Rheological characteristics of faecal sludge from VIP latrines and implications on pit emptying.

This work aims at characterizing the rheological properties of faecal sludge from Ventilated Improved Pit (VIP) latrines and their implication on pit emptying. Faecal sludge was sampled from 3 pit latrines located in the eThekwini Municipality (Durban, South Africa). Samples were taken at different positions within the pit. For each of the samples, measurements in the rheometer in triplicates were performed in order to determine their rheological properties, and their moisture and ash content were measured also in triplicates. Experiments in the rheometer were performed for samples for which its moisture content was modified. In order to better understand the influence of water addition into the pit. During pit emptying, calculations were carried out from the experimental data, based in the criteria set in the Omni-Ingestor initiative, carried out by the Bill & Melinda Gates Foundation. Faecal sludge exhibited a shear thinning behaviour, i.e. a decrease in viscosity with increasing shear rate, and presented a yield stress comprised between 500 to 1000 Pa. This needs to be surpassed in order to overcome the elastic resistance of the sludge to flow. Similar viscosities were found for the samples from the different pits, irrespective of the position within the pit, except for the sample from the bottom of one of the pits for which it was not possible to induce a flow. This sample had a considerably lower moisture content (67% wet basis) compared to the other samples (around 80% wet basis), probably due to a higher biodegradation as it was the most aged sludge in the pit. According to the experimental results and calculations, the pumping requirements during pit emptying will decrease drastically by increasing the moisture content of the sludge. The addition of water into the pit would then facilitate the pit emptying operation by reducing the head and power required for pumping. However, this practice would require employing considerable amounts of water and handling higher volumes of sludge, which would lead to longer pit emptying times and increase the difficulty of the operation. For example, increasing the moisture content of the sludge from 75 to 90% will reduce the head and power of the pump by a factor 100, but will triplicate the amount of water in the sludge and, consequently, the time for pit emptying. Therefore, a compromise has to be made between increasing the pumping feasibility and adding water to the pit.

A phylogenetic test of sympatric speciation in the Hydrobatinae (Aves: Procellariiformes).

Phylogenetic relationships among species can provide insight into how new species arise. For example, careful consideration of both the phylogenetic and geographic distributions of species in a group can reveal the geographic models of speciation within the group. One such model, sympatric speciation, may be more common than previously thought. The Hydrobatinae (Aves: Procellariformes) is a diverse subfamily of Northern Hemisphere storm-petrels for which the taxonomy is unclear. Previous studies showed that Hydrobates (formally Oceanodroma) castro breeding in the Azores during the cool season is sister species to H. monteiroi, a hot season breeder at the same locations, which suggests sympatric speciation by allochrony. To test whether other species within the subfamily arose via sympatric speciation by allochrony, we sequenced the cytochrome b gene and five nuclear introns to estimate a phylogenetic tree using multispecies coalescent methods, and to test whether species breeding in the same geographic area are monophyletic. We found that speciation within the Hydrobatinae appears to have followed several geographic modes of divergence. Sympatric seasonal species in Japan likely did not arise through sympatric speciation, but allochrony may have played a role in the divergence of H. matsudairae, a cool season breeder, and H. monorhis, a hot season breeder. No other potential cases of sympatric speciation were discovered within the subfamily. Despite breeding in the same geographic area, hydrobatine storm-petrels breeding in Baja California (H. microsoma and H. melania) are each sister to a species breeding off the coast of Peru (H. tethys and H. markhami, respectively). In fact, antitropical sister species appear to have diverged at multiple times, suggesting allochronic divergence might be common. In addition, allopatry has likely played a role in divergence of H. furcata, a north Pacific breeder, and H. pelagius, a north Atlantic breeder. This study demonstrates that a variety of mechanisms of divergence have played a role in generating the diversity of the Hydrobatinae and supports the current taxonomy of the subfamily.

Characterization of ultrafiltration of undiluted and diluted stored urine.

Urine ultrafiltration (UF) was studied in terms of flux, permeability, resistance and fouling. Two types of samples were used: stored urine representing the feedstock obtained from urine diversion dry toilets; and diluted stored urine representing the feedstock obtained from urinals. Three different filtration experiment sets were adopted in this study. For the first case, pressure was set in an ascending order, i.e. from 10 to 60 kPa during filtration of stored urine. For the second case, pressure was set in a descending order, i.e. from 60 to 10 kPa for the same feed stream. The third case involved filtration of diluted urine with pressure in ascending order, i.e. from 10 to 60 kPa. The results indicated that diluted urine had higher flux than undiluted urine with maximum values of 43 and 26 L·m-2·h-1 respectively. Cake formation was the dominating fouling mechanism during urine filtration with a contribution of about 90% to the total hydraulic resistance. The contribution of chemically irreversible fouling was low (-2%), unless operating from high to low pressures. Indeed, irreversible fouling appeared to be greater during the experiments starting at higher pressure. Although undiluted urine had a higher fouling potential compared to diluted urine, the specific cake resistance was higher for diluted urine, probably due to a denser cake caused by lower particle sizes in that sample. The permeate obtained after urine filtration had much lower suspended solids content compared to the feedstock, with rejections up to 99%. The concentration of the ionic species remained unchanged, and 75% of the organic compounds and dissolved solids remained in the permeate. Urine UF could then be used as pre-treatment to remove suspended solids.

Effect of ageing on the physicochemical properties of human faeces in the context of onsite sanitation.

Sanitation systems involving onsite storage of faecal matter cause excreta to undergo transformation and primary treatment in-situ. However, little is known about the transformation pathway followed by fresh faeces while contained in situ. The current paper investigated this transformation under ambient conditions during a 16-week in-situ-storage period. Moisture content, drying kinetics, rheological, physicochemical, and thermal properties were analysed to determine the effects of ageing. The faeces experienced dehydration, mainly affecting moisture-dependent characteristics. Moisture content decreased from 79%wt to 26%wt, and water activity of 0.67, which corresponds mainly to the removal of interstitial bound water, reducing mass by 72%. The decreasing moisture content expectantly reduced drying ability, flowability and thermal properties (heat capacity and thermal conductivity). During this period, negligible biodegradation was recorded (volatile solids reduced by 3%), resulting in consistent chemical oxygen demand, particle size, carbon content and calorific values. Ammonium and nitrates decreased, but total nitrogen remained unchanged. Therefore, ageing affects nitrogen chemical forms and not nutrient composition. The findings demonstrate the benefits of source separation and in particular ventilated storage as a passive way to pre-treat and recover resources from faecal material.

Trans-oceanic host dispersal explains high seabird tick diversity on Cape Verde islands.

Parasites represent ideal models for unravelling biogeographic patterns and mechanisms of diversification on islands. Both host-mediated dispersal and within-island adaptation can shape parasite island assemblages. In this study, we examined patterns of genetic diversity and structure of Ornithodoros seabird ticks within the Cape Verde Archipelago in relation to their global phylogeography. Contrary to expectations, ticks from multiple, geographically distant clades mixed within the archipelago. Trans-oceanic colonization via host movements probably explains high local tick diversity, contrasting with previous research that suggests little large-scale dispersal in these birds. Although host specificity was not obvious at a global scale, host-associated genetic structure was found within Cape Verde colonies, indicating that post-colonization adaptation to specific hosts probably occurs. These results highlight the role of host metapopulation dynamics in the evolutionary ecology and epidemiology of avian parasites and pathogens.

Convective drying kinetics of faecal sludge from VIP latrines.

In striving to achieve sustainable sanitation, one challenge is to ensure hygienic treatment of faecal sludge from on-site sanitation. Thermal drying is an important treatment step for moisture removal and disinfection. Improved understanding of the drying process is crucial for the proper design of treatment technologies for faecal sludge. In this study, faecal sludge from ventilated improved pit (VIP) latrines from Durban, South Africa, were dried in a convective drying thermobalance by varying the temperature from 40 to 80 °C, the relative humidity from 0 to 25% and the air velocity from 0.3 to 1.2 mm/s. The faecal sludge samples were in the form of a thin layer and pellets with different diameters from 8 to 14 mm. Kinetic parameters were determined from the experimental data, were compared to classical drying models in literature and were then used to develop a correlative drying model. Drying rates ranged between 1 and 40 g/min/m2, leading to drying times comprised between 100 and 300 min. The drying kinetics increased as temperature was higher, and pellet diameter and relative humidity were lower. Temperature had the greatest influence on the drying kinetics (in both the constant and falling rate periods), followed by the effect of pellet diameter. The drying kinetics were affected in a moderate way by the relative humidity in the constant rate period and part of the falling rate period. The air velocity had a slight effect of drying kinetics during the constant rate period, but this becomes insignificant during the falling rate period. The effective diffusivities increased from 7.81 × 10-8 to 1.97 × 10-7 m2/s by increasing the temperature from 40° to 80 °C, leading to an activation energy of 23 kJ/mol. These values are typical from those found for wastewater sludge. The sludge exhibited a critical moisture content varying between 2.4 and 3.2 g/g db during drying without a clear trend as a function of the operating conditions and suggested that sludge was composed of considerably more bound moisture than unbound. The experimental data fitted the most closely to the Page model and, based on this, a new model was proposed for the prediction of drying times across the range of explored temperatures and pellet diameters in this study. The results of the proposed model fitted the experimental data with acceptable accuracy, so that the developed model could be employed as an analytical tool for the design, operation and optimisation of drying equipment.

Mechanisms of global diversification in the brown booby (Sula leucogaster) revealed by uniting statistical phylogeographic and multilocus phylogenetic methods.

Recent theoretical and empirical research suggests that statistical models based on coalescent theory can improve both phylogeographic and phylogenetic inference. An approach that involves elements of both statistical phylogeography (e.g. Isolation with Migration analyses) and multilocus phylogenetic inference (e.g. *beast) may be particularly useful when applied to populations with relatively old divergence times. Here, we use such an approach in the globally distributed brown booby (Sula leucogaster). We sampled 215 individuals from all major breeding areas and genotyped them at eight microsatellite and three nuclear intron loci. We found that brown booby populations were highly differentiated and that colonies can be grouped into four major genetic populations (Caribbean Sea, Central Atlantic Ocean, Indo-Central Pacific and Eastern Pacific). These populations apparently diverged in the absence of gene flow and, with one exception, currently exchange few to no migrants. The Eastern Pacific population diverged from all other populations approximately one million years ago [90% highest posterior density: 330,000-2,000,000 years ago] and exhibits a distinct male plumage, relative to other populations. However, recent gene flow from the Indo-Central Pacific into the Eastern Pacific appears to have occurred, suggesting that approximately one million years of genetic isolation and divergence in male plumage colour are not sufficient to prevent interbreeding. Gene flow following secondary contact of the Indo-Central Pacific and Eastern Pacific populations was not detected in previous mitochondrial DNA (mtDNA) studies, and the contrast between the mtDNA results and our current results highlights the advantage of a multilocus phylogeographic approach.

A multilocus phylogeny of the Sulidae (Aves: Pelecaniformes).

Gene trees will often differ from the true species history, the species tree, as a result of processes such as incomplete lineage sorting. New methods such as Bayesian Estimation of the Species Tree (BEST) use the multispecies coalescent to model lineage sorting, and directly infer the species tree from multilocus DNA sequence data. The Sulidae (Aves: Pelecaniformes) is a family of ten booby and gannet species with a global distribution. We sequenced five nuclear intron loci and one mitochondrial locus to estimate a species tree for the Sulidae using both BEST and by concatenating nuclear loci. We also used fossil calibrated strict and relaxed molecular clocks in BEAST to estimate divergence times for major nodes in the sulid phylogeny. Individual gene trees showed little phylogenetic conflict but varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. On the other hand, both the BEST and concatenated species trees were highly resolved, strongly supported, and topologically consistent with each other. The three sulid genera (Morus, Sula, Papasula) were monophyletic and the relationships within genera were mostly consistent with both a previously estimated mtDNA gene tree and the mtDNA gene tree estimated here. However, our species trees conflicted with the mtDNA gene trees in the relationships among the three genera. Most notably, we find that the endemic and endangered Abbott's booby (Papasula abbotti) is likely basal to all other members of the Sulidae and diverged from them approximately 22 million years ago.

Comparative phylogeography of brown (Sula leucogaster) and red-footed boobies (S. sula): the influence of physical barriers and habitat preference on gene flow in pelagic seabirds.

To test the hypothesis that both physical and ecological barriers to gene flow drive population differentiation in tropical seabirds, we surveyed mitochondrial control region variation in 242 brown boobies (Sula leucogaster), which prefer inshore habitat, and 271 red-footed boobies (S. sula), which prefer pelagic habitat. To determine the relative influence of isolation and gene flow on population structure, we used both traditional methods and a recently developed statistical method based on coalescent theory and Bayesian inference (Isolation with Migration). We found that global population genetic structure was high in both species, and that female-mediated gene flow among ocean basins apparently has been restricted by major physical barriers including the Isthmus of Panama, and the periodic emergence of the Sunda and Sahul Shelves in Southeast Asia. In contrast, the evolutionary history of populations within ocean basins differed markedly between the two species. In brown boobies, we found high levels of population genetic differentiation and limited gene flow among colonies, even at spatial scales as small as 500km. Although red-footed booby colonies were also genetically differentiated within ocean basins, coalescent analyses indicated that populations have either diverged in the face of ongoing gene flow, or diverged without gene flow but recently made secondary contact. Regardless, gene flow among red-footed booby populations was higher than among brown booby populations. We suggest that these contrasting patterns of gene flow within ocean basins may be explained by the different habitat preferences of brown and red-footed boobies.

Effect of drying on the physical and chemical properties of faecal sludge for its reuse.

This work explores the effect of drying on the chemical and physical properties of faecal sludge, and evaluates the reuse potential of the dried material. For the purpose of this study, the nutrient content, calorific value and thermal properties were determined for faecal sludge samples dried at different moisture contents and under different drying and operating conditions. The results show that drying does not affect the nutrient content and calorific value, but it induces modifications of the chemical form of nitrogen and the thermal properties. The dried product was demonstrated to be suitable for reuse as an agricultural product and biofuel. In agriculture, dried faecal sludge could be used as an organic fertilizer with a particularly high phosphorous content and a slow release of nitrogen and phosphorous. As a biofuel, dried faecal sludge would have similar characteristics as wood.

Assessment of anti-TNF-alpha efficacy in rheumatoid arthritis: is 3 months sufficient?

OBJECTIVES: The optimal therapeutic trial duration of anti-TNF-alpha therapy is currently unknown. The British Society for Rheumatology (BSR) guidance states that non-response at 3 months warrants re-evaluation of treatment and recommends not to persist beyond 6 months. The National Institute for Health and Clinical Excellence (NICE) specifies treatment continuation if response is achieved by 6 months, yet the European League against Rheumatism (EULAR) and the American College of Rheumatology (ACR) maintain a 3 month cut-off. No evidence exists to support a 6 month therapeutic trial over 3 months. Thus, we undertook a study to evaluate the proportion of patients who failed to meet NICE response criteria at 3 months but obtained this by 6 months, and to identify predictive factors for this. METHODS: Patients who commenced anti-TNF-alpha therapy for RA were studied, counting those who switched to a second or third agent separately for each instigation of therapy (n = 244). Response at 3 and 6 months was defined according to NICE criteria as a >or=1.2 reduction in Disease Activity Score (DAS28). RESULTS: Of the 189 patients with available 3 month DAS28 responses, 149 fulfilled response criteria. Of the 40 who failed, 27 continued treatment, of whom 21 were available for follow-up at 6 months. Out of the 21 patients, 12 (57%; 95% CI 36, 78) achieved a response at this time. This data set was too small to investigate predictors of response at 6 months. CONCLUSIONS: A substantial proportion of patients who fail NICE response criteria at 3 months and continue on treatment to 6 months achieve a response. These results support a 6 month therapeutic trial over 3 months.

Population genetic structure in Atlantic and Pacific Ocean common murres (Uria aalge): natural replicate tests of post-Pleistocene evolution.

Understanding the factors that influence population differentiation in temperate taxa can be difficult because the signatures of both historic and contemporary demographics are often reflected in population genetic patterns. Fortunately, analyses based on coalescent theory can help untangle the relative influence of these historic and contemporary factors. Common murres (Uria aalge) are vagile seabirds that breed in the boreal and low arctic waters of the Northern Hemisphere. Previous analyses revealed that Atlantic and Pacific populations are genetically distinct; however, less is known about population genetic structure within ocean basins. We employed the mitochondrial control region, four microsatellite loci and four intron loci to investigate population genetic structure throughout the range of common murres. As in previous studies, we found that Atlantic and Pacific populations diverged during the Pleistocene and do not currently exchange migrants. Therefore, Atlantic and Pacific murre populations can be used as natural replicates to test mechanisms of population differentiation. While we found little population genetic structure within the Pacific, we detected significant east-west structuring among Atlantic colonies. The degree that population genetic structure reflected contemporary population demographics also differed between ocean basins. Specifically, while the low levels of population differentiation in the Pacific are at least partially due to high levels of contemporary gene flow, the east-west structuring of populations within the Atlantic appears to be the result of historic fragmentation of populations rather than restricted contemporary gene flow. The contrasting results in the Atlantic and Pacific Oceans highlight the necessity of carefully considering multilocus nonequilibrium population genetic approaches when reconstructing the demographic history of temperate Northern Hemisphere taxa.

DNA fragmentation in chronic glomerulonephritis: an immunohistological analysis.

BACKGROUND: Experimental data suggest that apoptosis plays an important pathophysiological role in glomerulonephritis by restoring tissue structure after proliferation of intrinsic renal cells and infiltration of leukocytes. Relatively little is known of apoptosis in human glomerulonephritis, particularly in predicting renal function during follow-up. METHODS: In order to colocalize different markers for cell damage in renal tissue from patients with different forms of glomerulonephritis (GN), a series of semithin sections from 34 kidney biopsies were studied retrospectively. Normal kidney from a nephrectomy specimen with a small renal adenocarcinoma served as a control. DNA fragmentation, expression of tissue transglutaminase II, BAX and BCL-2 were visualized immunohistochemically. In some renal biopsies, immunohistochemical staining for activated caspase 3 was performed. Proinflammatory markers (C-reactive protein, leukocytes), serum creatinine, creatinine clearance, total proteinuria, albuminuria, alpha(1)-microglobulin and IgG excretion were determined at the time of biopsy. Serum creatinine and total proteinuria were assessed 6 and 12 months after renal biopsy. RESULTS: Nuclei with different degrees of DNA fragmentation were mainly found in epithelial cells of tubules, but also in glomerular cells, regardless of the form of GN studied. Transglutaminase II expression was found only in cells with a strong staining for DNA fragmentation. DNA fragmentation localized to glomerular cells was more pronounced in proliferative than in non-proliferative forms of GN, being most abundant in patients with rapid progressive GN. Staining for activated caspase 3 in selected biopsies confirmed the presence of apoptosis. BAX and BCL-2 staining was detected within the same cells, but exhibited a different intracellular distribution. In proliferative GN, the extent of DNA damage in tubular epithelial cells significantly corresponds with the concentration of serum creatinine (p < 0.04) and with urinary excretion of alpha(1)-microglobulin (p < 0.01) at the time of biopsy. A significant correlation (p < 0.01) was seen between glomerular DNA fragmentation and follow-up total proteinuria 12 months after biopsy for proliferative forms of GN. The damaged glomerular area (e.g. mesangial sclerosis) significantly correlated with DNA fragmentation in proliferative, but not in nonproliferative GN at the time of biopsy. Furthermore, glomerular damaged showed a significant correlation with tubular DNA damage in proliferative GN. CONCLUSION: In glomerular cells, apoptosis may be important for the clearance of proliferating cells whereas in tubules, cell damage showed dependence on the degree of tubular injury mediated by inflammation and/or proteinuria. Although the degree of apoptosis in tubular cells correlates with serum creatinine in proliferative GN at the time of biopsy, it is of limited use to predict future renal function.

Branched chain amino acids induce apoptosis in neural cells without mitochondrial membrane depolarization or cytochrome c release: implications for neurological impairment associated with maple syrup urine disease.

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain alpha-keto acid dehydrogenase that can result in neurodegenerative sequelae in human infants. In the present study, increased concentrations of MSUD metabolites, in particular alpha-keto isocaproic acid, specifically induced apoptosis in glial and neuronal cells in culture. Apoptosis was associated with a reduction in cell respiration but without impairment of respiratory chain function, without early changes in mitochondrial membrane potential and without cytochrome c release into the cytosol. Significantly, alpha-keto isocaproic acid also triggered neuronal apoptosis in vivo after intracerebral injection into the developing rat brain. These findings suggest that MSUD neurodegeneration may result, at least in part, from an accumulation of branched chain amino acids and their alpha-keto acid derivatives that trigger apoptosis through a cytochrome c-independent pathway.

Response and Commentary.

Barbarism and Religion, the first half of J. G. A. Pocock's study of Gibbon's Decline and Fall, was designed to set Gibbon's work in context, using a method developed in Cambridge, by juxtaposing his narrative to many others-to some but not all of which he explicitly referred. Helena Rosenblatt has misunderstood Pocock's intent, which was to show that Gibbon's work took much of its inspiration for its treatment of Christianity from a world of ecclesiastical scholarship. Though Pierre Force shows that Voltaire's erudition was richer than has been thought, he and Gibbon pursued very different forms of learning. And though Pocock agrees with Jonathan Israel that many thinkers challenged traditional Christian theology, their efforts were far more varied than Israel holds. Brief studies of Jean LeClerc and Ralph call Israel's vision of radical Enlightenment into question.

Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release.

The distribution of protein kinase C activity and specific phorbol ester binding sites between soluble and particulate fractions of isolated guinea-pig cerebral cortical synaptosomes is examined following preincubation with phorbol esters. Half-maximal decrease in cytosolic activity requires 10 nM 4 beta-phorbol myristoyl acetate. Specific [3H]phorbol dibutyrate binding sites are translocated from cytoplasmic to particulate fractions in parallel with protein kinase C activity. Depolarization of the plasma membrane by 30 mM KCl does not cause translocation of protein kinase C. 1 microM 4 beta-phorbol myristoyl acetate and 1 microM 4 beta-phorbol didecanoate (but not 1 microM 4 alpha-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4 beta-phorbol myristoyl acetate is ineffective at 20 nM. 1 microM 4 beta-phorbol myristoyl acetate slightly increases the cytosolic free Ca2+ concentration of polarized synaptosomes, but not that following partial depolarization. 4 beta-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca2+-dependent glutamate release induced by sub-optimal ionomycin concentrations, but is without effect on the release induced by maximal ionomycin. It is concluded that phorbol esters stereospecifically enhance the Ca2+-sensitivity of glutamate release, but that higher concentrations may be required than for protein kinase C translocation in the same preparation. Instead the enhancement may be related to the rapid inactivation of protein kinase C which occurs with phorbol esters.

Phosphorylation of synapsin I and MARCKS in nerve terminals is mediated by Ca2+ entry via an Aga-GI sensitive Ca2+ channel which is coupled to glutamate exocytosis.

Ca2+ entry is a prerequisite for both exocytosis and the phosphorylation of synapsin I and MARCKS proteins in mammalian cerebrocortical synaptosomes. The novel spider toxin Aga-GI completely blocks KCl-evoked glutamate exocytosis but only partially inhibits KCl-evoked cytoplasmic Ca2+ elevations, thus revealing at least two pathways for KCl-induced Ca2+ entry. Aga-GI completely attenuates KCl-induced phosphorylation of synapsin I and MARCKS proteins. We therefore conclude that both exocytosis and the phosphorylation of synapsin I and MARCKS proteins are specifically coupled to Ca2+ entry via a subset of voltage dependent Ca2+ channels at the nerve terminal which are sensitive to Aga-GI.

The calcium channel coupled to the exocytosis of L-glutamate from cerebellar granule cells is inhibited by the spider toxin, Aga-GI.

The increase in cytosolic calcium, [Ca2+]c, evoked with 50 mM KCl in cerebellar granule cells consists of four components; (1) a rapidly inactivating transient or spike; (2) a nifedipine-sensitive non-inactivating plateau; (3) an Aga-GI (spider toxin) sensitive non-inactivating plateau; (4) a residual non-inactivating plateau insensitive to nifedipine and Aga-GI. None of these components is blocked by synthetic arginine polyamine toxin, spermine, (+)-MK-801 hydrogen maleate, D(-)-2-amino-5-phosphonopentanoic acid or omega-conotoxin-GVIA. The proposed P-type channel antagonist, omega-agatoxin-IVA, has a limited but non-significant effect on the elevated plateau [CA2+]c.L-type Ca2+ channels are located primarily on the soma whereas the component of the plateau which is blocked specifically by Aga-GI is localized primarily on the cell neurites. The latter component is coupled to the exocytosis of endogenous glutamate evoked with 50 mM KCl.

Glutamate exocytosis from cerebellar granule cells: the mechanism of a transition to an L-type Ca2+ channel coupling.

When cerebellar granule cells in the presence of 1.3 mM calcium chloride (Ca2+) are depolarized by high potassium chloride (KCl), the release of endogenous glutamate is coupled to a high threshold Ca2+ channel blocked by the spider toxin omega Agatoxin-glutamate-release-inhibitor (Aga-GI) and insensitive to the L-type voltage-dependent Ca2+ channel-inhibitor nifedipine. A prolonged KCl depolarization in the absence of Ca2+ followed by addition of 5 mM Ca2+ results in an enhanced nifedipine-sensitive Ca2+ entry; glutamate exocytosis retains sensitivity to tetanus toxin and bafilomycin A1, is now totally inhibited by nifedipine and shows greatly reduced sensitivity to AGA-GI. Single cell Ca2+ imaging indicates that the L-type channel modulating release is preferentially located at somatic regions rather than neurites. A different pattern of vesicle endocytosis monitored with the fluorescent indicator FM1-43 is seen in response to the two depolarization protocols. Furthermore, vesicles loaded during depolarization with high KCl in the presence of 5 mM Ca2+ extensively exocytose dye in a nifedipine-insensitive manner in response to a second similar stimulation but release little dye in response to stimulus with high KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+. In contrast, vesicles loaded by stimulating with KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+ can be released by a second similar stimulus and this release is sensitive to nifedipine. Nifedipine sensitivity is not induced in cerebellar synaptosomes subjected to stimulation with high KCl in the absence of Ca2+ followed by the re-addition of 5 mM Ca2+. The results indicate that different populations of channels and vesicles may be functional during two depolarization protocols.