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Beyond buffer zone protection: a comparative study of park and buffer zone products' importance to villagers living inside Royal Chitwan National Park and to villagers living in its buffer zone.

Even after 30 years of strict de jure protection, today's de facto extraction of products from Nepal's Royal Chitwan National Park (RCNP) and their great economic importance to local households suggests that this reality should be explicitly internalised in managing this world heritage park. Several studies have quantified local people's use of protected areas and estimated the value of such areas to them. However, few studies incorporate economic analyses to investigate the effect of management interventions on local communities' resource use and collection behaviour. In Nepal, buffer zones and especially buffer zone community forestry are seen as means to define and demarcate places, where local people may legally extract goods that are either identical to or relevant substitutes for products that are collected in protected areas. The intention is to resolve park-people conflicts over resource use. This article presents the findings of an in-depth study of the importance of natural resources to the livelihoods of 18 households. One village was located inside RCNP with no realistic alternatives to Park resources, while the other is located in the buffer zone with equal distance to the Park, a national forest and their community forest. For each household, the collection of products, allocation of time, and purchase and sale of goods were recorded daily through 12 consecutive months and economic values were calculated on the basis of local market prices and recorded quantities. The study shows that products from RCNP are of great importance to the livelihoods of local people. Furthermore, we find that products collected in the national forest substitute products from the Park, while the substitution effect of the community forest is small. Accordingly, the study illustrates that, irrespective of buffer zone community forestry, there is still a gap between local people's need for supplementing natural resources and their rights to satisfy them on a legal basis, which is likely to be unsustainable in the longer term. This calls for a thorough evaluation of actual park-people relations and how these may be improved through local participation that goes beyond the current form of buffer zone community forestry and the admitted 7-14 annual days of open access grass cutting within the park.

Agriculture↗

Ruminal buffers: temporal effects on buffering capacity and pH of ruminal fluid from cows fed a high concentrate diet.

In vitro characteristics of several buffers and alkalinizing agents commonly utilized to reduce ruminal acid load were evaluated. Ruminal fluid was collected from five cows consuming a diet containing concentrate and sorghum silage in a 68:32 ratio (DM basis). This fluid was incubated with either NaHCO3, a natural sodium sesquicarbonate, a multielement buffer or MgO (7.1 g/L of ruminal fluid), or no buffer for 48 h; flasks were removed and analyzed for pH, buffering capacity, and buffer value index every 12 h during the 48-h incubation. The buffer value index accounts simultaneously for alterations in pH and buffering capacity. Compared with the unbuffered control, all buffering compounds increased ruminal fluid buffer value index. However, the buffer value index separated these buffering compounds into two categories. The NaH-CO3 and sodium sesquicarbonate exhibited similar buffer value indexes; both were markedly higher than those for the multielement buffer and MgO. Although NaHCO3 and sodium sesquicarbonate each increased both ruminal fluid pH and buffering capacity sharply, the multielement buffer only increased pH and buffering capacity moderately. The increase in buffer value index for MgO primarily was due to an increase in pH. Both NaHCO3 and sodium sesquicarbonate were fully active within the first 12 h of incubation; activity of multielement buffer and MgO reached a plateau at 24 h. Compared with the multielement buffer and MgO, NaHCO3 and sodium sesquicarbonate should be more beneficial in preventing short-term postprandial increases in ruminal fluid hydrogen ion concentration; because of their slower release rates, the multielement buffer and MgO should help stabilize ruminal acid-base status, but efficacy might be reduced because of passage out of the rumen.

Acid-Base Equilibrium↗

Assessment of the myotoxicity of pharmaceutical buffers using an in vitro muscle model: effect of pH, capacity, tonicity, and buffer type.

The purpose of the present study was to investigate the myotoxicity of three buffers containing carboxylic acid groups (i.e., acetate, succinate, and citrate) as a function of their pH, capacity, and tonicity. The myotoxicity of these buffers in the range of pH 2-6 and 0.001-0.1 M buffer capacity was assessed using cumulative creatine kinase (CK) release from an isolated rodent muscle model following injection. Phenytoin and 0.9% NaCl injection were used as positive and negative controls, respectively. Buffer solutions were prepared. A lower pH and higher buffer capacity was linked to increased myotoxicity for the acetate buffers. However, for succinate and citrate buffers, pH appeared to influence the extent of myotoxicity, whereas buffer capacity did not seem to have an effect. When either NaCl or trehalose was used as a tonicity-adjusting agent at pH 6, isotonic 0.01 M buffer solutions dramatically lowered the cumulative CK release compared to those that were not isotonic. Isotonic succinate buffers displayed the lowest myotoxicity, whereas citrate buffers displayed the highest values. Citrate buffers containing three carboxylic acid groups showed higher myotoxicity than succinate buffers and acetate buffers at 0.001 and 0.01 M buffer capacities, whereas acetate buffer produced higher cumulative CK release than citrate and succinate buffers at 0.1 M buffer capacity. The myotoxicity of pharmaceutical buffers containing carboxylic acid groups appears to be directly affected by lowering the pH of the solution.

Acetates↗

A buffer value index to evaluate effects of buffers on ruminal milieu in cows fed high or low concentrate, silage, or hay diets.

Our objective was to develop a buffer value index that would incorporate alterations in both ruminal fluid pH and buffering capacity as indicators of the influence of dietary buffering and alkalinizing agents on ruminal acid-base status. This index was evaluated using ruminal fluid from four lactating Holstein cows fed either sorghum silage or alfalfa hay in high or low concentrate diets. Ruminal fluid was incubated in vitro for 1, 2, 3, 4, or 5 h with no buffer or with 7.1 g of either NaHCO3, sodium sesquicarbonate, or a multielement buffer added per liter of ruminal fluid. Ruminal fluid pH was lower for diets based on high concentrate or alfalfa; buffering capacity between pH 5 and 7 was greater for high concentrate diets but was not affected by forage type. Ruminal fluid pH was higher for sesquicarbonate than for NaHCO3, the multielement buffer, or the control; however, ruminal fluid H+ concentration was similar between sesquicarbonate and NaHCO3, and both were lower than for the multielement buffer. Hydrogen ion concentration for the multielement buffer was lower than for the control. Buffering capacity was highest for NaHCO3, followed by sesquicarbonate, the multielement buffer, and the control. The buffer value index, which accounted for alterations in both H+ concentration and buffering capacity, was highest for NaCHO3, followed by sesquicarbonate, the multielement buffer, and the control. The poor response to the multielement buffer may be attributable to our relatively short incubation interval (less than 5 h). Dietary buffers increase both ruminal fluid pH and buffering capacity; both of these responses are beneficial.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗

DNA and buffers: are there any noninteracting, neutral pH buffers?

The interaction of DNA with various neutral pH, amine-based buffers has been analyzed by free solution capillary electrophoresis, using a mixture of a plasmid-sized DNA molecule and a small DNA oligonucleotide as the reporter system. The two DNAs migrate as separate, nearly Gaussian-shaped peaks in 20-80 mM TAE (TAE, Tris-acetate-EDTA; Tris, tris[hydroxymethyl]aminomethane) buffer. The separation between the peaks gradually increases with increasing TAE buffer concentration because of differences in solvent friction between large and small DNA molecules. The two DNAs form complexes with the borate ions in TBE (Tris-borate-EDTA) buffer, with mobilities that depend on the DNA/borate ratio. In 45 mM TBE buffer, the two DNAs comigrate as a single sharp peak, with a mobility that is faster than either of the constituent DNAs in the same buffer. Hence, the mixed DNA-borate complex is stabilized by the binding of additional borate ions, possibly forming bridges between the different DNAs. The mixed DNA-borate complex is gradually dissociated into its component DNAs by increasing the TBE concentration, possibly because the borate binding sites become saturated at high buffer concentrations. Other neutral pH, amine-based buffers, such as Mops (3-[N-morpholino]propanesulfonic acid), Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), Bes (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), Tes (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), and tricine (N-tris[hydroxymethyl]methylglycine) also form complexes with DNA, giving distorted peaks in the electropherograms. The combined results indicate that borate buffers and most neutral pH, amine-based buffers interact with DNA.

Acetates↗

Automatic initialisation of buffer composition estimation for on-line analysis of unknown buffer solutions.

An automatic initialisation procedure for extracting useful information about buffer composition from a titration experiment is presented in this paper. The initialisation procedure identifies which buffering components are present in the sample from a relatively long list of buffers expected in the system monitored. The procedure determines approximate pKa values of the buffers and evaluates their maximum and minimum concentrations. This information is then used to start an optimisation procedure to fit the model of the buffer components to the titration data and to accurately determine buffer concentrations and pKa values. The procedure has been integrated as a software layer around the buffer capacity optimum model builder (BOMB) that fits a buffer-capacity model to a measured buffer-capacity curve to estimate model properties (pKa values and concentrations). The reliability and robustness of the resulting buffer capacity software (BCS) were tested using a titrimetric analyser simulator (TAS). The BCS was then validated off-line and on-line.

Journal Article↗

The buffer capacity and buffer systems of human whole saliva measured without loss of CO2.

The buffer capacity of unstimulated (UWS) and stimulated (SWS) whole-mouth saliva involves three major buffer systems. The aim was to determine the buffer capacity of UWS and SWS at specific pH in the interval from pH 7.5 down to pH 3.0. The contribution of each of the buffer systems was also determined under conditions resembling those in the mouth. UWS and SWS were collected from 20 healthy volunteers; the saliva was collected under paraffin oil in order to avoid loss of CO2. The buffer capacity of UWS and SWS in samples with and without bicarbonate (HCO3-) and CO2 were measured at various pH by acid titration in a closed system at 36 C. The mean concentrations of the buffer systems in UWS (mean flow rate 0.55 ml/min) were 4.4 mmol/l HCO3-, 4.5 mmol/l phosphate (of which 1.3 mmol/l was present in the form of HPO4(2-)), 1876 microg/ml protein; the saliva pH was 6.8 and the P(CO2) 29.3 mmHg. The corresponding mean concentrations in SWS (mean flow rate 1.66 ml/min) were 9.7 mmol/l HCO3-, 3.8 mmol/l phosphate (of which 1.9 mmol/l was present in the form of HPO4(2-)), 1955 microg/ml protein; pH 7.2 and P(CO2) 25.7 mmHg, The highest buffer capacity of UWS and SWS was 6.0 and 8.5 mmol H+ /(1 saliva*pH unit) at pH 6.25, respectively. At saliva pH in the range from pH 7 down to pH 5, the following had significant impact on buffer capacity: the HCO3- concentration (p < 0.001), the flow rate (p < 0.01), and the pH of the saliva (p < 0.05). At acidic pH in the range from pH 5 down to pH 4, however, only the protein concentration had a significant impact on buffer capacity (p < 0.01).

Adult↗

Isoelectric buffers for capillary electrophoresis. 2. Bismorpholine derivative of a carboxylic acid as a low molecular weight isoelectric buffer.

A new compound class of synthetic isoelectric buffers is introduced, designed as a small molecule with one fully or prevailingly dissociated acidic group (such as sulfonic or carboxylic) and two partly pronated (buffering) basic amino groups attached onto a hydrophilic UV-transparent backbone. As an example, a new isoelectric compound 2,2-bis(4-morpholinylmethyl)propanoic acid (BMMPA) was synthesized by attaching two morpholine groups onto a molecule of pivalic acid. It was characterized as having an isoelectric point pI = 6.5 and exhibiting satisfactory buffering capacity at the pI. Solutions of BMMPA are transparent down to the low-UV spectral region, thus making it a potentially suitable buffer for a number of separation methods. Its use in capillary electrophoresis was demonstrated in a separation system for indirect photometric detection of anions based on an electrolyte with the anionic dye Orange G as the indirect detection probe and using BMMPA as a buffer. The use of an isoelectric buffering compound brings the advantages of a buffered electrolyte without the concomitant introduction of co-ions that would be detrimental to the indirect detection process. Submicromole per liter limits of detection for a number of inorganic and small organic ions were achieved. Optimal structural properties of the isoelectric buffer with respect to its buffering properties are discussed.

Journal Article↗

Facilitation through buffer saturation: constraints on endogenous buffering properties.

Synaptic facilitation (SF) is a ubiquitous form of short-term plasticity, regulating synaptic dynamics on fast timescales. Although SF is known to depend on the presynaptic accumulation of Ca(2+), its precise mechanism is still under debate. Recently it has been shown that at certain central synapses SF results at least in part from the progressive saturation of an endogenous Ca(2+) buffer (Blatow et al., 2003), as proposed by Klingauf and Neher (1997). Using computer simulations, we study the magnitude of SF that can be achieved by a buffer saturation mechanism (BSM), and explore its dependence on the endogenous buffering properties. We find that a high SF magnitude can be obtained either by a global saturation of a highly mobile buffer in the entire presynaptic terminal, or a local saturation of a completely immobilized buffer. A characteristic feature of BSM in both cases is that SF magnitude depends nonmonotonically on the buffer concentration. In agreement with results of Blatow et al. (2003), we find that SF grows with increasing distance from the Ca(2+) channel cluster, and increases with increasing external Ca(2+), [Ca(2+)](ext), for small levels of [Ca(2+)](ext). We compare our modeling results with the experimental properties of SF at the crayfish neuromuscular junction, and find that the saturation of an endogenous mobile buffer can explain the observed SF magnitude and its supralinear accumulation time course. However, we show that the BSM predicts slowing of the SF decay rate in the presence of exogenous Ca(2+) buffers, contrary to experimental observations at the crayfish neuromuscular junction. Further modeling and data are required to resolve this aspect of the BSM.

Animals↗

Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency.

Control of acidity and preventing intracellular acidosis are recognized as critical properties of an effective organ preservation solution. Buffer capacity and efficiency are therefore important for comparing the relative merits of preservation fluids for optimum hypothermic storage, but these parameters are not available for the variety of organ preservation solutions of interest in transplantation today. Moreover, buffer capacity is dependent upon both concentration and pH such that buffer capacity is not easily predicted for a complex solution containing multiple buffer species. Using standard electrometric methods to measure acid dissociation constants, this study was undertaken to determine the maximum and relative buffer capacities of a variety of new and commonly used hypothermic preservation solutions as a function of temperature. The reference data provided by these measurements show that comparative buffer capacity and efficiency vary widely between the commonly used solutions. Moreover, the fluids containing zwitterionic sulfonic acid buffers such as Hepes possess superior buffering for alpha-stat pH regulation in the region of physiological importance.

Acid-Base Equilibrium↗

Theory and computer programs for calculating solution pH, buffer formula, and buffer capacity for multiple component system at a given ionic strength and temperature.

PURPOSE: A theory and computer programs running on Microsoft Excel for Windows for calculation of solution pH, buffer formula, and buffer capacity at a given ionic strength and temperature were developed. The theory does not limit the category of buffer components, the number of buffer components, or the number of ionizations for each buffer component. The usefulness of the programs was examined. METHODS: The formulas for 7 single component buffer solutions and 2 multiple component buffer solutions composed of citrate, phosphate, Tris, borate, and glycine were calculated. The solution pH values were measured at 25, 40, 55 and 70 degrees C for comparison with the calculated pH values. RESULTS: Of the 108 predictions made, 96 were of pH values with +/- 0.1 pH unit of the measured values, at temperatures ranging from 25 degrees C to 70 degrees C and at ionic strengths ranging from 0.1 M to 0.5 M. CONCLUSIONS: These programs will be useful for identifying appropriate buffer solutions at various temperatures and/or ionic strengths.

Buffers↗

The impact of buffers and surfactants from micellar electrokinetic chromatography on matrix-assisted laser desorption ionization (MALDI) mass spectrometry of peptides. Effect of buffer type and concentration on mass determination by MALDI-time-of-flight mass spectrometry.

This paper describes the effect of various buffers, surfactants, and organic additives commonly encountered in capillary zone electrophoresis and micellar electrokinetic chromatography on the molecular weight determination of peptides by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Signal-to-noise ratio generally decreased with increasing buffer concentration without affecting mass accuracy, but the type of buffer was also important. Good spectra were obtained with an ammonium acetate buffer up to a concentration of 500 mM without impacting ionization of either peptides or other mobile phase constituents. Ionization of organic additives, such as anionic surfactants, non-ionic surfactants, and cyclodextrins was buffer dependent and presented a problem when the mass of the additive was in the range of the peptide mass. Brij-35, Tween-80, and cyclodextrins all produced prominent spectra of their own in the presence of sodium or potassium containing buffers, but not with ammonium acetate. Cationization of these neutral species with sodium or potassium ions allowed them to acquire a positive charge and produce spectra. In contrast, the ammonium ion appears to be a poor cationizating agent. Ionization of neutral surfactants was suppressed in ammonium acetate without impacting the spectra of peptides. Ammonium acetate buffers containing 30 mM sodium dodecyl phosphate also gave spectra with good signal intensity and no interference from the surfactant. Suppression of peptide ionization in MALDI was a problem when methanol, tetrabutyl amine, or poly(vinyl alcohol) were used with either ammonium acetate, sodium phosphate, and N-(2-hydroxyethyl)piperazine-N-(2-ethansulfonic acid).

Buffers↗

Effect of initial buffer composition on pH changes during far-from-equilibrium freezing of sodium phosphate buffer solutions.

PURPOSE: This study aims to assess the pH changes induced by salt precipitation during far-from-equilibrium freezing of sodium phosphate buffers as a function of buffer composition, under experimental conditions relevant to pharmaceutical applications-sample volumes larger than a few microliters, experiencing large degrees of undercooling and supersaturation. METHODS: Buffer solutions were prepared by dissolving the monosodium and disodium phosphate salts in the appropriate ratios to obtain initial buffer concentrations in the range of 8-100 mM and pH values between 5.7 and 7.4 at 25 degrees C. Temperature and pH were monitored in situ during cooling to -10 degrees C (at a rate of 0.3 to 0.5 degrees C/min) and for 10-20 min after the sample reached the final temperature. Salt crystallization was confirmed by ion analysis and x-ray powder diffraction. RESULTS: Precipitation of Na2HPO4, 12H2O caused abrupt pH decreases after the onset of ice crystallization, at temperatures between -0.5 and -4.0 degrees C. Decreasing the initial buffer concentration and/or initial pH resulted in higher final pH values at -10 degrees C, farther removed from the equilibrium value of 3.6. At an initial pH of 7.4, the 50 and 100 mM buffer solutions reached a pH of 4.2 +/- 0.1 at -10 degrees C, whereas the 8 mM solutions reached a pH of 5.2 +/- 0.2. Solutions having an initial pH of 5.7 and initial buffer concentrations of 8 and 100 mM experienced less pH shifts upon freezing to -10 degrees C, with final pH values of 5.1 +/- 0.1 and 4.7 +/- 0.1, respectively. CONCLUSIONS: Precipitation-induced pH shifts are dependent on the concentrations (activities) of precipitating ions, and are determined by both initial pH and salt concentration. The ion activity product is a meaningful parameter when describing salt precipitation in solutions prepared by mixing salts containing precipitating and nonprecipitating ions.

Buffers↗

Biocompatibility and buffers: effect of bicarbonate-buffered peritoneal dialysis fluids on peritoneal cell function.

BACKGROUND: Conventional peritoneal dialysis fluids (PDF) have been shown to compromise the function of both leukocytes and human peritoneal mesothelial cells (HPMC). Various in vitro studies have identified the low initial pH in combination with high lactate content, as well as the hyperosmolality and high glucose concentration present in currently used solutions as the primary determinants of their bioincompatibility. Bicarbonate buffered PDF (at neutral pH) display improved in vitro biocompatibility as compared to conventional, lactate buffered PDF. However, little information is currently available regarding the potential impact of PDF on the function of human peritoneal fibroblasts (HPFB), the major cell population present in peritoneal interstitium. METHODS: The current study compares the effect of bicarbonate and lactate buffered PDF in a model system of resting peritoneal mesothelial cells and fibroblasts cultured from human omentum. Interleukin-1 beta-stimulated IL-6 release from HPMC and HPFB was used as the cell functional parameter. RESULTS: While short (30 min) pre-exposure to lactate buffered PDF significantly reduced the IL-1 beta-stimulated IL-6 release from HPMC during a subsequent recovery period (24 hr), a significant decrease in HPMC IL-6 secretion with bicarbonate buffered PDF was only observed after prolonged (> or = 60 min) exposure. In contrast, no significant IL-6 inhibition was detected with HPFB pre-exposed to PDF for up to 90 minutes. A significant suppression of HPFB IL-6 secretion was only observed in coincubation experiments (24 hr) with dilutions of both types of PDF. CONCLUSIONS: These results indicate that (i) bicarbonate buffered PDF are less inhibitory to peritoneal cell function as compared to conventional, lactate buffered PDF; and (ii) HPFB may be more resistant than HPMC to bioincompatible PDF.

Bicarbonates↗

Effect of moderately acidic pH on heat resistance of Clostridium sporogenes spores in phosphate buffer and in buffered pea puree.

The effect of pH in the range 5.0 to 7.0 on the thermal destruction of spores of Clostridium sporogenes putrefactive anaerobe 3679 was examined by three methods: a capillary tube method in which spores were suspended in phosphate buffers, a thermoresistometer method in which spores were suspended in buffered pea puree adjusted to the same set of pH values, and a thermal death time can method in which spores were again suspended in buffered pea puree. The results indicated that increasing acidity is, in general, accompanied by decreasing heat resistance, although the pH effect was more pronounced at the higher than at the lower processing temperatures. Certain pH values appear to be critical, as they produced, in all three sets of experiments, effects which would not be predicted by the overall relationship between acidity and spore heat resistance. Differences between heat resistance in phosphate buffer as compared with that in pea puree adjusted to the same pH were also noted. D-values in buffer were found to be lower than those in pea puree, except at the highest temperatures coupled with the lowest pH values. The differences between buffer D-value and pea puree D-value were found to increase with increasing pH and with decreasing temperature. On the other hand, at all pH values examined, z-values determined in buffer were somewhat higher than those determined in pea puree adjusted to the same pH.

Buffers↗

Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties.

Using a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel ("sea-level electrophoresis"), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris-boric-acid-EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA system, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer.

Boric Acids↗

Effect of dialysate and substitution fluid buffer on buffer flux in hemodiafiltration.

The effect of differing dialysate and substitution fluid buffer types and concentrations on acid-base balance have not been assessed in patients treated with hemodiafiltration for ESRD. To determine bicarbonate, acetate, lactate and total buffer flux, mass balance studies were performed in patients treated with hemodiafiltration using four different combinations of dialysate and substitution fluids. Driving force for bicarbonate flux was assessed in all treatments. Bicarbonate flux depended on bicarbonate driving force and ultrafiltration rate. Bicarbonate flux was negative in all treatment combinations, even when the driving force was positive. Acetate flux was positive in all treatment combinations, but the net magnitude was small. Lactate flux, when lactate containing substitution fluid was used, varied with dialysate buffer employed during treatment. Overall buffer flux depended on the bicarbonate driving force, ultrafiltration rate, and varied with the type of substitution and dialysate buffer employed. The types and concentrations of buffer used in dialysate and substitution fluid have important effects on the acid-base balance of patients treated with hemodiafiltration. The long-term implications of different therapeutical choices in these patients is unknown.

Acetates↗

Calcium dynamics, buffering, and buffer saturation in the boutons of dentate granule-cell axons in the hilus.

The axons of dentate gyrus granule cells form synapses in the hilus. Ca(2+) signaling was investigated in the boutons of these axons using confocal fluorescence imaging. Boutons were loaded with various concentrations of the Ca(2+) indicator Oregon Green BAPTA-1 by patch-clamping the cell bodies and allowing the dye to diffuse into the axon. Resting free [Ca(2+)] started at 74 nm, rose to approximately 1 microm immediately after an action potential, and then decayed to rest with a time constant of 43 msec (all extrapolated to a dye concentration of zero). Action potential-induced [Ca(2+)] rises were smaller in larger boutons, consistent with a size-independent Ca(2+) channel density of 45/microm(2). Action potential-induced [Ca(2+)] changes varied with dye concentration in a manner consistent with kappa(E) approximately 20 for the ratio of endogenous buffer-bound Ca(2+) to free Ca(2+). During trains of action potentials, [Ca(2+)] increments summed supralinearly by more than that expected from dye saturation. The amount of endogenous Ca(2+) buffering declined as [Ca(2+)] rose, and this saturation indicated a buffer with a dissociation constant of approximately 500 nm and a concentration of approximately 130 microm. This is similar to the dissociation constant of calbindin-D28K, a Ca(2+)-binding protein that is abundant in dentate granule cells. Thus, calbindin-D28K is a good candidate for the Ca(2+) buffer revealed by these experiments. The saturation of endogenous buffer can generate short-term facilitation by amplifying [Ca(2+)] changes during repetitive activity. Buffer saturation may also be relevant to the presynaptic induction of long-term potentiation at synapses formed by dentate granule cells.

Action Potentials↗