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J D Clements

Publications and source records attributed to J D Clements.

At least 37 records · Page 2Linked to original sources
PubMed2000

Functional mapping of protective domains and epitopes in the rotavirus VP6 protein.

The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed in Escherichia coli was examined. Mice (BALB/c) immunized with two 9-microg doses of each of the chimeras and 10 microg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P </= 0.01) following challenge. Because CD produced almost complete protection, we prepared four E. coli-expressed, MBP-fused chimeras containing overlapping fragments of the CD region (i.e., CD1, CD2, CD3, and CD4) whose lengths ranged from 61 to 67 amino acid residues. Following i.n. immunization, CD1, CD2, and CD4 induced significant (P </= 0.004) protection (88, 84, and 92% reduction, respectively). In addition, 11 peptides (18 to 30 residues) of the CD region with between 0 and 13 overlapping amino acids were synthesized. Two 50-microg doses of each peptide with LT(R192G) were administered i.n. to BALB/c mice. Five peptides were found to elicit significant (P </= 0.02) protection. Moreover, a 14-amino-acid region within peptide 6 containing a putative CD4(+) T-cell epitope was found to confer nearly complete protection, suggesting a protective role for CD4(+) T cells. Mice that were protected by fragments BC and CD1 and four of the five protective synthetic peptides did not develop measurable rotavirus antibodies in serum or stool, implying that protection induced by these domains was not dependent on antibody. Together, these observations suggest that multiple regions of VP6 can stimulate protection, a region of VP6 as small as 14 amino acids containing a CD4(+) T-cell epitope can stimulate nearly complete protection, and protection mediated by a subset of epitopes in the VP6 protein does not require antibodies in BALB/c mice.

Amino Acid Sequence↗
PubMed1999

The role of cAMP in mucosal adjuvanticity of Escherichia coli heat-labile enterotoxin (LT).

Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) produced by Vibrio cholerae have been shown to function as potent mucosal adjuvants. A number of studies have examined the effects of different mutations at either the active site or the protease site of LT and CT and the influence of those mutations on toxicity and adjuvanticity. However, different observations reported by various groups using a variety of animal models with different antigens or different routes of immunization have provided contradictory findings and evoked many questions regarding the underlying mechanisms of mucosal adjuvanticity of LT and CT. In this study, the role of cAMP in mucosal adjuvanticity was examined by comparing three LT active site mutants (S61F, A69G, E112K), a protease site mutant (R192G) and recombinant LT-B for toxicity, cAMP activity and mucosal adjuvanticity using tetanus toxoid (TT) as a model antigen. While all mutants examined showed reduced toxicity, the effects of each mutation on its ability to function as an adjuvant varied. Following intranasal immunization, native LT as well as protease and active site mutants of LT induced serum anti-TT IgG and their responses were virtually indistinguishable from one another. In addition, LT-B was also able to enhance production of serum anti-TT IgG, though at a level significantly lower than that achieved by native LT and mutants. Following oral immunization, the best serum anti-TT IgG responses were obtained with native LT and mutants that retained the ability to induce accumulation of cAMP. Despite the nearly identical serum anti-TT IgG responses following intranasal immunization, there was a strong correlation between the ability to induce accumulation of cAMP in cultured Caco-2 cells and the ability to elicit production of antigen-specific Th1 or Th2 cytokines.

Adjuvants, Immunologic↗
PubMed1999

Postsynaptic expression of long-term potentiation in the rat dentate gyrus demonstrated by variance-mean analysis.

1. Long-term potentiation (LTP) of synaptic transmission is the putative mechanism underlying learning and memory. Despite intensive study, it remains controversial whether LTP is expressed at a pre- or postsynaptic locus. A new approach was used to investigate this question at excitatory synapses from the medial perforant path (MPP) onto granule cells in the hippocampal dentate gyrus. The variance of the evoked synaptic amplitude was plotted against mean synaptic amplitude at several different Cd2+ concentrations. The slope of the variance-mean plot estimates the average amplitude of the response following the release of a single vesicle of transmitter (Qav). A presynaptic modulation should not affect Qav, but a postsynaptic modulation should alter it. 2. The variance-mean technique was tested by applying the analysis before and after three different synaptic modulations: (i) a reduction in Qav by the addition of the competitive antagonist CNQX; (ii) a reduction in the average probability of transmitter release (Pav) by the addition of baclofen; and (iii) an increase in the number of active synaptic terminals (N) by increasing the stimulus strength. CNQX reduced the average synaptic amplitude and Qav to the same extent, consistent with a postsynaptic action. In contrast, neither a change in N nor Pav altered Qav. This confirms that the variance-mean technique can distinguish between a pre- and a postsynaptic site of modulation. 3. Induction of LTP increased EPSC amplitude by 50 +/- 0.4 % (n = 5) and, in the same cells, increased Qav by 47 +/- 0.6 %. There was no significant difference between the increase in EPSC amplitude and the increase in Qav. Thus, LTP of the MPP input to dentate granule cells can be explained by an increase in the postsynaptic response to transmitter.

Algorithms↗
PubMed1999

Photolytic manipulation of [Ca2+]i reveals slow kinetics of potassium channels underlying the afterhyperpolarization in hippocampal pyramidal neurons.

The identity of the potassium channel underlying the slow, apamin-insensitive component of the afterhyperpolarization current (sIAHP) remains unknown. We studied sIAHP in CA1 pyramidal neurons using simultaneous whole-cell recording, calcium fluorescence imaging, and flash photolysis of caged compounds. Intracellular calcium concentration ([Ca2+]i) peaked earlier and decayed more rapidly than sIAHP. Loading cells with low concentrations of the calcium chelator EGTA slowed the activation and decay of sIAHP. In the presence of EGTA, intracellular calcium decayed with two time constants. When [Ca2+]i was increased rapidly after photolysis of DM-Nitrophen, both apamin-sensitive and apamin-insensitive outward currents were activated. The apamin-sensitive current activated rapidly (<20 msec), whereas the apamin-insensitive current activated more slowly (180 msec). The apamin-insensitive current was reduced by application of serotonin and carbachol, confirming that it was caused by sIAHP channels. When [Ca2+]i was decreased rapidly via photolysis of diazo-2, the decay of sIAHP was similar to control (1. 7 sec). All results could be reproduced by a model potassium channel gated by calcium, suggesting that the channels underlying sIAHP have intrinsically slow kinetics because of their high affinity for calcium.

Animals↗
PubMed1999

Quantal amplitude and quantal variance of strontium-induced asynchronous EPSCs in rat dentate granule neurons.

1. Excitatory postsynaptic currents (EPSCs) were recorded from granule cells of the dentate gyrus in acute slices of 17- to 21-day-old rats (22-25 C) using tissue cuts and minimal extracellular stimulation to selectively activate a small number of synaptic contacts. 2. Adding millimolar Sr2+ to the external solution produced asynchronous EPSCs (aEPSCs) lasting for several hundred milliseconds after the stimulus. Minimally stimulated aEPSCs resembled miniature EPSCs (mEPSCs) recorded in the same cell but differed from them in ways expected from the greater range of dendritic filtering experienced by mEPSCs. aEPSCs had the same stimulus threshold as the synchronous EPSCs (sEPSCs) that followed the stimulus with a brief latency. aEPSCs following stimulation of distal inputs had a slower mean rise time than those following stimulation of proximal inputs. These results suggest that aEPSCs arose from the same synapses that generated sEPSCs. 3. Proximally elicited aEPSCs had a mean amplitude of 6.7 +/- 2.2 pA (+/- s.d., n = 23 cells) at -70 mV and an amplitude coefficient of variation of 0. 46 +/- 0.08. 4. The amplitude distributions of sEPSCs never exhibited distinct peaks. 5. Monte Carlo modelling of the shapes of aEPSC amplitude distributions indicated that our data were best explained by an intrasite model of quantal variance. 6. It is concluded that Sr2+-evoked aEPSCs are uniquantal events arising at synaptic terminals that were recently invaded by an action potential, and so provide direct information about the quantal amplitude and quantal variance at those terminals. The large quantal variance obscures quantization of the amplitudes of evoked sEPSCs at this class of excitatory synapse.

Animals↗
PubMed1999

Bacterial toxins as mucosal adjuvants.

The use of mucosally administered killed bacteria or viruses as vaccines has a number of attractive features over the use of viable attenuated organisms, including safety, cost, storage and ease of delivery. Unfortunately, mucosally administered killed organisms are not usually effective as vaccines. The use of LT(R192G), a genetically detoxified derivative of LT, as a mucosal adjuvant enables the use of killed bacteria or viruses as vaccines by enhancing the overall humoral and cellular host immune response to these organisms, especially the Th1 arm of the immune response. With this adjuvant, protective responses equivalent to those elicited by live attenuated organisms can be achieved with killed organisms without the potential side effects. These findings have significant implications for vaccine development and further support the potential of LT(R192G) to function as a safe, effective adjuvant for mucosally administered vaccines. There are a number of unresolved issues regarding the use of LT and CT mutants as mucosal adjuvants. Both active-site and protease-site mutants of LT and CT have been constructed and adjuvanticity reported for these molecules in various animal models and with different antigens. There needs to be a side-by-side comparison of CT, LT, active-site mutants, protease-site mutants and recombinant B subunits regarding the ability to induce specific, targeted immunological outcomes as a function of route of immunization and nature of the co-administered antigen. Those side-by-side comparisons have not been carried out and there is a substantial body of evidence indicating that the outcomes may very well be different. With that information, vaccine strategies could be designed employing the optimum adjuvant/antigen formulation and route of administration for a variety of bacterial and viral pathogens. Also lacking is an understanding of the underlying cellular and intracellular signaling pathways activated by these different molecules and an understanding of the mechanisms of adjuvanticity at the cellular level. These are important issues because they take us beyond the phenomenological observations of "enhanced immunity" to a more clear understanding of the mechanisms of adjuvant activity.

Adjuvants, Immunologic↗
PubMed1999

Effectiveness of a vaccine composed of heat-killed Candida albicans and a novel mucosal adjuvant, LT(R192G), against systemic candidiasis.

The incidence of fungal infections caused by the opportunistic yeast Candida albicans has increased significantly in recent years. The ability to vaccinate selected patients against the organism would be advantageous. In this paper we describe a potential anti-C. albicans vaccine consisting of heat-killed C. albicans (HK-CA) in combination with the novel mucosal adjuvant LT(R192G), a genetically detoxified form of the heat-labile toxin of enterotoxigenic Escherichia coli. Groups of male CBA/J mice were immunized intranasally on three occasions at weekly intervals with 2 x 10(7) HK-CA per dose, alone or in conjunction with 10 micrograms of LT(R192G) per dose. Two weeks following the last application of antigen, some animals were challenged intravenously (i.v.) with 10(4), 10(5), or 10(6) viable C. albicans to assess protection as measured by survival and/or culture. Some groups of animals were footpad tested with C. albicans mannan to assess delayed-type hypersensitivity (DTH), and all the animals were bled for antibody assays. In two independent studies, all the animals immunized with HK-CA plus LT(R192G) were able to eradicate 10(4) C. albicans completely, as determined by kidney culture 4 weeks after challenge. Animals immunized with HK-CA only had reduced levels of C. albicans compared to the adjuvant or saline-only control. Greatly enhanced survival was observed when mice immunized with HK-CA plus LT(R192G) were challenged with 10(5) live C. albicans as well. Animals immunized with HK-CA plus LT(R192G) developed a significant DH response, while those given HK-CA alone developed only marginal DH responses. High immunoglobulin G (IgG) levels to cytoplasmic antigens developed in mice immunized with HK-CA plus LT(R192G), but they were found only after i.v. challenge. Addition of adjuvant shifted the antibody isotype production in i.v.-challenged animals to a response dominated by IgG2a. Clearly, intranasal immunization with killed C. albicans in conjunction with LT(R192G) afforded significant levels of protection. This novel approach offers new possibilities for the development of an effective vaccine against candidiasis for use in humans.

Adjuvants, Immunologic↗
PubMed1999

In vivo expression and immunoadjuvancy of a mutant of heat-labile enterotoxin of Escherichia coli in vaccine and vector strains of Vibrio cholerae.

Vibrio cholerae secretes cholera toxin (CT) and the closely related heat-labile enterotoxin (LT) of Escherichia coli, the latter when expressed in V. cholerae. Both toxins are also potent immunoadjuvants. Mutant LT molecules that retain immunoadjuvant properties while possessing markedly diminished enterotoxic activities when expressed by E. coli have been developed. One such mutant LT molecule has the substitution of a glycine residue for arginine-192 [LT(R192G)]. Live attenuated strains of V. cholerae that have been used both as V. cholerae vaccines and as vectors for inducing mucosal and systemic immune responses directed against expressed heterologous antigens have been developed. In order to ascertain whether LT(R192G) can act as an immunoadjuvant when expressed in vivo by V. cholerae, we introduced a plasmid (pCS95) expressing this molecule into three vaccine strains of V. cholerae, Peru2, ETR3, and JRB14; the latter two strains contain genes encoding different heterologous antigens in the chromosome of the vaccine vectors. We found that LT(R192G) was expressed from pCS95 in vitro by both E. coli and V. cholerae strains but that LT(R192G) was detectable in the supernatant fraction of V. cholerae cultures only. In order to assess potential immunoadjuvanticity, groups of germfree mice were inoculated with the three V. cholerae vaccine strains alone and compared to groups inoculated with the V. cholerae vaccine strains supplemented with purified CT as an oral immunoadjuvant or V. cholerae vaccine strains expressing LT(R192G) from pCS95. We found that mice continued to pass stool containing V. cholerae strains with pCS95 for at least 4 days after oral inoculation, the last day evaluated. We found that inoculation with V. cholerae vaccine strains containing pCS95 resulted in anti-LT(R192G) immune responses, confirming in vivo expression. We were unable to detect immune responses directed against the heterologous antigens expressed at low levels in any group of animals, including animals that received purified CT as an immunoadjuvant. We were, however, able to measure increased vibriocidal immune responses against vaccine strains in animals that received V. cholerae vaccine strains expressing LT(R192G) from pCS95 compared to the responses in animals that received V. cholerae vaccine strains alone. These results demonstrate that mutant LT molecules can be expressed in vivo by attenuated vaccine strains of V. cholerae and that such expression can result in an immunoadjuvant effect.

Adhesins, Bacterial↗
PubMed1999

Antibody-independent protection against rotavirus infection of mice stimulated by intranasal immunization with chimeric VP4 or VP6 protein.

This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-microg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-micrograms doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient microMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody.

ATP-Binding Cassette Transporters↗
PubMed1998

N- and P/Q-type Ca2+ channels mediate transmitter release with a similar cooperativity at rat hippocampal autapses.

The relationship between extracellular Ca2+ concentration and EPSC amplitude was investigated at excitatory autapses on cultured hippocampal neurons. This relationship was steeply nonlinear, implicating the cooperative involvement of several Ca2+ ions in the release of each vesicle of transmitter. The cooperativity was estimated to be 3.1 using a power function fit and 3.3 using a Hill equation fit. However, simulations suggest that these values underestimate the true cooperativity. The role of different Ca2+ channel subtypes in shaping the Ca2+ dose-response relationship was studied using the selective Ca2+ channel blockers omega-agatoxin GIVA (omega-Aga), which blocks P/Q-type channels, and omega-conotoxin GVIA (omega-CTx), which blocks N-type channels. Both blockers broadened the dose-response relationship, and the Hill coefficient was reduced to 2.5 by omega-Aga and to 2.6 by omega-CTx. This broadening is consistent with a nonuniform distribution of Ca2+ channel subtypes across presynaptic terminals. The similar Hill coefficients in omega-Aga or omega-CTx suggest that there was no difference in the degree of cooperativity for transmitter release mediated via N- or P/Q-type Ca2+ channels. A model of the role of calcium in transmitter release is developed. It is based on a modified Dodge-Rahamimoff equation that includes a nonlinear relationship between extracellular and intracellular Ca2+ concentration, has a cooperativity of 4, and incorporates a nonuniform distribution of Ca2+ channel subtypes across presynaptic terminals. The model predictions are consistent with all of the results reported in this study.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1998

Activation kinetics of AMPA receptor channels reveal the number of functional agonist binding sites.

AMPA and NMDA receptor channels are closely related molecules, yet they respond to glutamate with distinct kinetics, attributable to differences in ligand binding and channel gating steps (for review, see Edmonds et al., 1995). We used two complementary approaches to investigate the number of functional binding sites on AMPA channels on outside-out patches from cultured hippocampal neurons. The activation kinetics of agonist binding were measured during rapid steps into low concentrations of selective AMPA receptor agonists and during steps from a competitive AMPA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione, into a saturating concentration of agonist. Both approaches revealed sigmoidal kinetics, which suggests that multiple agonist binding steps or antagonist unbinding steps are needed for channel activation. A kinetic model with two independent binding sites gave a better fit to the activation phase than models with one or three independent sites. A more refined analysis incorporating cooperative interaction between the two binding sites significantly improved the fits to the responses. The affinity of the first binding step was two to three times higher than the second step. These results demonstrate that binding of two agonist molecules are needed to activate AMPA receptors, but the two binding sites are not identical and independent. Because NMDA receptors require four ligand molecules for activation (two glycine and two glutamate; Benveniste and Mayer, 1991; Clements and Westbrook, 1991), it may be that some binding sites on AMPA receptors are functionally silent.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1998

LT(R192G), a non-toxic mutant of the heat-labile enterotoxin of Escherichia coli, elicits enhanced humoral and cellular immune responses associated with protection against lethal oral challenge with Salmonella spp.

In the current study we examined the ability of a novel mucosal adjuvant, LT(R192G), to enhance the humoral and cellular immune responses against killed Salmonella spp. and to affect protection against lethal oral challenge with wild-type organisms. Mice orally immunized with killed S. dublin in conjunction with LT(R192G) were protected against lethal oral challenge and had higher IFN-gamma, IL-2 and IgG responses than did mice orally immunized with killed S. dublin alone which were not protected. This study demonstrates that the function of LT(R192G) in protection against typhoid-like disease is to upregulate/enhance the Th1 arm of the immune response against killed organisms. When used as a mucosal adjuvant, LT(R192G) enables the use of killed bacteria or viruses as vaccines by enhancing the overall humoral and cellular host immune response to these organisms, especially the Th1 arm of the immune response. These findings have significant implications for vaccine development and further support the potential of LT(R192G) to function as a safe, effective adjuvant for mucosally administered vaccines.

Administration, Oral↗
PubMed1998

Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene.

The authors have designed and constructed a plant-optimize synthetic gene encoding the Escherichia coli heat-labile enterotoxin B subunit (LT-B), for use in transgenic plants as an edible vaccine against enterotoxigenic E. coli. Expression of the synthetic LT-B gene in potato plants under the control of a constitutive promoter yielded increased accumulation of LT-B in leaves and tubers, as compared to the bacterial LT-B gene. The plant-derived LT-B assembled into native pentameric structures as evidenced by its ability to bind ganglioside. The authors demonstrated immunogenicity by feeding mice the raw tubers and comparing the anti-LT-B serum IgG and faecal IgA to that produced in mice gavaged with bacterial LT-B. Mice were fed three weekly doses of 5 g tuber tissue containing either 20 or 50 micrograms LT-B, or gavaged weekly with 5 micrograms of LT-B from recombinant E. coli. One week after the third dose, mice immunized with potato LT-B had higher levels of serum and mucosal anti-LT-B than those gavaged with bacterial LT-B. Mice were challenged by oral administration of 25 micrograms LT, and protection assessed by comparing the gut/carcass mass ratios. Although none of the mice were completely protected, the higher dose potato vaccine compared favourably with the bacterial vaccine. These findings show that an edible vaccine against E. coli LT-B is feasible.

Administration, Oral↗
PubMed1998

Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato.

Compared with vaccine delivery by injection, oral vaccines offer the hope of more convenient immunization strategies and a more practical means of implementing universal vaccination programs throughout the world. Oral vaccines act by stimulating the immune system at effector sites (lymphoid tissue) located in the gut. Genetic engineering has been used with variable success to design living and non-living systems as a means to deliver antigens to these sites and to stimulate a desired immune response. More recently, plant biotechnology techniques have been used to create plants which contain a gene derived from a human pathogen; the resultant plant tissues will accumulate an antigenic protein encoded by the foreign DNA. In pre-clinical trials, we found that antigenic proteins produced in transgenic plants retained immunogenic properties when purified; if injected into mice the antigen caused production of protein-specific antibodies. Moreover, in some experiments, if the plant tissues were simply fed to mice, a mucosal immune response occurred. The present study was conducted as a proof of principle to determine if humans would also develop a serum and/or mucosal immune response to an antigen delivered in an uncooked foodstuff.

Adolescent↗
PubMed1998

Rotavirus 2/6 viruslike particles administered intranasally with cholera toxin, Escherichia coli heat-labile toxin (LT), and LT-R192G induce protection from rotavirus challenge.

We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 microg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced equivalent geometric mean titers of rotavirus-specific serum antibody and intestinal immunoglobulin G (IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and LT-R192G were totally protected (100%) from rotavirus challenge, while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91% protection from challenge. The availability of a safe, effective mucosal adjuvant such as LT-R192G will increase the practicality of administering recombinant vaccines mucosally.

Administration, Intranasal↗
PubMed1997

Nonuniform distribution of Ca2+ channel subtypes on presynaptic terminals of excitatory synapses in hippocampal cultures.

Several subtypes of Ca2+ channel support the release of glutamate at excitatory synapses. We investigated the pattern of colocalization of these subtypes on presynaptic terminals in hippocampal cultures. N-type (conotoxin GVIA-sensitive) or P/Q-type (agatoxin IVA-sensitive) Ca2+ channels were blocked selectively, and the reduction in transmitter release probability (Pr) was measured with MK-801. The antagonists completely blocked release at some terminals, reduced Pr at others, and failed to affect the remainder. In contrast, nonselective reduction of presynaptic Ca2+ influx by adding Cd2+ or lowering external Ca2+ reduced Pr uniformly at all terminals. We conclude from these results that the mixture of N-type and P/Q-type channels varies markedly between terminals on the same afferent. The distribution of Ca2+ channel subtypes was the same for high and low Pr terminals. Given that Ca2+ channel subtypes are affected differentially by neuromodulators, these findings lead to the possibility of terminal-specific modulation of synaptic function.

Animals↗
PubMed1997

Detection of spontaneous synaptic events with an optimally scaled template.

Spontaneous synaptic events can be difficult to detect when their amplitudes are close to the background noise level. Here we report a sensitive new technique for automatic detection of small asynchronous events. A waveform with the time course of a typical synaptic event (a template) is slid along the current or voltage trace and optimally scaled to fit the data at each position. A detection criterion is calculated based on the optimum scaling factor and the quality of the fit. An event is detected when this criterion crosses a threshold level. The algorithm automatically compensates for changes in recording noise. The sensitivity and selectivity of the method were tested using real and simulated data, and the influence of the template parameter settings was investigated. Its performance was comparable to that obtained by visual event detection, and it was more sensitive than previously described threshold detection techniques. Under typical recording conditions, all fast synaptic events with amplitudes of at least three times the noise standard deviation (3 sigma) could be detected, as could 75% of events with amplitudes of 2 sigma. The scaled template technique is implemented within a commercial data analysis application and can be applied to many standard electrophysiological data file formats.

Animals↗