Discovery of a Thermostable GH10 Xylanase with Broad Substrate Specificity from the Arctic Mid-Ocean Ridge Vent System.

A two-domain GH10 xylanase-encoding gene (amor_gh10a) was discovered from a metagenomic data set, generated after in situ incubation of a lignocellulosic substrate in hot sediments on the sea floor of the Arctic Mid-Ocean Ridge (AMOR). AMOR_GH10A comprises a signal peptide, a carbohydrate-binding module belonging to a previously uncharacterized family, and a catalytic glycosyl hydrolase (GH10) domain. The enzyme shares the highest sequence identity (42%) with a hypothetical protein from a Verrucomicrobia bacterium, and its GH10 domain shares low identity (24 to 28%) with functionally characterized xylanases. Purified AMOR_GH10A showed thermophilic and halophilic properties and was active toward various xylans. Uniquely, the enzyme showed high activity toward amorphous cellulose, glucomannan, and xyloglucan and was more active toward cellopentaose than toward xylopentaose. Binding assays showed that the N-terminal domain of this broad-specificity GH10 binds strongly to amorphous cellulose, as well as to microcrystalline cellulose, birchwood glucuronoxylan, barley ß-glucan, and konjac glucomannan, confirming its classification as a novel CBM (CBM85).IMPORTANCE Hot springs at the sea bottom harbor unique biodiversity and are a promising source of enzymes with interesting properties. We describe the functional characterization of a thermophilic and halophilic multidomain xylanase originating from the Arctic Mid-Ocean Ridge vent system, belonging to the well-studied family 10 of glycosyl hydrolases (GH10). This xylanase, AMOR_GH10A, has a surprisingly wide substrate range and is more active toward cellopentaose than toward xylopentaose. This substrate promiscuity is unique for the GH10 family and could prove useful in industrial applications. Emphasizing the versatility of AMOR_GH10A, its N-terminal domain binds to both xylans and glycans, while not showing significant sequence similarities to any known carbohydrate-binding module (CBM) in the CAZy database. Thus, this N-terminal domain lays the foundation for the new CBM85 family.

Validation of the register-based lifetime antimicrobial usage measurement for finisher batches based on comparison with recorded antimicrobial usage at farm level.

Assessing the relationship between antimicrobial usage (AMU) and antimicrobial resistance (AMR) requires the accurate and precise utilisation of register data. Therefore, validation of register-based data is essential for evaluating the quality and, subsequently, the internal validity of studies based on the data. In this study, different smoothing methods for Veterinary Medicine Statistic Program database (VetStat)-records were validated by comparing these with farm-records. Comparison between measurements included accuracy as; completeness and correctness, and precision as; a relative difference of the error, correlation with Fisher's z transformation and reliability coefficient. The most valid methods of those examined were then used in re-analyses of the abundance of AMR genes in 10 finisher batches from a previous study. Improved accuracy was found when detailed smoothing methods were applied. Although the precision also increased, the effect was not as pronounced, as the usage estimate of all smoothing methods deviated moderately compared with the farm-registrations. Applying the most valid methods to the 10 finisher batches increased estimates of statistical model fit for aminoglycosides, lincosamides, tetracyclines and decreased estimates of statistical model fit for macrolides. The estimates of statistical model fit for sulfonamides and broad-spectrum penicillins remained the same. Through refined data transformation, VetStat-records can be used to calculate a daily amount of AMU per pig reflecting the true usage accurately and moderately precisely, which is the foundation for calculating lifetime AMU.

The association between measurements of antimicrobial use and resistance in the faeces microbiota of finisher batches.

The objectives were to present three approaches for calculating antimicrobial (AM) use in pigs that take into account the rearing period and rearing site, and to study the association between these measurements and phenotypical resistance and abundance of resistance genes in faeces samples from 10 finisher batches. The AM use was calculated relative to the rearing period of the batches as (i) 'Finisher Unit Exposure' at unit level, (ii) 'Lifetime Exposure' at batch level and (iii) 'Herd Exposure' at herd level. A significant effect on the occurrence of tetracycline resistance measured by cultivation was identified for Lifetime Exposure for the AM class: tetracycline. Furthermore, for Lifetime Exposure for the AM classes: macrolide, broad-spectrum penicillin, sulfonamide and tetracycline use as well as Herd Unit Exposure for the AM classes: aminoglycoside, lincosamide and tetracycline use, a significant effect was observed on the occurrence of genes coding for the AM resistance classes: aminoglycoside, lincosamide, macrolide, ß-lactam, sulfonamide and tetracycline. No effect was observed for Finisher Unit Exposure. Overall, the study shows that Lifetime Exposure is an efficient measurement of AM use in finisher batches, and has a significant effect on the occurrence of resistance, measured either by cultivation or metagenomics.

Microcirculatory dysfunction and tissue oxygenation in critical illness.

Severe sepsis is defined by organ failure, often of the kidneys, heart, and brain. It has been proposed that inadequate delivery of oxygen, or insufficient extraction of oxygen in tissue, may explain organ failure. Despite adequate maintenance of systemic oxygen delivery in septic patients, their morbidity and mortality remain high. The assumption that tissue oxygenation can be preserved by maintaining its blood supply follows from physiological models that only apply to tissue with uniformly perfused capillaries. In sepsis, the microcirculation is profoundly disturbed, and the blood supply of individual organs may therefore no longer reflect their access to oxygen. We review how capillary flow patterns affect oxygen extraction efficacy in tissue, and how the regulation of tissue blood flow must be adjusted to meet the metabolic needs of the tissue as capillary flows become disturbed as observed in critical illness. Using the brain, heart, and kidney as examples, we discuss whether disturbed capillary flow patterns might explain the apparent mismatch between organ blood flow and organ function in sepsis. Finally, we discuss diagnostic means of detecting capillary flow disturbance in animal models and in critically ill patients, and address therapeutic strategies that might improve tissue oxygenation by modifying capillary flow patterns.

Expression studies of six human obesity-related genes in seven tissues from divergent pig breeds.

Obesity has reached epidemic proportions globally and has become the cause of several major health risks worldwide. Presently, more than 100 loci have been related to obesity and metabolic traits in humans by genome-wide association studies. The complex genetic architecture behind obesity has triggered a need for the development of better animal models than rodents. The pig has emerged as a very promising biomedical model to study human obesity traits. In this study, we have characterized the expression patterns of six obesity-related genes, leptin (LEP), leptin receptor (LEPR), melanocortin 4 receptor (MC4R), fat mass and obesity associated (FTO), neuronal growth regulator 1 (NEGR)1 and adiponectin (ADIPOQ), in seven obesity-relevant tissues (liver; muscle; pancreas; hypothalamus; and retroperitoneal, subcutaneous and mesenteric adipose tissues) in two pig breeds (production pigs and Göttingen minipigs) that deviate phenotypically and genetically from each other with respect to obesity traits. We observe significant differential expression for LEP, LEPR and ADIPOQ in muscle and in all three adipose tissues. Interestingly, in pancreas, LEP expression is only detected in the fat minipigs. FTO shows significant differential expression in all tissues analyzed, and NEGR1 shows significant differential expression in muscle, pancreas, hypothalamus and subcutaneous adipose tissue. The MC4R transcript can be detected only in hypothalamus. In general, the expression profiles of the investigated genes are in accordance with those observed in human studies. Our study shows that both the differences between the investigated breeds and the phenotypic state with respect to obesity/leanness play a large role for differential expression of the obesity-related genes.

Platelet activation and aggregation: the importance of thrombin activity--a laboratory model.

This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated by tissue factor evaluated by means of impedance aggregometry. Citrated whole blood from healthy volunteers and haemophilia A patients with the addition of inhibitors of the contact pathway and fibrin polymerization was evaluated. In healthy persons, a second wave of platelet aggregation was found to coincide with the thrombin burst and to be abolished by thrombin inhibitors. In this system, platelet aggregation in severe haemophilia A (n = 10) was found to be significantly decreased as compared with healthy individuals (912 ± 294 vs. 1917 ± 793 AU × min, P = 0.003), most probably due to the weak level of thrombin generation. For the first time, analysis of platelet aggregation as induced by endogenously generated thrombin was demonstrated. The new method makes it possible to explore the influence of the coagulation system on platelet function. In contrast to the general understanding, the data suggest that the impaired thrombin generation in haemophilia may affect platelet activation. Future studies will address whether our results may contribute to understanding differences in bleeding phenotypes and response to haemostatic substitution observed among patients.

The quantitative effect of antimicrobial usage in Danish pig farms on the abundance of antimicrobial resistance genes in slaughter pigs.

Research has long established the connection between antimicrobial use (AMU) and antimicrobial resistance (AMR) in production animals, and shown that the ceasing of AMU reduces AMR. Our previous study of Danish slaughter-pig production found a quantitative relationship between lifetime AMU and abundance of antimicrobial resistance genes (ARGs). This study aimed to generate further quantitative knowledge on how changes in AMU in farms influence the abundance of ARGs both with immediate effect and over time. The study included 83 farms that were visited from 1 to 5 times. From each visit, a pooled faecal sample was produced. The abundance of ARGs was obtained by metagenomics. We used two-level linear mixed models for estimating the effect of AMU on the abundance of ARGs against six antimicrobial classes. The lifetime AMU of each batch was calculated from usage during their three rearing periods; as piglets, weaners and slaughter pigs (rearing pathway). AMU at farm level was estimated as the mean lifetime AMU of the sampled batches from each farm. At batch level, AMU was measured as the deviation between the batch-specific lifetime AMU and the general mean lifetime AMU at the farm. For peroral tetracycline and macrolide use there was a significant quantitative linear effect on the abundance of ARGs in batches within individual farms, indicating an immediate effect of changed AMU from batch to batch within farms. These estimated effects between batches within farms were approximately 1/2-1/3 of the effect estimated between farms. For all antimicrobial classes, the effect of the mean farm-level AMU and the abundance of ARGs present in the faeces of slaughter pigs was significant. This effect was identified only for peroral use, except for lincosamides, where the effect was for parenteral use. The results also indicated that the abundance of ARGs against a specific antimicrobial class also increased by the peroral usage of one or several other antimicrobial classes, except for ARGs against beta-lactams. These effects were generally lower than the AMU effect of the specific antimicrobial class. Overall, the farm peroral mean lifetime AMU affected the abundance of ARGs at antimicrobial class level and abundance of ARGs of other classes. However, the difference of AMU of the slaughter-pig batches affected only the abundance of ARGs at the same antimicrobial class level in the same antimicrobial class. The results do not exclude that parenteral usage of antimicrobials may have an effect on the abundance of ARGs.

Accuracy of cryptorchidism diagnoses and corrective surgical treatment registration in the Danish National Patient Registry.

PURPOSE: In recent years several Danish studies of the etiology, time trends and long-term health consequences of cryptorchidism have relied on diagnoses and surgical treatments registered in the National Patient Registry. We evaluated the diagnostic accuracy of these registry data. MATERIALS AND METHODS: According to the Danish National Patient Registry, 16,168 males were diagnosed with cryptorchidism and 9,244 surgical treatments for cryptorchidism were performed between January 1, 1995 and October 10, 2009. We randomly selected 500 diagnosed cases, of which 284 had been managed surgically. We requested the medical records from the departments making the diagnoses and performing the surgery. RESULTS: We successfully retrieved medical records for 452 diagnosed cases (90%) and 249 operations (88%). Overall positive predictive value of a registry diagnosis of cryptorchidism was 80% (95% CI 77-84) using the testicular position described by the physician performing the clinical examination as the gold standard. Similarly the positive predictive value of the surgical treatment registration was 99% (95% CI 98-100) using the type of procedure performed. CONCLUSIONS: The data on cryptorchidism in the Danish National Patient Registry are quite accurate. In etiological research the limited misclassification will in most cases only slightly attenuate estimates of the true relative association. Thus, the registry has the potential to serve as a valuable research tool, although caution should be exercised when studying time trends or geographical differences.

Does breastfeeding influence future sperm quality and reproductive hormones?

No human study has investigated the possible impact of breastfeeding on semen quality and levels of reproductive hormones, but a recent study of another hypothesis indicated an association with oligozoospermia. We investigated the association between breastfeeding, semen quality and levels of reproductive hormones. From a Danish pregnancy cohort established in 1984-1987, 347 sons were selected according to maternal smoking during pregnancy and followed up with questionnaires, semen analysis and blood sampling in 2005-2006. Complete data were available for 269 men aged 18-21 years. Breastfeeding was not statistically significantly associated with sperm concentration, total sperm count, sperm motility or morphology, oligozoospermia, follicle-stimulating hormone, inhibin B, luteinizing hormone, sex hormone-binding globulin (SHBG), the calculated level of free testosterone, free oestradiol, the free testosterone/free oestradiol ratio or the follicle-stimulating hormone/inhibin B ratio. Total testosterone and total oestradiol was 16% (p = 0.01) and 14% (p = 0.06), respectively, lower among men never breastfed in comparison with men breastfed exclusively for 1 month or longer. When taking SHBG into account, neither free testosterone nor free oestradiol was different between the two groups. This study shows no association between breastfeeding and sperm quality or reproductive hormones and a strong association is unlikely. A larger study would be needed to detect more subtle effects.

Maternal alcohol consumption during pregnancy and semen quality in the male offspring: two decades of follow-up.

BACKGROUND: Concurrent alcohol exposure has been associated with reduced fecundity, but no studies have estimated the effect of prenatal alcohol exposure on male fecundity. The aim of this study was to investigate the association between maternal alcohol consumption during pregnancy, semen quality and levels of reproductive hormones in young, adult men. METHODS: From a Danish pregnancy cohort established in 1984-1987, 347 sons were selected for a follow-up study conducted in 2005-2006. Semen and blood samples were analyzed for conventional semen characteristics and reproductive hormones, respectively, and results were related to prospectively self-reported information on maternal alcohol consumption during pregnancy. RESULTS: The sperm concentration decreased with increasing prenatal alcohol exposure. The adjusted mean sperm concentration among sons of mothers drinking >or=4.5 drinks per week during pregnancy was 40 (95% CI: 25-60) millions/ml. This concentration was approximately 32% lower compared with men exposed to <1.0 drink per week, who had a sperm concentration of 59 (95% CI: 44-77) millions/ml. The semen volume and the total sperm count were also associated with prenatal alcohol exposure; sons prenatally exposed to 1.0-1.5 drinks per week had the highest values. No associations were found for sperm motility, sperm morphology or any of the reproductive hormones, including testosterone. CONCLUSIONS: These results indicate that prenatal exposure to alcohol may have a persisting adverse effect on Sertoli cells, and thereby sperm concentration. If these associations are causal they could explain some of the reported differences between populations and long-term changes in semen quality.

Predicting effects of changed antimicrobial usage on the abundance of antimicrobial resistance genes in finisher' gut microbiomes.

It is accepted that usage of antimicrobials (AMs) in food animals causes the emergence and spread of antimicrobial resistance (AMR) in this sector, while also contributing to the burden of AMR in humans. Curbing the increasing occurrence of AMR in food animals requires in-depth knowledge of the quantitative relationship between antimicrobial usage (AMU) and AMR to achieve desired resistance reductions from interventions targeting AMU. In the observational study, the relationships between lifetime AMU in 83 finisher batches from Danish farms and the AMR gene abundances of seven antimicrobial classes in their gut microbiomes were quantified using multi-variable linear regression models. These relationships and the national lifetime AMU in pigs were included in the predictive modelling that allowed for testing of scenarios with changed lifetime AMU for finishers produced in Denmark in 2014. A total of 50 farms from the observational study were included in validating the observational study and the predictive modelling. The results from the observational study showed that the relationship was linear, and that the parenteral usage of AMs had a high effect on specific AM-classes of resistance, whereas the peroral usage had a lower but broader effect on several classes. Three different scenarios of changed lifetime AMU were simulated in the predictive modelling. When all tetracycline usage ceased, the predicted interval reductions of aminoglycoside, lincosamide and tetracycline resistance were 4-42 %, 0-8 % and 9-18 %, respectively. When the peroral tetracycline usage of the 10 % highest users was replaced with peroral macrolide usage, the tetracycline resistance fell by 1-2 % and the macrolide and MLSb resistance increased by 5-8 %. When all extended-spectrum penicillin usage was replaced with parenteral lincosamide usage, the beta-lactam resistance fell by 2-7 %, but the lincosamide usage and resistance increased by 194 % and 10-45 %, respectively. The external validation provided results within the 95 % CI of the predictive modelling outcome at national level, while the external validation at farm level was less accurate. In conclusion, interventions targeting AMU will reduce AMR abundance, though differently depending on the targeted AM-class and provided the reduction of one AM-class usage is not replaced with usage of another AM-class. Predicting several classes of AMR gene abundance simultaneously will support stakeholders when deciding on interventions targeting AMU in the finisher production to avoid adverse and unforeseen effects on the AMR abundance. This study provides a sound predictive modelling framework for further development, including the dynamics of AMU on AMR in finishers at national level.

Temporal changes in pH within the phagocytic vacuole of the polymorphonuclear neutrophilic leukocyte.

Although previous workers have established that the pH of the phagocytic vacuole of the polymorphonuclear (PMN) leukocyte changes from neutral to acid, the time course of conversion has not been investigated. The present experiments were initiated to study pH changes immediately after phagocytosis. Peritoneal exudates were induced in rats; 4 h later, yeast stained with pH indicators was injected intraperitoneally, and the exudate was retrieved at 30-s intervals and examined by light microscopy. Results revealed that (a) within 3 min, pH dropped to approximately 6.5, as indicated by the change in color of neutral red-stained yeast; (b) within 7-15 min, pH dropped progressively to approximately 4.0, as indicated by color change in bromcresol green-stained yeast; (c) pH did not fall below 4, since no color change was observed up to 24 h when bromphenol blue-stained yeast was used. The finding that intravacuolar acidity increases rapidly after phagocytosis is undoubtedly important with respect to PMN leukocyte function in killing and digesting microorganisms, for many PMN leukocyte granule enzymes (i.e., peroxidase and lysosomal enzymes) are activated at acid pH ( approximately 4.5). It follows that temporal changes in pH and maximal pH depression should be considered in studies of intraleukocytic microbicidal mechanisms, since a defect in these factors could result in impaired PMN leukocyte function.

Phospholipid flipping involves a central cavity in P4 ATPases.

P4 ATPase flippases translocate phospholipids across biomembranes, thus contributing to the establishment of transmembrane lipid asymmetry, a feature important for multiple cellular processes. The mechanism by which such phospholipid flipping occurs remains elusive as P4 ATPases transport a giant substrate very different from that of other P-type ATPases such as Na+/K+- and Ca2+-ATPases. Based on available crystal structures of cation-transporting P-type ATPases, we generated a structural model of the broad-specificity flippase ALA10. In this model, a cavity delimited by transmembrane segments TM3, TM4, and TM5 is present in the transmembrane domain at a similar position as the cation-binding region in related P-type ATPases. Docking of a phosphatidylcholine headgroup in silico showed that the cavity can accommodate a phospholipid headgroup, likely leaving the fatty acid tails in contact with the hydrophobic portion of the lipid bilayer. Mutagenesis data support this interpretation and suggests that two residues in TM4 (Y374 and F375) are important for coordination of the phospholipid headgroup. Our results point to a general mechanism of lipid translocation by P4 ATPases, which closely resembles that of cation-transporting pumps, through coordination of the hydrophilic portion of the substrate in a central membrane cavity.

Fast and one-step folding of closely and distantly related homologous proteins of a four-helix bundle family.

Bovine acyl-coenzyme A binding protein is a four-helix bundle protein belonging to a group of homologous eukaryote proteins that binds medium and long-chain acyl-coenzyme A esters with a very high affinity. The three-dimensional structure of both the free and the ligated protein together with the folding kinetics have been described in detail for the bovine protein and with four new sequences reported here, a total of 16 closely related sequences ranging from yeasts and plants to human are known. The kinetics of folding and unfolding in different concentrations of guanidine hydrochloride together with equilibrium unfolding have been measured for bovine, rat and yeast acyl-coenzyme A binding protein. The bovine and rat sequences are closely related whereas the yeast is more distantly related to these. In addition to the three natural variants, kinetics of a bovine mutant protein, Tyr31 --> Asn, have been studied. Both the folding and unfolding rates in water of the yeast protein are 15 times faster than those of bovine. The folding rates in water of the two mammalian forms, rat and bovine, are similar, though still significantly different. A faster unfolding rate both for rat and the bovine mutant protein results from a lower stability of the native states of these. These hydrophobic regions, mini cores, have been identified in the three-dimensional structure of the bovine protein and found to be formed primarily by residues that have been conserved throughout the entire eukaryote evolution from yeasts to both plants and mammals as seen in the sample of 16 sequences. The conserved residues are found to stabilize helix-helix interactions and serve specific functional purposes for ligand binding. The fast one-step folding mechanism of ACBP has been shown to be a feature that seems to be maintained throughout evolution despite numerous differences in sequence and even dramatic differences in folding kinetics and protein stability. The protein study raises the question to what extent does the conserved hydrophobic residues provide a scaffold for an efficient one-step folding mechanism.

Design and evaluation of a handheld impedance plethysmograph for measuring heart rate variability.

Heart rate variability (HRV) analysis from 10s ECGs has been shown to be reliable. However, the short examination time warrants a user-friendly system that can be used for ad-hoc examinations without normal preparation, unlike ECG. A handheld device has been developed that can measure ultra-short HRV from impedance plethysmographic recordings of the pulse wave in distal superficial arteries. The prototype device was made user-friendly through a compact, pen-like design and the use of integrated metal electrodes that were especially designed for dry operation. The main signal processing was performed by a digital signal processor, where the discrete heart beats were detected using a correlation algorithm that could adapt to individual pulse wave shapes to account for biological variation. The novel device was evaluated in 20 mainly young volunteers, using 10 s time-correlated ECG recordings as the reference method. Agreement between the two methods in measuring heart rate and root mean square of successive differences in the heart beat interval (RMSSD) was analysed using correlation coefficients (Pearson's R2), mean differences with 95% confidence intervals and 95% limits of agreement, and Bland-Altman plots. The correlation between the two methods was R2 = 1.00 and R2 = 0.99 when heart rate and RMSSD were measured, respectively. The Bland-Altman plots showed suitable agreement between the novel device and standard 10 s ECGs, which was substantiated by 95% limits of agreement of the difference of +/- 0.1 beats min(-1) and approximately +/- 10 ms for heart rate and RMSSD, respectively. Therefore the evaluation showed no significant systematic error of the novel device compared with ECG.

Amino acid sequence of bovine protein Z: a vitamin K-dependent serine protease homolog.

The amino acid sequence of protein Z has been determined from sequence analysis performed on fragments obtained by chemical and enzymatic degradations. The polypeptide consists of a single chain containing 396 amino acid residues (Mr 43 677). Comparison with the vitamin K-dependent plasma proteins reveals an extensive homology. The N-terminal part, containing 13 gamma-carboxyglutamic acid and one beta-hydroxyaspartic acid residue, is extensively homologous to and of similar length to the light chain of factor X. The remainder of protein Z is homologous to the serine proteases and of similar size to the heavy chain of factor Xa, but of the active site residues only aspartic acid-102 is present. Histidine-57 and serine-195 are replaced in protein Z by threonine and alanine, respectively. The physiological function of protein Z is still uncertain.

Modulation of the responses to the GABA-mimetics, THIP and piperidine-4-sulphonic acid, by agents which interact with benzodiazepine receptors. An electrophysiological study on cultured mouse neurones.

Electrophysiological recordings from mouse neurones in tissue culture have been used to investigate how agents which interact with the benzodiazepine receptor modulate neuronal responses to gamma-aminobutyric acid (GABA) and its mimetics, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and piperidine-4-sulphonic acid (P4S). Experiments were performed in a physiological medium, pH 7.35 at 34-36 degrees C. gamma-Aminobutyric acid, THIP and P4S were applied by iontophoresis to neuronal somata. Responses were assessed by current-clamp or voltage-clamp recordings. Midazolam (an agonist at the benzodiazepine receptor) and the beta-carboline, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM; an inverse agonist at the BZ receptor), were applied by pressure ejection from blunt pipettes. The potency order of the agonists was GABA greater than P4S greater than THIP. Midazolam (10(-7)-10(-5) M) potentiated responses to all three agonists to a similar extent with a shift to the left of the dose-response curve. The drug DMCM (10(-6)-10(-5) M) decreased the responses to all three agonists to a similar extent. The DMCM-induced depression was of a non-competitive nature. It has previously been proposed that THIP is a partial agonist and P4S an antagonist at the GABA receptor coupled to the benzodiazepine receptor, or that the benzodiazepine-receptor-coupled and electrophysiological GABA receptors are distinct. In the present study, responses to the three agonists were modulated to a comparable extent following manipulation of the benzodiazepine receptor. It is therefore unnecessary to invoke the above explanations to account for these results.

Developmental increase in asynchronous GABA release in cultured hippocampal neurons.

Developmental changes in GABAergic synaptic transmission were examined in cultured hippocampal neurons using patch-clamp recordings and Ca(2+) imaging. In paired recordings, tetanization of the presynaptic GABAergic neuron with 80 pulses at either 40 or 80Hz was accompanied by tetanic depression of inhibitory postsynaptic responses. In neurons that had been cultured for more than two weeks, asynchronous inhibitory postsynaptic currents often appeared during the tetanus and continued for several seconds following stimulation. There was little asynchronous activity in neurons that had been cultured for shorter times. However, no age-related changes were observed in the amplitude of single synchronous inhibitory postsynaptic currents, paired-pulse depression or post-tetanic potentiation of inhibitory postsynaptic currents. Following equimolar replacement of extracellular Ca(2+) with strontium ions (Sr(2+)), single autaptic inhibitory postsynaptic currents were depressed in amplitude and asynchronous inhibitory postsynaptic currents were present on the decaying phase. Sr(2+)-induced asynchronous inhibitory postsynaptic currents showed no dependence on age in culture. Imaging of Ca(2+) in single GABAergic boutons was performed by including Fluo-3 in the patch pipette. During action potential firing induced by stimulating at 80Hz for 1s, intracellular calcium [Ca(2+)](i) increased rapidly in individual boutons. Following the stimulus, [Ca(2+)](i) decayed back to baseline within 10-15s. The half-time of decay increased from 1. 7+/-0.2s at 15days in vitro to 4.0+/-0.2s at 30days in vitro (P<0. 05), with a developmental profile that closely matched the increase in asynchronous inhibitory postsynaptic currents. We propose that the increase in tetanus-induced asynchronous GABA-release during the first month of synapse maturation in vitro is caused by a slowing of the Ca(2+)-clearing mechanisms in the GABAergic boutons. This results in larger and more prolonged elevations of [Ca(2+)](i) during tetanic stimulation, which leads to enhanced asynchronous transmitter release. We propose that the results of this study demonstrate a potentially important aspect of synapse maturation during development, and also imply that GABA release is up-regulated in conditions of decreased Ca(2+) buffering and clearing.

Electrophysiological studies in cultured mouse CNS neurones of the actions of an agonist and an inverse agonist at the benzodiazepine receptor.

The action of agents which bind with the benzodiazepine (BZ) receptor has been investigated by use of intracellular recordings from dissociated mouse neurones grown in tissue culture. The agents tested were midazolam (an agonist at the BZ receptor) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM-an inverse agonist at the BZ receptor). These were applied to the neurone under study by one of the following methods: iontophoresis; pressure application of known concentrations from blunt pipettes; directly in the perfusing medium. On only very few occasions did midazolam or DMCM have a direct effect on the membrane potential (EM) or conductance (GM) of the impaled neurone. For the neurones where direct effects were present, there was no consistent pattern of response. Neither substance affected the threshold for action potential generation. The effect of midazolam and DMCM on responses evoked by iontophoretic application of gamma-aminobutyric acid (GABA) was also investigated. Three parameters were used to quantify GABA responses: the depolarization (VGABA); the increase in GM (gGABA) measured with constant current pulses; using voltage clamp, the GABA current (IGABA). The GABA response should be quantified by a parameter which is linearly related to the number of GABA-operated channels which are conducting at any instant. VGABA does not fulfil this criterion. gGABA is an appropriate parameter, but is difficult to determine for large responses where the membrane is nearly short circuited. IGABA measured during voltage clamp fulfils this criterion. Midazolam (greater than 10(-6) M) reliably potentiated GABA responses with a parallel shift to the left of the dose-response curve. This is in agreement with biochemical studies where BZs increase the affinity of the GABA receptor for its ligand. The effect of DMCM on GABA responses was more variable. In the majority of cases GABA responses were reduced by DMCM. The threshold dose for this depression was usually around 10(-6) M, but was sometimes as low as 10(-8) M. Dose-response curves of IGABA or gGABA showed the inhibition to be of a non-competitive nature. The maximum inhibition achieved was around 70%. For a given neurone, and at doses which did not necessarily cause a reduction of the response to GABA, DMCM could antagonize the potentiating action of midazolam on GABA responses. A possible interpretation is that more than one BZ site per receptor complex must be occupied by a BZ agonist (or inverse agonist) before the functional changes for the complex as a whole can occur. Desensitization to GABA was increased by midazolam.