinylated vector proteins were incubated with living parasites in physiological conditions. After extensive washing, the parasites were subjected to SDS-PAGE electrophoresis and the polypeptides were electroblotted onto nitrocellulose membrane. Staining with avidin-horseradish peroxidase revealed only biotin-labelled proteins from the vector which were bound to the parasite. A multitude of tissue-specific proteins of Glossina palpalis gambiensis and G. morsitans morsitans proteins, able to bind to cultured procyclic trypanosomes of Trypanosoma brucei spp., has been demonstrated. The relevance of these interactions in relation to the developmental journey of the trypanosome in the tsetse fly is briefly discussed.

Functional morphologies of retinal ganglion cells in the turtle.

Retinal ganglion cells in the turtle, Pseudemys scripta elegans, were examined by intracellular recording with a protocol of stationary and moving lights. Responses were apportioned among OFF, ON, and ON-OFF categories, and directional selectivity. Cells were injected with Neurobiotin, then later conjugated with avidin-horseradish peroxidase in standard procedure. Morphological analysis of the stained cells included measurements of soma and dendritic field sizes, dendritic stratification, number of cell processes, dendritic branchings, and dendritic symmetry ratios. ON and ON-OFF cells are at least bistratified, sometimes tristratified, in both sublaminae A and B whether directionally selective or not. OFF cells, in contrast, are monostratified, or at least confined to sublamina A. Morphological parameters of somal and dendritic field areas, branch point densities, and dendritic field asymmetries do not predict directional selectivity. Membrane polarization accompanying moving stimulation is discussed in terms of shunting inhibition and recording site.

Relationships between morphology and physiology of pyramid-pyramid single axon connections in rat neocortex in vitro.

1. Double intracellular recordings were made from 1163 pairs of pyramidal neurones in layer V-VI of the rat somatomotor cortex in vitro using sharp electrodes filled with biocytin. Monosynaptically connected pairs of cells were identified when an action potential in one could elicit a constant latency excitatory postsynaptic potential (EPSP) in the other and the cells were filled with biocytin. Labelled cells were subsequently identified histologically with avidin-horseradish peroxidase. 2. Thirty-four pairs of cells were found to be monosynaptically connected. Fifteen of these pairs were sufficiently stable for electrophysiological recordings and three of these were recovered sufficiently to permit full morphological reconstruction. 3. The EPSP recorded between the first pair of pyramids varied in amplitude between 0 and 3 mV (mean 1.33 +/- 1.06 mV) and fluctuated considerably (coefficient of variation, 0.796). This was largely due to a high incidence of apparent failures of transmission. On reconstruction two boutons from the presynaptic pyramid axon were in close apposition to the proximal portions of basal dendrites of the postsynaptic cell. 4. In the second pair of pyramids the EPSP had a mean amplitude of 1.06 mV, and displayed a 10-90% rise time of 2.8 ms and a width at half-amplitude of 23 ms. This EPSP did not alter significantly with changes in membrane potential at the soma. The presynaptic axon closely apposed the distal apical dendrite of the postsynaptic cell in eight places. 5. In the third pair of pyramids, the EPSPs, recorded at a relatively depolarized membrane potential, were long lasting and could elicit slow dendritic spikes with long and variable latencies. These slow spikes suggested that the postsynaptic recording site was dendritic and on reconstruction a possible location was identified on the apical dendrite. A total of five presynaptic boutons closely apposed three separate, proximal branches of the postsynaptic apical dendrite. 6. These results provide the first illustration of a morphological basis for variations in functional properties of pyramid-pyramid connections in the neocortex.

Detection of botulinum toxin using an evanescent wave immunosensor

Using fluorescein isothiocyanate (FITC)-streptavidin, quartz fibre-immobilized antibody (FiAb) and the evanescent wave component of a light beam, detection of Botulinum Toxin-B (BoTX) is described. Exposure of 3-aminopropyltriethoxysilane/glutaraldehyde (APTS/GA) treated quartz fibres to increasing amounts of anti-BoTX Ab indicated toxin binding to increase in a linear fashion up to approximately 125 ng added Ab. Quantitation of bound BoTX and FiAb by Dot-Blot analysis using avidin-Horseradish peroxidase (HRP) conjugation indicated the presence of 0·27 and 0·67 pmoles, respectively. Inclusion of nonbiotinylated BoTX in sampling mixtures reduced fluorescence in a dose-dependent manner over a narrow concentration range (0–300 ng). Exposure of FiAb to a variety of venoms resulted in no reduction of BoTX binding suggesting detection of BoTX via immobilized anti-BoTX Ab to be very specific.

Human coronavirus OC43 interacts with major histocompatibility complex class I molecules at the cell surface to establish infection.

Human coronaviruses have been associated with common colds, diarrhea and enterocolitis, and have been implicated in multiple sclerosis. HLA class I molecules may play a critical role as receptor for OC43 because monoclonal antibody (mAb)W6/32 to HLA-A, -B and -C specificities completely blocks infectivity in human rhabdomyosarcoma (RD) cells. The role of HLA class 1 antigen as the virus receptor was examined using HLA-A3.1 stably transfected human plasma cells and untransfected HMY.C1R cells which do not express HLA-A and -B molecules. When the cells (5 x 10(6)) were infected at a multiplicity of one, the HLA.A3 transfected cells produced 10(8) PFU of virus whereas no replication occurred in the HMY.C1R cells mAb W6/32 reduced the virus yield by 99.9%. Cell membranes from HMY.C1R, HMY.A3 cells and chicken erythrocytes were biotinylated as live cells. Immunoprecipitation with polyclonal antiviral antibody to detect binding of biotinylated cell membranes to virus revealed that biotinylated HMY.A3 membranes coprecipitated with virus-antibody complexes when the immunoprecipitates were electrophoresed on SDS-PAGE gel, electroblotted and stained with Avidin-horseradish peroxidase. The results provide direct evidence that OC43 virus can recognize HLA class I as receptor on the cell surface.

Large, deep layer pyramid-pyramid single axon EPSPs in slices of rat motor cortex display paired pulse and frequency-dependent depression, mediated presynaptically and self-facilitation, mediated postsynaptically

1. In slices of rat sensorimotor cortex, dual intracellular recordings were obtained from 1,952 pairs of deep layer pyramidal neurons. Where action potentials in one neurone elicited excitatory postsynaptic potentials (EPSPs, n = 56) in the other, responses to different presynaptic firing rates and patterns and at different postsynaptic membrane potentials were recorded and on some occasions both neurons were filled with biocytin. 2. Slices were fixed, sectioned again at 60 microns, and incubated with Avidin horseradish peroxidase (HRP), which was then visualized using the 3,3'-diaminobenzidine tetrahydrochloride (DAB) method. All neurones reported here that were identified histologically were pyramidal cells with their somata in the deep layers (V and VI). 3. One in 70 of the tests performed revealed a synaptic connection, 25 of which were studied in detail. Mean EPSP amplitude was 1.67 +/- 1.66 (SD) mV, with some single sweep events as large as 9 mV. For some of the smaller EPSPs the amplitude distributions contained a clear peak around 0 mV, the coefficient of variation (CV) was large, and paired pulse facilitation was apparent. EPSPs with large average amplitudes displayed no apparent failures of transmission, EPSP amplitudes were fairly evenly distributed around the mean, CVs were small, and paired pulse depression was apparent in 2.5 mM extracellular Ca2+. When single sweeps were selected according to the size of the first EPSP, large second EPSPs were found to follow small first EPSPs and small second EPSPs to follow large first EPSPs. Paired pulse effects appeared, in the majority of tests, to be due to a change in presynaptic release probability. 4. Two EPSPs were recorded in three different extracellular Ca2+ concentrations. In 1 mM Ca2+, the first EPSP of a short interval pair was small and paired pulse facilitation was apparent. In 5 mM Ca2+, first EPSPs were between 2.5 and 4 times larger than in 1 mM Ca2+ and paired pulse depression was apparent. In all Ca2+ concentrations however, averaged third and fourth EPSPs of brief bursts were of similar amplitudes and smaller than second EPSPs. If presynaptic inhibition does contribute to paired pulse effects here, it is not overcome by a combination of raised extracellular Ca2+ and repetitive presynaptic firing. 5. These EPSPs displayed a wide range of time courses. The mean 10-90% rise time was 2.49 +/- 1.08 ms, the mean width at half amplitude was 15.39 +/- 5.42 ms (n = 22), and the mean EPSP latency was 1.59 +/- 0.68 ms (n = 18). (ABSTRACT TRUNCATED AT 400 WORDS)