Molecular and organizational diversity intersect to generate functional synaptic heterogeneity within and between excitatory neuronal subtypes.

Synaptic heterogeneity is a hallmark of complex nervous systems that enables reliable and responsive communication in neural circuits. In this study, we investigated the contributions of voltage-gated calcium channels (VGCCs) to synaptic heterogeneity at two closely related Drosophila glutamatergic motor neurons, one low- and one high-Pr. We find that VGCC levels are highly predictive of heterogeneous release probability among individual active zones (AZs) of low- or high-Pr inputs, but not between neuronal subtypes. Underlying organizational differences in the AZ cytomatrix, VGCC composition, and a more compact arrangement of VGCCs alter the relationship between VGCC levels and Pr at AZs of low- vs. high -Pr inputs, explaining this apparent paradox. We further find that the CAST/ELKS AZ scaffolding protein Bruchpilot differentially regulates VGCC levels at low- and high-Pr AZs following acute glutamate receptor inhibition, indicating that synapse-specific organization also impacts adaptive plasticity. These findings reveal intersecting levels of molecular and spatial diversity with context-specific effects on heterogeneity in synaptic strength and plasticity.

Dual-modality endomicroscopy with co-registered fluorescence and phase contrast.

We describe a dual-modality laser scanning endomicroscope that provides simultaneous fluorescence contrast based on confocal laser endomicroscopy (CLE) and phase-gradient contrast based on scanning oblique back-scattering microscopy (sOBM). The probe consists of a 2.6mm-diameter micro-objective attached to a 30,000-core flexible fiber bundle. The dual contrasts are inherently co-registered, providing complementary information on labeled and un-labeled sample structure. Proof of principle demonstrations are presented with ex-vivo mouse colon tissue.

Peptidases specific for proline-containing peptides and their unusual peptide-dependent regulation in Oenococcus oeni.

AIMS: Growth of the lactic acid bacterium (LAB) Oenococcus oeni, which is involved in malolactic fermentation during the winemaking process, is stimulated by peptides originating from yeast. In this study, we investigated the impact of peptides on O. oeni growth, peptidase activity and the expression of genes encoding the studied peptidases. METHODS AND RESULTS: Low levels of PepN activity and very high levels of PepI activity were observed in O. oeni, whereas levels of PepX activity were intermediate. The level of biosynthesis of these O. oeni peptidases was shown to depend on peptides present in the culture medium. These results were confirmed by transcriptional analyses of putative pep genes. The mechanism of repression by peptides did not involve a CodY-like regulator. CONCLUSIONS: Peptides from yeast decrease the levels of enzymatic activity and relative gene expression of O. oeni peptidases. Peptidases specific for proline-containing peptides are important for O. oeni nitrogen metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: We report here for the first time that the enzymes involved in the assimilation of proline-containing peptides by O. oeni differ from the well-described proteolytic system of milk LAB. This may reflect a specific adaptation to the wine environment.

Oligopeptide assimilation and transport by Oenococcus oeni.

AIMS: Oenococcus oeni is a slow-growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino-acid medium. We aimed to characterize the oligopeptide use of this micro-organism. METHODS AND RESULTS: Several peptides of two to eight amino-acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. CONCLUSIONS: Oenococcus oeni was able to transport the oligopeptides with two to five amino-acid residues tested and to hydrolyse them further. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.

Improvements in routine internal monitoring--an overview of the IDEA project.

The IDEA project aimed to improve the assessment of incorporated radionuclides through developments of advanced in vivo and bioassay monitoring techniques and making use of such enhancements for improvements in routine monitoring. Many of these findings are not new in the sense that they are being already employed in advanced laboratories or for specialised applications. The primary goal was to categorise those new developments regarding their potential and eligibility for the routine monitoring community. Attention has been given to in vivo monitoring techniques with respect to detector characteristics and measurement geometry to improve measurement efficiency with special attention to low energy gamma emitters. Calibration-specifically supported by or through methods of numerical simulation-have been carefully analysed to reduce overall measurement uncertainties and explore ways to accommodate the individual variability based on characteristic features of a given person. For bioassay measurements at low detection limits, inductively coupled plasma mass spectroscopy offers significant advantages both in accuracy, speed, and sample preparation. Specifically, the determination of U and Th in urine and the associated models have been investigated. Finally, the scientific achievements have been analysed regarding their potential to offer benefits for routine monitoring. These findings will be presented in greater detail in other papers at this conference, whereas this paper intends to give an overview and put both the scientific achievements as well as the derived benefits into perspective.

Implementation of bioassay methods to improve assessment of incorporated radionuclides.

The present work which was carried out in the framework of an EU project (IDEA: Internal Dosimetry-Enhancements in Application; Contract Number: FIKR CT2001 00164) shall provide commonly acceptable guidelines for optimum performance of ICP-MS measurements with focus on urinary measurements of uranium, thorium and actinides. From the results of this work it is recommended that, whenever feasible, 24 h urine sampling should be conducted to avoid large uncertainties in the quantitation of daily urinary excretion values. For storage, urine samples should be acidified and kept frozen before analysis. Measurement of total uranium in urine by ICP-MS at physiological levels (<10 ng.l(-1)) requires no sample preparation besides UV photolysis and/or dilution. For the measurement of thorium in urine by ICP-MS, it can be concluded, that salt removal from the urine samples is not recommended. For the measurement of actinides in urine it is shown that ICP-MS is well-suited and a good alternative to alpha-spectrometry for isotopes with T1/2>5x10(4) years. In general, ICP-MS measurements are an easy, fast and cost-saving methodology. New improved measuring techniques (HR-SF-ICP-MS) with detection limits in urine of 150 pg.l(-1) (1.9 microBq.l(-1)) for 238U, 30 pg.l(-1) (2.4 microBq.l(-1)) for 235U and 100 pg.l(-1) (0.4 microBq.l(-1)) for (232)Th, respectively, meet all necessary requirements. This method should therefore become the routine technique for incorporation monitoring of workers and of members of the general public, in particular for uranium contamination.

Practical implications of procedures developed in IDEA project--comparison with traditional methods.

The idea of the IDEA project aimed to improve assessment of incorporated radionuclides through developments of more reliable and possibly faster in vivo and bioassay monitoring techniques and making use of such enhancements for improvements in routine monitoring. In direct in vivo monitoring technique the optimum choice of the detectors to be applied for different monitoring tasks has been investigated in terms of material, size and background in order to improve conditions namely to increase counting efficiency and reduce background. Detailed studies have been performed to investigate the manifold advantageous applications and capabilities of numerical simulation method for the calibration and optimisation of in vivo counting systems. This calibration method can be advantageously applied especially in the measurement of low-energy photon emitting radionuclides, where individual variability is a significant source of uncertainty. In bioassay measurements the use of inductively coupled plasma mass spectrometry (ICP-MS) can improve considerably both the measurement speed and the lower limit of detection currently achievable with alpha spectrometry for long-lived radionuclides. The work carried out in this project provided detailed guidelines for optimum performance of the technique of ICP-MS applied mainly for the determination of uranium and thorium nuclides in the urine including sampling procedure, operational parameters of the instruments and interpretation of the measured data. The paper demonstrates the main advantages of investigated techniques in comparison with the performances of methods commonly applied in routine monitoring practice.

Absorption, accumulation and biological effects of depleted uranium in Peyer's patches of rats.

The digestive tract is the entry route for radionuclides following the ingestion of contaminated food and/or water wells. It was recently characterized that the small intestine was the main area of uranium absorption throughout the gastrointestinal tract. This study was designed to determine the role played by the Peyer's patches in the intestinal absorption of uranium, as well as the possible accumulation of this radionuclide in lymphoid follicles and the toxicological or pathological consequences on the Peyer's patch function subsequent to the passage and/or accumulation of uranium. Results of experiments performed in Ussing chambers indicate that the apparent permeability to uranium in the intestine was higher (10-fold) in the mucosa than in Peyer's patches ((6.21+/-1.21 to 0.55+/-0.35)x10(-6)cm/s, respectively), demonstrating that the small intestinal epithelium was the preferential pathway for the transmucosal passage of uranium. A quantitative analysis of uranium by ICP-MS following chronic contamination with depleted uranium during 3 or 9 months showed a preferential accumulation of uranium in Peyer's patches (1355% and 1266%, respectively, at 3 and 9 months) as compared with epithelium (890% and 747%, respectively, at 3 and 9 months). Uranium was also detected in the mesenteric lymph nodes ( approximately 5-fold after contamination with DU). The biological effects of this accumulation of depleted uranium after chronic contamination were investigated in Peyer's patches. There was no induction of the apoptosis pathway after chronic DU contamination in Peyer's patches. The results indicate no change in the cytokine expression (Il-10, TGF-beta, IFN-gamma, TNF-alpha, MCP-1) in Peyer's patches and in mesenteric lymph nodes, and no modification in the uptake of yeast cells by Peyer's patches. In conclusion, this study shows that the Peyer's patches were a site of retention for uranium following the chronic ingestion of this radionuclide, without any biological consequences of such accumulation on Peyer's patch functions.

Potentialities of mass spectrometry (ICP-MS) for actinides determination in urine.

The applicability of inductively coupled plasma-mass spectrometry (ICP-MS) for determining actinides in urine was investigated. Performances of ICP-MS including detection limit and analysis time were studied and compared with alpha spectrometry performances. In the field of individual monitoring of workers, the comparison chart obtained in this study can be used as a guide for medical laboratories to select the most adequate procedure to be carried out depending on the case in question (the radioisotope to be measured, the required sensitivity, and the desired response time).

Validation of uranium determination in urine by ICP-MS.

A rapid procedure--dilution of urine+ICP-MS measurement--for the determination of uranium in urine was validated. Large ranges of concentration and isotopic composition were studied on urine samples excreted by occupationally exposed workers. The results were consistent with those obtained by fluorimetry and by alpha spectrometry after a purification procedure, two currently used techniques. However, the proposed procedure is limited for determination of the minor isotope 234U. Thus for worker monitoring, the conversion of 234U mass concentration into activity concentration can lead to an erroneous value of the effective dose, in particular for a contamination at very low level with highly enriched uranium. A solution to avoid this hazard is to perform a chemical purification prior to ICP-MS measurement to lower uncertainty and detection limit for 234U.

Determination of 232Th in urine by ICP-MS for individual monitoring purposes.

Thorium is naturally occurring in various ores used for industrial purposes and has numerous applications. This paper sets out to investigate urine analysis as a suitable monitoring approach for workers potentially exposed to thorium. Due to its biokinetic behavior and its low solubility, urinary concentrations are generally very low, requiring therefore high sensitivity analytical methods. An analytical procedure has been developed for detecting 232Th concentrations of below 1 mBq L(-1) quickly and easily. Due to the long half-life (1.41 x 10(10) y) of 232Th, the potential of a procedure based on urine sample dilution and ICP-MS (inductively coupled plasma-mass spectrometry) measurement was investigated first. Two dilution factors were chosen: 100, which is more suitable for long-term measurement trials, and 20, which increases sensitivity. It has been shown that a 100-fold dilution can be used to measure concentrations of below 1 mBq L(-1), whereas a 20-fold one can be used to reach concentrations of below 0.06 mBq L(-1). Then, on the basis of the limitation of the procedure based on urine dilution, the suitable field of application for the different procedures (100-fold and 20-fold dilution and also a chemical purification followed by an ICP-MS measurement) was determined in relation to monitoring objectives.

Quantitative in vivo analysis of the kinematics of carpal bones from three-dimensional CT images using a deformable surface model and a three-dimensional matching technique.

The purpose of this study was to obtain quantitative information of the relative displacements and rotations of the carpal bones during movement of the wrist. Axial helical CT scans were made of the wrists of 11 volunteers. The wrists were imaged in the neutral position with a conventional CT technique, and in 15-20 other postures (flexion-extension, radial-ulnar deviation) with a low-dose technique. A segmentation of the carpal bones was obtained by applying a deformable surface model to the regular-dose scan. Next, each carpal bone, the radius, and ulna in this scan was registered with the corresponding bone in each low-dose scan using a three-dimensional matching technique. A detailed definition of the surfaces of the carpal bones was obtained from the regular-dose scans. The low-dose scans provided sufficient information to obtain an accurate match of each carpal bone with its counterpart in the regular-dose scan. Accurate estimates of the relative positions and orientations of the carpal bones during flexion and deviation were obtained. This quantification will be especially useful when monitoring changes in kinematics before and after operative interventions, like mini-arthrodeses. This technique can also be applied in the quantification of the movement of other bones in the body (e.g., ankle and cortical spine).

Implementation of ICP-MS protocols for uranium urinary measurements in worker monitoring.

The uranium concentration in human urine spiked with natural uranium and rat urine containing metabolized depleted uranium was determined by ICP-MS. The use of ICP-MS was investigated without any chemical treatment or after the different stages of a purification protocol currently carried out for routine monitoring. In the case of spiked urine, the measured uranium concentrations were consistent with those certified by an intercomparison network in radiotoxicological analysis (PROCORAD) and with those obtained by alpha spectrometry in the case of the urine containing metabolized uranium. The quantitative information which could be obtained in the different protocols investigated shows the extent to which ICP-MS provides greater flexibility for setting up appropriate monitoring approaches in radiation protection routines and accidental situations. This is due to the combination of high sensitivity and the accuracy with which traces of uranium in urine can be determined in a shorter time period. Moreover, it has been shown that ICP-MS measurement can be used to quantify the 235U isotope, which is useful for characterizing the nature of the uranium compound, but difficult to perform using alpha spectrometry.

Network oscillations generated by balancing graded asymmetric reciprocal inhibition in passive neurons.

We describe a novel mechanism by which network oscillations can arise from reciprocal inhibitory connections between two entirely passive neurons. The model was inspired by the activation of the gastric mill rhythm in the crab stomatogastric ganglion by the modulatory commissural ganglion neuron 1 (MCN1), but it is studied here in general terms. One model neuron has a linear current-voltage (I-V) curve with a low (L) resting potential, and the second model neuron has a linear current-voltage curve with a high (H) resting potential. The inhibitory connections between them are graded. There is an extrinsic modulatory excitatory input to the L neuron, and the L neuron presynaptically inhibits the modulatory neuron. Activation of the extrinsic modulatory neuron elicits stable network oscillations in which the L and H neurons are active in alternation. The oscillations arise because the graded reciprocal synapses create the equivalent of a negative-slope conductance region in the I-V curves for the cells. Geometrical methods are used to analyze the properties of and the mechanism underlying these network oscillations.

Synchronization and oscillatory dynamics in heterogeneous, mutually inhibited neurons.

We study some mechanisms responsible for synchronous oscillations and loss of synchrony at physiologically relevant frequencies (10-200 Hz) in a network of heterogeneous inhibitory neurons. We focus on the factors that determine the level of synchrony and frequency of the network response, as well as the effects of mild heterogeneity on network dynamics. With mild heterogeneity, synchrony is never perfect and is relatively fragile. In addition, the effects of inhibition are more complex in mildly heterogeneous networks than in homogeneous ones. In the former, synchrony is broken in two distinct ways, depending on the ratio of the synaptic decay time to the period of repetitive action potentials (tau s/T), where T can be determined either from the network or from a single, self-inhibiting neuron. With tau s/T > 2, corresponding to large applied current, small synaptic strength or large synaptic decay time, the effects of inhibition are largely tonic and heterogeneous neurons spike relatively independently. With tau s/T < 1, synchrony breaks when faster cells begin to suppress their less excitable neighbors; cells that fire remain nearly synchronous. We show numerically that the behavior of mildly heterogeneous networks can be related to the behavior of single, self-inhibiting cells, which can be studied analytically.

Frequency control in synchronized networks of inhibitory neurons.

We analyze the control of frequency for a synchronized inhibitory neuronal network. The analysis is done for a reduced membrane model with a biophysically based synaptic influence. We argue that such a reduced model can quantitatively capture the frequency behavior of a larger class of neuronal models. We show that in different parameter regimes, the network frequency depends in different ways on the intrinsic and synaptic time constants. Only in one portion of the parameter space, called phasic, is the network period proportional to the synaptic decay time. These results are discussed in connection with previous work of the authors, which showed that for mildly heterogeneous networks, the synchrony breaks down, but coherence is preserved much more for systems in the phasic regime than in the other regimes. These results imply that for mildly heterogeneous networks, the existence of a coherent rhythm implies a linear dependence of the network period on synaptic decay time and a much weaker dependence on the drive to the cells. We give experimental evidence for this conclusion.