Activity-dependent bulk endocytosis proteome reveals a key presynaptic role for the monomeric GTPase Rab11.

Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle endocytosis during high-frequency stimulation, suggesting it should play key roles in neurotransmission during periods of intense neuronal activity. However, efforts in elucidating the physiological role of ADBE have been hampered by the lack of identified molecules which are unique to this endocytosis mode. To address this, we performed proteomic analysis on purified bulk endosomes, which are a key organelle in ADBE. Bulk endosomes were enriched via two independent approaches, a classical subcellular fractionation method and isolation via magnetic nanoparticles. There was a 77% overlap in proteins identified via the two protocols, and these molecules formed the ADBE core proteome. Bioinformatic analysis revealed a strong enrichment in cell adhesion and cytoskeletal and signaling molecules, in addition to expected synaptic and trafficking proteins. Network analysis identified Rab GTPases as a central hub within the ADBE proteome. Subsequent investigation of a subset of these Rabs revealed that Rab11 both facilitated ADBE and accelerated clathrin-mediated endocytosis. These findings suggest that the ADBE proteome will provide a rich resource for the future study of presynaptic function, and identify Rab11 as a regulator of presynaptic function.

A multiarm randomized field trial evaluating strategies for udder health improvement in Swiss dairy herds.

The aims of this study were to quantify the effectiveness of specialist advice about udder health in Swiss dairy herds and to compare 3 different udder health improvement strategies against a negative control group. In 2010, 100 Swiss dairy herds with a high (between 200,000 and 300,000 cells/mL) yield-corrected bulk milk somatic cell count (YCBMSCC) were recruited for a 1-yr multiarm randomized field trial. The herds were visited between September and December 2011 to evaluate udder health-management practices and then randomly allocated into 1 of 4 study arms containing 25 herds each. The negative control study arm received neither recommendations for improving udder health nor any active support. The remaining 75 farmers received a herd-specific report with recommendations to improve udder health management. The positive control study arm received no further active support during 2012. The veterinarian study arm received additional support in the form of monthly visits by their herd veterinarian. Finally, the study group study arm received support in the form of bimonthly study group meetings where different topics concerning udder health were discussed. One year later, implementation of recommendations and changes in udder health were assessed. Of the recommendations given, 44.3% were completely implemented, 23.1% partially, and 32.6% were not implemented. No differences in implementation of recommendations were noted between the 3 study arms. At study enrollment, farmers were asked for the study arm of their preference but were subsequently randomly assigned to 1 of the 4 study arms. Farmers that were assigned to the study arm of their preference implemented more recommendations than farmers assigned to a study arm not of their preference. No decrease in the within-herd prevalence of cows that had a high (≥200,000 cells/mL) composite somatic cell count was observed in herds that had a YCBMSCC ≥200,000 cells/mL at the start of intervention. However, the 3 study arms with intervention (positive control, the veterinarian, and the study groups) prevented an increase in the within-herd prevalence of cows that had a high somatic cell count in herds with a low YCBMSCC at the start of the intervention compared with the negative control study arm. In the year after sending the report, herds assigned to the study group study arm had a reduced incidence rate of treated mastitis cases in comparison with the year before sending the report.

Increasing use of third-generation cephalosporins for pneumonia in the emergency department: may some prescriptions be avoided?

Third-generation cephalosporins are used to treat inpatients with community-acquired pneumonia. Some of these prescriptions may be avoided, i.e. replaced by agents less likely to promote ESBL-mediated resistance. Our objectives were to assess the recent trend of third-generation cephalosporins use for pneumonia in the emergency department, and the proportion of avoidable prescriptions. This was a retrospective study of patients treated for community-acquired pneumonia in an emergency department, and subsequently hospitalized in non ICU wards. Third-generation cephalosporin prescriptions were presumed unavoidable if they met both criteria: (i) age ≥ 65 yr or comorbid condition, and (ii) allergy or intolerance to penicillin, or failure of penicillin first-line therapy, or treatment with penicillin in three previous months. Prescriptions were otherwise deemed avoidable. The proportion of patients treated with a third generation cephalosporin increased significantly from 13.9 % (6.9-24.1 %) in 2002 to 29.5 % (18.5-42.6 %) in 2012 (OR = 1.07 [1.01-1.14] , P = 0.02). This increase was independent from other factors associated with the prescription of a third-generation cephalosporin (immunocompromising condition, antibacterial therapy in three previous months, fluid resuscitation and REA-ICU class). Treatment with third-generation cephalosporin was avoidable in 118 out of 147 patients (80.3 % [72.7-86.2 %]). On day 7 after admission in the ED, treatment with third-generation cephalosporins was stopped or de-escalated in, respectively, 17 % and 32 % of patients. Antibiotic stewardship programs should be implemented to restrict the third-generation cephalosporins use for pneumonia in the emergency department.

A novel immunoassay using recombinant allergens simplifies peanut allergy diagnosis.

BACKGROUND: Double-blind placebo-controlled food challenge (DBPCFC) is currently considered the gold standard for peanut allergy diagnosis. However, this procedure that requires the hospitalization of patients, mostly children, in specialized centers for oral exposure to allergens may cause severe reactions requiring emergency measures. Thus, a simpler and safer diagnosis procedure is needed. The aim of this study was to evaluate the diagnostic performance of a new set of in vitro blood tests for peanut allergy. METHODS: The levels of IgE directed towards peanut extract and recombinant peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 7, and Ara h 8 were measured in 3 groups of patients enrolled at 2 independent centers: patients with proven peanut allergy (n=166); pollen-sensitized subjects without peanut allergy (n=61), and control subjects without allergic disease (n=10). RESULTS: Seventy-nine percent of the pollen-sensitized patients showed IgE binding to peanut, despite their tolerance to peanut. In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/l. Use of a threshold of 0.23 kU/l for recombinant Ara h 2 increased specificity (96%) at the cost of sensitivity (93%). CONCLUSION: A simple blood test can be used to diagnose peanut allergy with a high level of precision. However, DBPCFC will remain useful for the few cases where immunological and clinical observations yield conflicting results.

Activity-dependent bulk synaptic vesicle endocytosis--a fast, high capacity membrane retrieval mechanism.

Central nerve terminals are placed under considerable stress during intense stimulation due to large numbers of synaptic vesicles (SVs) fusing with the plasma membrane. Classical clathrin-dependent SV endocytosis cannot correct for the large increase in nerve terminal surface area in the short term, due to its slow kinetics and low capacity. During such intense stimulation, an additional SV retrieval pathway is recruited called bulk endocytosis. Recent studies have shown that bulk endocytosis fulfils all of the physiological requirements to remedy the acute changes in nerve terminal surface area to allow the nerve terminal to continue to function. This review will summarise the recent developments in the field that characterise the physiology of bulk endocytosis which show that it is a fast, activity-dependent and high capacity mechanism that is essential for the function of central nerve terminals.

Differential labelling of bulk endocytosis in nerve terminals by FM dyes.

Bulk endocytosis is triggered in central nerve terminals during intense physiological stimulation. This endocytosis pathway can be labelled by the dye FM1-43 but not its more hydrophilic counterpart FM2-10. This selective labelling was proposed to be due to the retention of FM1-43, but not FM2-10, in slowly retrieving structures after washout of the dye. However, this explanation assumed that bulk endocytosis was a slow process that persisted after stimulation. We have recently shown that the great majority of bulk endocytosis occurs during stimulation, therefore another explanation for the specific labelling of this pathway by FM1-43 must be found. In this paper we show that the ability of FM dyes to label bulk endocytosis is dependent on the concentration of dye used and not their washout properties. When the loading concentration of FM1-43 was reduced 10-fold, its ability to label bulk endocytosis was lost. Conversely when the loading concentration of FM2-10 was increased 10-fold, it now labelled the pathway. This suggests that a difference in affinity of bulk endosome membranes for FM1-43 and FM2-10 underlies the disparity in labelling.

Functional reconstitution of mammalian 'chloride intracellular channels' CLIC1, CLIC4 and CLIC5 reveals differential regulation by cytoskeletal actin.

Chloride intracellular channels (CLICs) are soluble, signal peptide-less proteins that are distantly related to Omega-type glutathione-S-transferases. Although some CLICs bypass the classical secretory pathway and autoinsert into cell membranes to form ion channels, their cellular roles remain unclear. Many CLICs are strongly associated with cytoskeletal proteins, but the role of these associations is not known. In this study, we incorporated purified, recombinant mammalian CLIC1, CLIC4 and (for the first time) CLIC5 into planar lipid bilayers, and tested the hypothesis that the channels are regulated by actin. CLIC5 formed multiconductance channels that were almost equally permeable to Na(+), K(+) and Cl(-), suggesting that the 'CLIC' nomenclature may need to be revised. CLIC1 and CLIC5, but not CLIC4, were strongly and reversibly inhibited (or inactivated) by 'cytosolic' F-actin in the absence of any other protein. This inhibition effect on channels could be reversed by using cytochalasin to disrupt the F-actin. We suggest that actin-regulated membrane CLICs could modify solute transport at key stages during cellular events such as apoptosis, cell and organelle division and fusion, cell-volume regulation, and cell movement.

The dephosphins: dephosphorylation by calcineurin triggers synaptic vesicle endocytosis.

When nerve terminals in the brain are stimulated, a group of phosphoproteins called the dephosphins are coordinately dephosphorylated by calcineurin, the Ca(2+)-dependent protein phosphatase. Amazingly, the seven presently known dephosphins are not structurally related, yet each has been independently shown to be essential for synaptic vesicle endocytosis (SVE). Nowhere else in biology is there a similar example of the coordinated dephosphorylation of such a large group of proteins each sharing roles in the same biological response. This suggests that dephosphorylation and phosphorylation of the dephosphins is essential for SVE. Recent studies in synaptosomes have confirmed this view, with calcineurin-mediated dephosphorylation of the dephosphins essential for triggering SVE. The phosphorylation cycle of the dephosphins might regulate SVE by targeting the proteins to sites of action and by stimulating the assembly of several large essential endocytic protein complexes.

A predictive model to describe the effects of temperature, sodium lactate, and sodium diacetate on the inactivation of a serotype 4b strain of Listeria monocytogenes in a frankfurter slurry.

A modified Gompertz equation was used to model the effects of temperature (55, 60, and 65 degrees C), sodium lactate (0, 2.4, and 4.8%), and sodium diacetate (0, 0.125, and 0.25%) on inactivation of Listeria monocytogenes strain MFS 102 (serotype 4b) in frankfurter slurry. The effects of these factors were determined on the shouldering region (parameter A), maximum death rate (parameter B), and tailing region (parameter C) of microbial inactivation curves. Increased temperature or sodium diacetate concentrations increased the death rate, whereas increased sodium lactate concentrations decreased heat resistance. Complex two-way interactive effects were also observed. As both temperature and sodium lactate increased, the death rate decreased; however, as temperature and sodium diacetate increased, the death rate increased. The effect of the interaction between sodium lactate and sodium diacetate on the maximum death rate varied with temperature. Increases in both acidulants at temperatures above 56.7 degrees C decreased the death rate, whereas at temperatures below 56.7 degrees C, increases in both acidulants increased the death rate. To test for significant differences between treatments, D-values were calculated and compared. This comparison revealed that, in general, sodium lactate increased heat resistance and sodium diacetate decreased heat resistance of L. monocytogenes. This information is important for reducing and minimizing contamination during postprocessing thermal treatments.

Detection of Escherichia coli O157:H7 and Salmonella typhimurium using filtration followed by Fourier-transform infrared spectroscopy.

Fourier-transform infrared spectroscopy has been successfully used as a nondestructive method for identifying, distinguishing, and classifying pathogens. In this study, a less time-consuming Fourier-transform infrared procedure was developed to identify Escherichia coli O157:H7 and Salmonella Typhimurium. Samples containing 10(9) CFU/ml were prepared in tryptic soy broth and then serially diluted (up to eight times) to obtain bacterial solutions of 10(9) to 10 CFU/ml. These dilutions were incubated at 37 degrees C for 6 h, samples were filtered through a Metricel filter hourly (for 0 to 6 h), and spectra were obtained using a ZnSe contact attenuated total reflectance accessory on a Continu mum infrared microscope. Midinfrared spectra (4,000 to 700 cm(-1)) of Salmonella Typhimurium and E. coli O157:H7 were generated, and peak areas in the region of 1,589 to 1,493 cm(-1) were used to detect the pathogens. Initially, detection limits were between 10(6) and 10(7) CFU/ml without preenrichment, and samples starting with 500 CFU/ml were detectable following incubation for 6 h, when counts reached at least 10(6) CFU/ ml. Compared with results of previously published studies in which Fourier-transform infrared spectroscopy was used to identify select pathogens, this method is more rapid and less expensive for practical large-scale sample analysis.

[Economic consequences of biological monitoring and medical complications of injectable anticoagulants in France]. / Conséquences économiques du suivi biologique et des complications médicales en lien avec l'utilisation des anticoagulants injectables en France.

AIM: To assess the frequency of platelet monitoring and bleeding risks associated with the use of injectable anticoagulants in a real life setting and to estimate the associated costs. METHOD: An analysis of the 2013 data from a random sample of ≈600,000 patients registered in the French National Health Insurances reimbursement database was conducted to identify platelet counts performed during injectable anticoagulants exposure period and treatment interruptions due to heparin-induced thrombocytopenia or transfusion. Events were then valued to establish associated costs. RESULTS: Overall 15,985 adult patients representing a cumulated injectable anticoagulants exposure time of 12,264 months were selected. Treatment sequences involved unfractionated heparin (2.8%), low molecular weight heparin (86.9%), and fondaparinux (13.1%). Patients treated with unfractionated heparin were older (77 vs. 57 and 59 years) with longer treatment duration (32.6 vs. 25.1 and 21 days). After statistical adjustment, the average monthly number of platelet counts was 1.36-fold lower in patients treated with fondaparinux compared to low molecular weight heparin (P<0.0001). No difference was found between low molecular weight heparin and fondaparinux regarding the incidence of bleeding with transfusion (P=0.76) or hospitalized thrombocytopenia (P=0.82). Extrapolated for the whole country, the estimated costs for biological monitoring were € 21.6 million for low molecular weight heparin and € 0.9 million for fondaparinux. CONCLUSION: Significantly fewer platelet counts were performed among patients treated with fondaparinux than among patients receiving low molecular weight heparin without additional bleeding risk. This finding should be taken into account when assessing the costs of such treatments.

Ca(2+) influx inhibits dynamin and arrests synaptic vesicle endocytosis at the active zone.

Ca(2+) entry into nerve terminals through clusters of voltage-dependent Ca(2+) channels (VDCCs) at active zones creates a microdomain of elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)) that stimulates exocytosis. We show that this VDCC-mediated [Ca(2+)](i) elevation has no specific role in stimulating endocytosis but can inhibit endocytosis evoked by three different methods in isolated mammalian nerve terminals. The inhibition can be relieved by using either VDCC antagonists or fast, but not slow, binding intracellular Ca(2+) chelators. The Ca(2+)-dependent inhibition of endocytosis is mimicked in vitro by a low-affinity inhibition of dynamin I vesiculation of phospholipids. Increased [Ca(2+)](i) also inhibits dynamin II GTPase activity and receptor-mediated endocytosis in non-neuronal cells. VDCC-meditated Ca(2+) entry inhibits dynamin-mediated endocytosis at the active zone and provides neurons with a mechanism to clear recycling vesicles to nonactive zone regions during periods of high activity.

Behavior of Listeria monocytogenes in pH-modified chicken salad during refrigerated storage.

The growth and inactivation kinetics of L. monocytogenes were evaluated at pH 4.0, 4.6, and 5.2 during storage at 5.0 degrees C, 7.2 degrees C, and 21.1 degrees C (41 degrees F, 45 degrees F, and 70 degrees F). Using commercially produced pasteurized chicken salad, the authors adjusted the pH levels with acetic acid or sodium acetate. Samples of 25 g each of the pH-modified salad were inoculated to approximately 1 x 10(6) cells per gram with a three-strain mixture of L. monocytogenes and stored for up to 119 days. Samples were enumerated for L. monocytogenes according to the Food and Drug Administration-modified most-probable-number (MPN) procedure, and log MPN was plotted against time. Inactivation was seen at all pH levels and at all temperatures. At 21.1 degrees C, a 6-log reduction was seen after 14 days at pH 4.0, after 52 days at pH 4.6, and after 38 days at pH 5.2. Inactivation at 21.1 degrees C began within hours or days at pH 4.0, and after a lag phase of 10 to 12 days at pH 4.6 and 5.2. Inactivation was slower in cold-storage temperatures. At 7.2 degrees C, a microbial reduction of 1.1 log (pH 5.2) and > 3 log (pH 4.0 and 4.6) was observed at 119 days. At 5 degrees C, a 7.5-log reduction was observed at 24 days at pH 4.0. At pH levels of 4.6 and 5.2, however, only a 4-log reduction was found at 119 days. The data generated in this study may be used to develop predictive models that could specifically address the interactions of pH and storage temperature on the viability of L. monocytogenes in prepared salads.

21.1.1, a novel activation marker of T and B cells.

A new rat mAb designated mAb 21.1.1 was raised against a T cell hybridoma of mouse origin, T2D4. This antibody, an IgG2b, immunoprecipitates from the membrane extracts of iodinated T2D4 cells a 56-kDa glycoprotein of apparent pI 4.6 which gives a 34-kDa polypeptide after treatment with endoglycosidase F. MAb 21.1.1 reacts with an antigen expressed on murine mitogen-activated thymocytes and T cells, and on B cells stimulated by anti-IgM antibodies. Cells isolated from the spleen, lymph nodes and bone marrow are negative, as are purified resting B cells or T cells. This antigen is strongly expressed on most day-16 fetal thymocytes whereas adult thymocytes are almost negative. mAb 21.1.1 may be useful for the study of activation and differentiation of T and B cells.

A live-cell assay for studying extracellular and intracellular endothelin-converting enzyme activity.

Endothelin-1 (ET-1) is formed from its precursor preproET-1 via the cleavage of the intermediate bigET-1 by endothelin-converting enzyme (ECE-1). However, the subcellular site at which this step occurs is not clear: It could occur intravesicularly along the secretory pathway or bigET-1 might be released and processed extracellularly. To address this point, we have developed an integrated autocrine system that uses a recombinant Chinese hamster ovary (CHO) luciferase reporter cell line that permanently expresses the human ET(A) receptor. Into these cells we transiently transfected human ECE-1a cDNA, either together with the human preproET-1 cDNA (as an endogenous source of bigET-1), or alone (in which case exogenous bigET-1 was added). Phosphoramidon inhibited the conversion of exogenous bigET-1 (IC50 = 5 to 30 micromol/L) much better than that of endogenous bigET-1 (IC50 > 1 mmol/L). Both conversions showed similar high yields (20% to 100%) that depended on the amount of ECE-1a expressed. Thus, ECE-1a has two equally relevant activities in this recombinant system for CHO cells: (1) an intracellular, probably intravesicular activity, corresponding to the ECE-1a-mediated step of ET-1 biosynthesis and (2) an extracellular activity at the plasma membrane. If this is also the case for endothelial cells, ECE-1a inhibitors would have to cross the plasma and vesicle membranes to be effective. The present system could be useful for screening such inhibitors.

Synaptic vesicle endocytosis: calcium works overtime in the nerve terminal.

The functions of Ca2+ are many and varied within cells, but in the nerve terminals of neurons it has had a very defined role. That is, the influx of extracellular Ca2+ through voltage-dependent Ca2+ channels stimulates neurotransmitter release by exocytosis. For years this was assumed to be the main role for Ca2+ in this specialized subcellular region. However recent studies have shown that Ca2+ also has multiple roles in synaptic-vesicle endocytosis. This review will present evidence for three Ca2+-dependent and -independent steps; a high-affinity Ca2+-dependent triggering step, a Ca2+-independent maintenance phase, and a low-affinity Ca2+-dependent inhibition step. How the control of endocytosis by Ca2+ might impact on different neuronal functions such as synaptic transmission, the nucleation of SV endocytosis, and the repair of damaged membrane is then discussed.

Stability of 70S ribosomes in relation to misreading and antibacterial activity of aminoglycosides.

The anomeric aminoglycosides, RU 21886 and RU 23468, which both have a 2-deoxystreptamine residue, stabilize 70S ribosomes to similar extents at low magnesium ion concentrations. Only RU 21886, however, has marked antibacterial and bactericidal activity and gives rise to a high level of misreading in cell-free protein synthesizing systems. It would thus appear that the ability to stabilize the association of the two ribosomal subunits does not necessarily lead to errors in translation.

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.

Early anuria prevention in human kidney transplantation. Advantage of fluid load under pulmonary arterial pressure monitoring during surgical period.

In human kidney transplantation, a high blood flow established through the graft immediately upon clamp release is usually associated with immediate satisfactory renal function. One hundred consecutive kidney transplant patients were thus provided with a large volume of fluid during surgery. To avoid pulmonary edema, fluid load was given under mean pulmonary arterial pressure (PAP) monitoring, and controlled ventilation was maintained during the early postoperative period. Whether initial PAP value was within normal range or elevated, all patients required an equivalent fluid load to reach the best hemodynamic condition upon clamp removal. The mean intraoperative fluid load consisted of 2406 +/- 968 ml of water with 22.8 +/- 9.4 g of sodium chloride, 5.9 +/- 1.8 units of albumin, and 2.6 +/- 1.8 units of packed red blood cells. Immediately before clamp release patients were given furosemide and mannitol. During the postoperative period, i.v. infusions consisted of water and sodium chloride (6 g/liter) to match urine output, provided that diuresis was equal to or above 400 ml/hr. If diuresis remained or decreased below this level, diuresis replacement was associated with PAP-controlled infusion of saline, albumin, and red blood cells if needed. Furosemide was eventually given if diuresis did not increase above 400 ml/hr with fluid loading. With this protocol a good early diuresis was established in 95% of the cases. Ten patients required dialysis before the 5th postoperative day, one of them because of fluid overload and anuria. Concurrently, a decreased mortality rate and an increased graft survival rate were observed.

Presynaptic calcium channels and field-evoked transmitter exocytosis from cultured cerebellar granule cells.

Regulated exocytosis from cultured rat cerebellar granule cells can be localized by the vesicle specific marker FM2-10 to specific sites, the highest density of which are at visible varicosities coinciding with neurite-neurite contacts. Exocytosis can be evoked by uniform electrical field pulses, which initiate tetrodotoxin-sensitive action potentials, or by elevated KCl. [3H]D-Aspartate is an authentic false transmitter in this preparation, judged by sensitivity of release to bafilomycin A1 and tetanus toxin. The coupling of presynaptic voltage-activated Ca2+ channels to [3H]D-aspartate exocytosis was determined during field stimulation. The peak cytoplasmic free Ca2+ concentration achieved in the varicosities was proportional to Ca2+ entry during a 10 strain of pulses. L-type Ca2+ channels did not contribute to either Ca2+ entry or [3H]D-aspartate exocytosis. The P-type Ca2+ channel antagonist omega-agatoxin-IVA (30 nM) only inhibited at 75% of the varicosities, although a mean 15% inhibition of Ca2+ entry caused a 39% inhibition of exocytosis. In contrast the N-type Ca2+ channel inhibitor omega-conotoxin-GVIA (1 microM), which inhibited at virtually all varicosities, caused mean inhibitions of Ca2+ entry and exocytosis of 26% and 24% respectively. The toxin omega-conotoxin-MVIIC (5 microM), which inhibits N-, P- and Q-type Ca2+ channels, was effective at all varicosities. The Q-type component of Ca2+ entry was calculated to be only 5-10%; however, the additional inhibition of exocytosis was 30%. Thus P-type and particularly Q-type channels appear to be more closely coupled to exocytosis than N-type Ca2+ channels. The residual Ca2+ entry following 5 microM omega-conotoxin-MVIIC is scarcely coupled to release. The omega-agatoxin-IVA and omega-conotoxin-GVIA inhibitions of both Ca2+ entry and exocytosis were additive and varied stochastically between individual varicosities. These results demonstrate that both Q- and P-type Ca2+ channels are highly efficient in their coupling to amino acid exocytosis, with N-type less efficient, and L-type channels not at all. The Ca2+ channel types coupled to exocytosis are also able to support exocytosis when evoked by either brief field-evoked action potentials or prolonged depolarization with KCl, indicating that these presynaptic channels, in contrast to those on the somata of the cells, can respond to widely different patterns of activation.