Three-dimensional quantitative muscle ultrasound in patients with facioscapulohumeral dystrophy and myotonic dystrophy.

INTRODUCTION/AIMS: Ultrasound imaging of muscle tissue conventionally results in two-dimensional sampling of tissue. For heterogeneously affected muscles, a sampling error using two-dimensional (2D) ultrasound can therefore be expected. In this study, we aimed to quantify and extend ultrasound imaging findings in neuromuscular disorders by using three-dimensional quantitative muscle ultrasound (3D QMUS). METHODS: Patients with facioscapulohumeral dystrophy (n = 31) and myotonic dystrophy type 1 (n = 16) were included in this study. After physical examination, including Medical Research Council (MRC) scores, the tibialis anterior muscle was scanned with automated ultrasound. QMUS parameters were calculated over 15 cm of the length of the tibialis anterior muscle and were compared with a healthy reference data set. RESULTS: With 3D QMUS local deviations from the healthy reference could be detected. Significant Pearson correlations (P < .01) between MRC score and QMUS parameters in male patients (n = 23) included the mean echo intensity (EI) (0.684), the standard deviation of EI (0.737), and the residual attenuation (0.841). In 91% of all patients, mean EI deviated by more than 1 standard deviation from the healthy reference. In general, the proportion of muscle tissue with a Z score >1 was about 50%. DISCUSSION: In addition to mean EI, multiple QMUS parameters reported in this study are potential biomarkers for pathology. Besides a moderate correlation of mean EI with muscle weakness, two other parameters showed strong correlations: standard deviation of EI and residual attenuation. Local detection of abnormalities makes 3D QMUS a promising method that can be used in research and potentially for clinical evaluation.

Conjugates of nucleobases with triazole-hydroxamic acids for the reactivation of acetylcholinesterase and treatment of delayed neurodegeneration induced by organophosphate poisoning.

A series of new uncharged conjugates of adenine, 3,6-dimetyl-, 1,6-dimethyl- and 6-methyluracil with 1,2,4-triazole-3-hydroxamic and 1,2,3-triazole-4-hydroxamic acid moieties were synthesized and studied as reactivators of organophosphate-inhibited cholinesterase. It is shown that triazole-hydroxamic acids can reactivate acetylcholinesterase (AChE) inhibited by paraoxon (POX) in vitro, offering reactivation constants comparable to those of pralidoxime (2-PAM). However, in contrast to 2-PAM, triazole-hydroxamic acids demonstrated the ability to reactivate AChE in the brain of rats poisoned with POX. At a dose of 200 mg/kg (i.v.), the lead compound 3e reactivated 22.6 ± 7.3% of brain AChE in rats poisoned with POX. In a rat model of POX-induced delayed neurodegeneration, compound 3e reduced the neuronal injury labeled with FJB upon double administration 1 and 3 h after poisoning. Compound 3e was also shown to prevent memory impairment of POX-poisoned rats as tested in a Morris water maze.

Real-time laser speckle contrast imaging measurement during normothermic machine perfusion in pretransplant kidney assessment.

OBJECTIVES: Normothermic machine perfusion (NMP) provides a platform for pre-transplant kidney quality assessment that is essential for the use of marginal donor kidneys. Laser speckle contrast imaging (LSCI) presents distinct advantages as a real-time and noncontact imaging technique for measuring microcirculation. In this study, we aimed to assess the value of LSCI in visualizing renal cortical perfusion and investigate the additional value of dual-side LSCI measurements compared to single aspect measurement during NMP. METHODS: Porcine kidneys were obtained from a slaughterhouse and then underwent NMP. LSCI was used to measure one-sided cortical perfusion in the first 100 min of NMP. Thereafter, the inferior renal artery branch was occluded to induce partial ischemia and LSCI measurements on both ventral and dorsal sides were performed. RESULTS: LSCI fluxes correlated linearly with the renal blood flow (R2 = 0.90, p < 0.001). After renal artery branch occlusion, absence of renal cortical perfusion could be visualized and semiquantified by LSCI. The overall ischemic area percentage of the ventral and dorsal sides was comparable (median interquartile range [IQR], 38 [24-43]% vs. 29 [17-46]%, p = 0.43), but heterogenous patterns between the two aspects were observed. There was a significant difference in oxygen consumption (mean ± standard deviation [SD], 2.57 ± 0.63 vs. 1.83 ± 0.49 mLO2 /min/100 g, p < 0.001), urine output (median [IQR], 1.3 [1.1-1.7] vs. 0.8 [0.6-1.3] mL/min, p < 0.05), lactate dehydrogenase (mean ± SD, 768 ± 370 vs. 905 ± 401 U/L, p < 0.05) and AST (mean ± SD, 352 ± 285 vs. 462 ± 383 U/L, p < 0.01) before and after renal artery occlusion, while no significant difference was found in creatinine clearance, fractional excretion of sodium, total sodium reabsorption and histological damage. CONCLUSIONS: LSCI fluxes correlated linearly with renal blood flow during NMP. Renal cortical microcirculation and absent perfusion can be visualized and semiquantified by LSCI. It provides a relative understanding of perfusion levels, allowing for a qualitative comparison between regions in the kidney. Dual-side LSCI measurements are of added value compared to single aspect measurement and renal function markers.

Three-dimensional quantitative muscle ultrasound in a healthy population.

INTRODUCTION/AIMS: Quantitative muscle ultrasound offers biomarkers that aid in the diagnosis, detection, and follow-up of neuromuscular disorders. At present, quantitative muscle ultrasound methods are 2D and are often operator and device dependent. The aim of this study was to combine an existing device independent method with an automated ultrasound machine and perform 3D quantitative muscle ultrasound, providing new normative data of healthy controls. METHODS: In total, 123 healthy volunteers were included. After physical examination, 3D ultrasound scans of the tibialis anterior muscle were acquired using an automated ultrasound scanner. Image postprocessing was performed to obtain calibrated echo intensity values based on a phantom reference. RESULTS: Tibialis anterior muscle volumes of 61.2 ± 24.1 mL and 53.7 ± 22.7 mL were scanned in males and females, respectively. Echo intensity correlated with gender**, age**, fat fraction*, histogram kurtosis**, skewness* and standard deviation** (*P < .05, **P < .01). Outcome measures did not differ significantly for different acquisition presets. The 3D quantitative muscle ultrasound revealed the non-uniformity of echo intensity values over the length of the tibialis anterior muscle. DISCUSSION: Our method extended 2D measurements and confirmed previous findings. Our method and reported normative data of (potential) biomarkers can be used to study neuromuscular disorders.

Ca2+-Activated K+ Channels Reduce Network Excitability, Improving Adaptability and Energetics for Transmitting and Perceiving Sensory Information.

Ca2+-activated K+ channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modeling, we discover how visual information transfer in mutants lacking the BK channel (dSlo- ), SK channel (dSK- ), or both (dSK- ;; dSlo- ) is shaped in the female fruit fly (Drosophila melanogaster) R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K+ conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. We show how these dynamics inescapably increase the energy cost of information and promote the mutants' distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca2+-activated K+ channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information.SIGNIFICANCE STATEMENT In this study, we directly link in vivo and ex vivo experiments with detailed stochastically operating biophysical models to extract new mechanistic knowledge of how Drosophila photoreceptor-interneuron-photoreceptor (R-LMC-R) circuitry homeostatically retains its information sampling and transmission capacity against chronic perturbations in its ion-channel composition, and what is the cost of this compensation and its impact on optomotor behavior. We anticipate that this novel approach will provide a useful template to other model organisms and computational neuroscience, in general, in dissecting fundamental mechanisms of homeostatic compensation and deepening our understanding of how biological neural networks work.

Stabilization of the Highly Hydrophobic Membrane Protein, Cytochrome bd Oxidase, on Metallic Surfaces for Direct Electrochemical Studies.

The cytochrome bd oxidase catalyzes the reduction of oxygen to water in bacteria and it is thus an interesting target for electrocatalytic studies and biosensor applications. The bd oxidase is completely embedded in the phospholipid membrane. In this study, the variation of the surface charge of thiol-modified gold nanoparticles, the length of the thiols and the other crucial parameters including optimal phospholipid content and type, have been performed, giving insight into the role of these factors for the optimal interaction and direct electron transfer of an integral membrane protein. Importantly, all three tested factors, the lipid type, the electrode surface charge and the thiol length mutually influenced the stability of films of the cytochrome bd oxidase. The best electrocatalytic responses were obtained on the neutral gold surface when the negatively charged phosphatidylglycerol (PG) was used and on the charged gold surface when the zwitterionic phosphatidylethanolamine (PE) was used. The advantages of the covalent binding of the membrane protein to the electrode surface over the non-covalent binding are also discussed.

Physicochemical characterization of human cardiovascular deposits.

Detailed crystal chemical characterization of human pathological cardiovascular deposits (PCD) was conducted applying wide set of the instrumental methods (XRD, FTIR, Raman, SEM, different chemical analyses). There was some progress achieved in the understanding of it formation mechanism. The obtained data evidence that pathological cardiovascular deposits are presented by non-stoichiometric water-bearing B-type carbonated hydroxyapatite just like other apatites of the human body. But PCD apatite is characterized by higher concentration of B-type carbonate ion (up to ~ 6 wt%) which leads to the increasing influence of the carbonate-ion on the unit cell parameters in comparison with water and other substitutes. Another difference between PCD apatite and other pathogenic apatites of the human body is the smaller variations of the unit cell parameters, caused by smaller variations of the blood chemical composition. It was shown that apatite on the surface of PCD is characterized by the more non-stoichiometric composition compared to apatite inside these deposits. It is assumed that the formation mechanisms of the PCD apatite and the bone apatite may be similar.

The H2O2-Resistant Fe-S Redox Switch MitoNEET Acts as a pH Sensor To Repair Stress-Damaged Fe-S Protein.

Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, we demonstrated the involvement of mNT in a specific cytosolic pathway dedicated to the reactivation of oxidatively damaged cytosolic aconitase by cluster transfer. In vitro studies using apo-ferredoxin (FDX) reveal that mNT uses an Fe-based redox switch mechanism to regulate the transfer of its cluster. Using the "gold standard" cluster recipient protein, FDX, we show that this transfer is direct and that only one of the two mNT clusters is transferred when the second one is decomposed. Combining complementary biophysical and biochemical approaches, we show that pH affects both the sensitivity of the cluster to O2 and dimer stability. Around physiological cytosolic pH, the ability of mNT to transfer its cluster is tightly regulated by the pH. Finally, mNT is extremely resistant to H2O2 compared to ISCU and SufB, two other Fe-S cluster transfer proteins, which is consistent with its involvement in a repair pathway of stress-damaged Fe-S proteins. Taken together, our results suggest that the ability of mNT to transfer its cluster to recipient proteins is not only controlled by the redox state of its cluster but also tightly modulated by the pH of the cytosol. We propose that when pathophysiological conditions such as cancer and neurodegenerative diseases dysregulate cellular pH homeostasis, this pH-dependent regulation of mNT is lost, as is the regulation of cellular pathways under the control of mNT.

A synaptic mechanism for temporal filtering of visual signals.

The visual system transmits information about fast and slow changes in light intensity through separate neural pathways. We used in vivo imaging to investigate how bipolar cells transmit these signals to the inner retina. We found that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Individual cells transmit through multiple terminals varying in size, but smaller terminals generate faster and larger calcium transients to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Modeling global calcium dynamics triggering vesicle release indicated that variations in the volume of presynaptic compartments contribute directly to all these differences in response dynamics. These results indicate how one neuron can transmit different temporal components in the visual signal through synaptic terminals of varying geometries with different adaptational properties.

Pre-transplant kidney quality evaluation using photoacoustic imaging during normothermic machine perfusion.

Due to the shortage of kidneys donated for transplantation, surgeons are forced to use the organs with an elevated risk of poor function or even failure. Although the existing methods for pre-transplant quality evaluation have been validated over decades in population cohort studies across the world, new methods are needed as long as delayed graft function or failure in a kidney transplant occurs. In this study, we explored the potential of utilizing photoacoustic (PA) imaging during normothermic machine perfusion (NMP) as a means of evaluating kidney quality. We closely monitored twenty-two porcine kidneys using 3D PA imaging during a two-hour NMP session. Based on biochemical analyses of perfusate and produced urine, the kidneys were categorized into 'non-functional' and 'functional' groups. Our primary focus was to quantify oxygenation (sO2) within the kidney cortical layer of depths 2 mm, 4 mm, and 6 mm using two-wavelength PA imaging. Next, receiver operating characteristic (ROC) analysis was performed to determine an optimal cortical layer depth and time point for the quantification of sO2 to discriminate between functional and non-functional organs. Finally, for each depth, we assessed the correlation between sO2 and creatinine clearance (CrCl), oxygen consumption (VO2), and renal blood flow (RBF). We found that hypoxia of the renal cortex is associated with poor renal function. In addition, the determination of sO2 within the 2 mm depth of the renal cortex after 30 min of NMP effectively distinguishes between functional and non-functional kidneys. The non-functional kidneys can be detected with the sensitivity and specificity of 80% and 85% respectively, using the cut-off point of sO2 < 39%. Oxygenation significantly correlates with RBF and VO2 in all kidneys. In functional kidneys, sO2 correlated with CrCl, which is not the case for non-functional kidneys. We conclude that the presented technique has a high potential for supporting organ selection for kidney transplantation.

Photoacoustic imaging on its way toward clinical utility: a tutorial review focusing on practical application in medicine.

Significance: Photoacoustic imaging (PAI) enables the visualization of optical contrast with ultrasonic imaging. It is a field of intense research, with great promise for clinical application. Understanding the principles of PAI is important for engineering research and image interpretation. Aim: In this tutorial review, we lay out the imaging physics, instrumentation requirements, standardization, and some practical examples for (junior) researchers, who have an interest in developing PAI systems and applications for clinical translation or applying PAI in clinical research. Approach: We discuss PAI principles and implementation in a shared context, emphasizing technical solutions that are amenable to broad clinical deployment, considering factors such as robustness, mobility, and cost in addition to image quality and quantification. Results: Photoacoustics, capitalizing on endogenous contrast or administered contrast agents that are approved for human use, yields highly informative images in clinical settings, which can support diagnosis and interventions in the future. Conclusion: PAI offers unique image contrast that has been demonstrated in a broad set of clinical scenarios. The transition of PAI from a "nice-to-have" to a "need-to-have" modality will require dedicated clinical studies that evaluate therapeutic decision-making based on PAI and consideration of the actual value for patients and clinicians, compared with the associated cost.

Ultrasound-guided breast biopsy using an adapted automated cone-based ultrasound scanner: a feasibility study.

BACKGROUND: Among available breast biopsy techniques, ultrasound (US)-guided biopsy is preferable because it is relatively inexpensive and provides live imaging feedback. The availability of magnetic resonance imaging (MRI)-3D US image fusion would facilitate US-guided biopsy even for US occult lesions to reduce the need for expensive and time-consuming MRI-guided biopsy. In this paper, we propose a novel Automated Cone-based Breast Ultrasound Scanning and Biopsy System (ACBUS-BS) to scan and biopsy breasts of women in prone position. It is based on a previously developed system, called ACBUS, that facilitates MRI-3D US image fusion imaging of the breast employing a conical container filled with coupling medium. PURPOSE: The purpose of this study was to introduce the ABCUS-BS system and demonstrate its feasibility for biopsy of US occult lesions. METHOD: The biopsy procedure with the ACBUS-BS comprises four steps: target localization, positioning, preparation, and biopsy. The biopsy outcome can be impacted by 5 types of errors: due to lesion segmentation, MRI-3D US registration, navigation, lesion tracking during repositioning, and US inaccuracy (due to sound speed difference between the sample and the one used for image reconstruction). For the quantification, we use a soft custom-made polyvinyl alcohol phantom (PVA) containing eight lesions (three US-occult and five US-visible lesions of 10 mm in diameter) and a commercial breast mimicking phantom with a median stiffness of 7.6 and 28 kPa, respectively. Errors of all types were quantified using the custom-made phantom. The error due to lesion tracking was also quantified with the commercial phantom. Finally, the technology was validated by biopsying the custom-made phantom and comparing the size of the biopsied material to the original lesion size. The average size of the 10-mm-sized lesions in the biopsy specimen was 7.00 ± 0.92 mm (6.33 ± 1.16 mm for US occult lesions, and 7.40 ± 0.55 mm for US-visible lesions). RESULTS: For the PVA phantom, the errors due to registration, navigation, lesion tracking during repositioning, and US inaccuracy were 1.33, 0.30, 2.12, and 0.55 mm. The total error was 4.01 mm. For the commercial phantom, the error due to lesion tracking was estimated at 1.10 mm, and the total error was 4.11 mm. Given these results, the system is expected to successfully biopsy lesions larger than 8.22 mm in diameter. Patient studies will have to be carried out to confirm this in vivo. CONCLUSION: The ACBUS-BS facilitates US-guided biopsy of lesions detected in pre-MRI and therefore might offer a low-cost alternative to MRI-guided biopsy. We demonstrated the feasibility of the approach by successfully taking biopsies of five US-visible and three US-occult lesions embedded in a soft breast-shaped phantom.

Features of the Preparation and Luminescence of Langmuir-Blodgett Films Based on the Tb(III) Complex with 3-Methyl-1-phenyl-4-stearoylpyrazol-5-one and 2,2'-Bipyridine.

In this study, we investigated the effect of terbium ions (Tb3+) on the subphases of the limiting area of the molecule for the complex compound (CC) TbL3∙bipy (where HL is 3-methyl-1-phenyl-4-stearoylpyrazol-5-one and bipy is 2,2'-bipyridine). We examined the Langmuir monolayer and the change in the luminescence properties of TbL3∙bipy-based Langmuir-Blodgett films (LBFs). The analysis of the compression isotherms, infrared, and luminescence spectra of TbL3∙bipy LBFs was performed by varying the concentration of Tb3+ in the subphases. Our results demonstrate the partial dissociation of the CC at concentrations of C(Tb3+) < 5 × 10-4 M.

An Open-Source Framework for Automated High-Throughput Cell Biology Experiments.

Modern data analysis methods, such as optimization algorithms or deep learning have been successfully applied to a number of biotechnological and medical questions. For these methods to be efficient, a large number of high-quality and reproducible experiments needs to be conducted, requiring a high degree of automation. Here, we present an open-source hardware and low-cost framework that allows for automatic high-throughput generation of large amounts of cell biology data. Our design consists of an epifluorescent microscope with automated XY stage for moving a multiwell plate containing cells and a perfusion manifold allowing programmed application of up to eight different solutions. Our system is very flexible and can be adapted easily for individual experimental needs. To demonstrate the utility of the system, we have used it to perform high-throughput Ca2+ imaging and large-scale fluorescent labeling experiments.

Electrocatalytic evidence of the diversity of the oxygen reaction in the bacterial bd oxidase from different organisms.

Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed.

Quantitative Evaluation of an Automated Cone-Based Breast Ultrasound Scanner for MRI-3D US Image Fusion.

Breast cancer is one of the most diagnosed types of cancer worldwide. Volumetric ultrasound breast imaging, combined with MRI can improve lesion detection rate, reduce examination time, and improve lesion diagnosis. However, to our knowledge, there are no 3D US breast imaging systems available that facilitate 3D US - MRI image fusion. In this paper, a novel Automated Cone-based Breast Ultrasound System (ACBUS) is introduced. The system facilitates volumetric ultrasound acquisition of the breast in a prone position without deforming it by the US transducer. Quality of ACBUS images for reconstructions at different voxel sizes (0.25 and 0.50 mm isotropic) was compared to quality of the Automated Breast Volumetric Scanner (ABVS) (Siemens Ultrasound, Issaquah, WA, USA) in terms of signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and resolution using a custom made phantom. The ACBUS image data were registered to MRI image data utilizing surface matching and the registration accuracy was quantified using an internal marker. The technology was also evaluated in vivo. The phantom-based quantitative analysis demonstrated that ACBUS can deliver volumetric breast images with an image quality similar to the images delivered by a currently commercially available Siemens ABVS. We demonstrate on the phantom and in vivo that ACBUS enables adequate MRI-3D US fusion. To our conclusion, ACBUS might be a suitable candidate for a second-look breast US exam, patient follow-up, and US guided biopsy planning.

Impact of two of the world's largest protected areas on longline fishery catch rates.

Two of the largest protected areas on earth are U.S. National Monuments in the Pacific Ocean. Numerous claims have been made about the impacts of these protected areas on the fishing industry, but there has been no ex post empirical evaluation of their effects. We use administrative data documenting individual fishing events to evaluate the economic impact of the expansion of these two monuments on the Hawaii longline fishing fleet. Surprisingly, catch and catch-per-unit-effort are higher since the expansions began. To disentangle the causal effect of the expansions from confounding factors, we use unaffected control fisheries to perform a difference-in-differences analysis. We find that the monument expansions had little, if any, negative impacts on the fishing industry, corroborating ecological models that have predicted minimal impacts from closing large parts of the Pacific Ocean to fishing.

Coordinated Ras and Rac Activity Shapes Macropinocytic Cups and Enables Phagocytosis of Geometrically Diverse Bacteria.

Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialized cup-shaped protrusions. To effectively capture and internalize their targets, these cups are organized into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR, and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac, and phospholipid interactions to localize at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, we propose RGBARG shapes the protrusion by expanding Rac activation at the rim while suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of coordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.

Distinct roles for two histamine receptors (hclA and hclB) at the Drosophila photoreceptor synapse.

Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC(50) = 25 microM). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states ( approximately 25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC(50) = 14 and 1.2 microM, respectively), with much smaller single-channel conductances ( approximately 4 pS). Null mutations of hclA (ort(US6096)) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG "on" transients in hclB mutants transients were approximately twofold enhanced, whereas intracellular recordings from their LMCs revealed altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, was exclusively localized to the glia cells, whereas HCLA expression was confirmed in the LMCs. Our results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response.

Long-term imaging of calcium dynamics using genetically encoded calcium indicators and automatic tracking of cultured cells.

Calcium dynamics is crucial for many signaling pathways and cell functions. Understanding how calcium regulates cell function often requires long-term imaging of calcium dynamics. Here we report a methodological approach of long-term (5-10 h) imaging of calcium dynamics in cultured cells. The approach links calcium imaging using genetically encoded calcium indicators and semi-automatic tracking of individual cells. It can be used in a large variety of situations, ranging from the role of calcium in biological processes to cell heterogeneity and screening of drugs modifying signaling pathways.