FluoAnalysis: An Open-Source MATLAB Toolbox for Analysis of Calcium Imaging Measurements of Oscillatory Astrocytic and Neuronal Networks.

Calcium imaging, especially two-photon imaging, has become essential in neuroscience for studying neuronal and astrocytic activity under in vivo and in vitro conditions. Current advances in the development of calcium sensors as well as imaging hardware enable high-frequency measurements of calcium signals in hundreds of cells simultaneously. The analysis of these large datasets requires special tools and usually a certain level of programming experience. Despite advancements in calcium imaging analysis software development, significant gaps remain, particularly for data acquired at a high sampling rate that would allow for the spectral analysis of calcium signals. The FluoAnalysis MATLAB toolbox addresses these gaps by offering a comprehensive solution for analyzing simultaneously measured calcium imaging and electrophysiological data. It features both GUI-based and command-line approaches, emphasizing frequency domain analysis to reveal network-level oscillatory signals linked to single-cell activity. In addition, the toolbox puts special emphasis on differentiating between astrocytes and neurons, revealing the interactions between the network activity of the two major cell types of the brain. It facilitates a streamlined workflow for data loading, ROI identification, cell classification, fluorescence intensity calculation, spectral analysis, and report generation, supporting both manual and automated high-throughput analysis. This versatile platform enables the comprehensive analysis of large imaging datasets. In conclusion, the FluoAnalysis MATLAB toolbox provides a robust and versatile platform for the integrated analysis of calcium imaging and electrophysiological data, supporting diverse neuroscience research applications.

High-Frequency Imaging Reveals Synchronised Delta- and Theta-Band Ca2+ Oscillations in the Astrocytic Soma In Vivo.

One of the major breakthroughs of neurobiology was the identification of distinct ranges of oscillatory activity in the neuronal network that were found to be responsible for specific biological functions, both physiological and pathological in nature. Astrocytes, physically coupled by gap junctions and possessing the ability to simultaneously modulate the functions of a large number of surrounding synapses, are perfectly positioned to introduce synchronised oscillatory activity into the neural network. However, astrocytic somatic calcium signalling has not been investigated to date in the frequency ranges of common neuronal oscillations, since astrocytes are generally considered to be slow responders in terms of Ca2+ signalling. Using high-frequency two-photon imaging, we reveal fast Ca2+ oscillations in the soma of astrocytes in the delta (0.5-4 Hz) and theta (4-8 Hz) frequency bands in vivo in the rat cortex under ketamine-xylazine anaesthesia, which is known to induce permanent slow-wave sleep. The high-frequency astrocytic Ca2+ signals were not observed under fentanyl anaesthesia, excluding the possibility that the signals were introduced by motion artefacts. We also demonstrate that these fast astrocytic Ca2+ signals, previously considered to be exclusive to neurons, are present in a large number of astrocytes and are phase synchronised at the astrocytic network level. We foresee that the disclosure of these high-frequency astrocytic signals may help with understanding the appearance of synchronised oscillatory signals and may open up new avenues of treatment for neurological conditions characterised by altered neuronal oscillations.

Evaluation of Ultrasound-based Surveillance for Hepatocellular Carcinoma in Patients at Risk: Results From a German Multicenter Retrospective Cohort Study.

Background and Aims: Hepatocellular carcinoma (HCC) surveillance in patients at risk is strongly recommended and usually performed by ultrasound (US) semiannually with or without alfa-fetoprotein (AFP) measurements. Quality parameters except for surveillance intervals have not been strictly defined. We aimed to evaluate surveillance success and risk factors for surveillance failure. Methods: Patients with ≥1 US prior to HCC diagnosis performed at four tertiary referral hospitals in Germany between 2008 and 2019 were retrospectively analyzed. Surveillance success was defined as HCC detection within Milan criteria. Results: Only 47% of 156 patients, median age 63 (interquartile range: 57-70) years, 56% male, and 96% with cirrhosis, received recommended surveillance modality and interval. Surveillance failure occurred in 29% and was significantly associated with lower median model for end-stage liver disease (MELD) score odds ratio (OR) 1.154, 95% confidence interval (CI): 1.027-1.297, p=0.025) and HCC localization within right liver lobe (OR: 6.083, 95% CI: 1.303-28.407, p=0.022), but not with AFP ≥200 µg/L. Patients with surveillance failure had significantly more intermediate/advanced tumor stages (93% vs. 6%, p<0.001), fewer curative treatment options (15% vs. 75%, p<0.001) and lower survival at 1 year (54% vs. 75%, p=0.041), 2 years (32% vs. 57%, p=0.019) and 5 years (0% vs. 16%, p=0.009). Alcoholic and non-alcoholic fatty liver disease (OR: 6.1, 95% CI: 1.7-21.3, p=0.005) and ascites (OR: 3.9, 95% CI: 1.2-12.6, p=0.021) were independently associated with severe visual limitations on US. Conclusions: US-based HCC surveillance in patients at risk frequently fails and its failure is associated with unfavorable patient-related outcomes. Lower MELD score and HCC localization within right liver lobe were significantly associated with surveillance failure.

Dual Role for Astroglial Copper-Assisted Polyamine Metabolism during Intense Network Activity.

Astrocytes serve essential roles in human brain function and diseases. Growing evidence indicates that astrocytes are central players of the feedback modulation of excitatory Glu signalling during epileptiform activity via Glu-GABA exchange. The underlying mechanism results in the increase of tonic inhibition by reverse operation of the astroglial GABA transporter, induced by Glu-Na+ symport. GABA, released from astrocytes, is synthesized from the polyamine (PA) putrescine and this process involves copper amino oxidase. Through this pathway, putrescine can be considered as an important source of inhibitory signaling that counterbalances epileptic discharges. Putrescine, however, is also a precursor for spermine that is known to enhance gap junction channel communication and, consequently, supports long-range Ca2+ signaling and contributes to spreading of excitatory activity through the astrocytic syncytium. Recently, we presented the possibility of neuron-glia redox coupling through copper (Cu+/Cu2+) signaling and oxidative putrescine catabolism. In the current work, we explore whether the Cu+/Cu2+ homeostasis is involved in astrocytic control on neuronal excitability by regulating PA catabolism. We provide supporting experimental data underlying this hypothesis. We show that the blockade of copper transporter (CTR1) by AgNO3 (3.6 µM) prevents GABA transporter-mediated tonic inhibitory currents, indicating causal relationship between copper (Cu+/Cu2+) uptake and the catabolism of putrescine to GABA in astrocytes. In addition, we show that MnCl2 (20 µM), an inhibitor of the divalent metal transporter DMT1, also prevents the astrocytic Glu-GABA exchange. Furthermore, we observed that facilitation of copper uptake by added CuCl2 (2 µM) boosts tonic inhibitory currents. These findings corroborate the hypothesis that modulation of neuron-glia coupling by copper uptake drives putrescine → GABA transformation, which leads to subsequent Glu-GABA exchange and tonic inhibition. Findings may in turn highlight the potential role of copper signaling in fine-tuning the activity of the tripartite synapse.

Transabdominal ultrasonography to reduce the burden of X-ray imaging in prophylactic pancreatic stent localization after ERCP-A prospective trial.

BACKGROUND: Before performing endoscopy to remove prophylactic pancreatic stents placed in patients with high risk of post-endoscopic retrograde cholangiopancreatography pancreatitis (PEP), X-ray imaging is recommended to confirm the stents position in the pancreatic duct. OBJECTIVES: The aim of the present study was to investigate the feasibility of prophylactic pancreatic stent detection by transabdominal ultrasonography, to reduce the burden of X-ray imaging, which is currently the golden standard. METHODS: All patients who received a pancreatic stent for PEP prophylaxis were included in the present prospective trial. First, stent position was determined by transabdominal ultrasonography. Afterwards, it was verified by X-ray imaging. Retained stents were removed by esophagogastroduodenoscopy. Dislocated stents needed no further intervention. RESULTS: Fourty-one patients were enrolled in this study. All prophylactic pancreatic stents were straight 6 cm long 5 Fr stents with external flap. All stents were removed between day 1 and 10 (median: 3 days) in all cases. In 34 of 41 cases (83.0%), the pancreatic stent was still in place on the day of examination. Twenty-nine of 34 (85.3%) stents were detected correctly by transabdominal ultrasonography. Overlying gas prevented visualization of the pancreas in 3/41 (7.3%) cases. Sensitivity of sonographic detection of the stent was 93.5% (29/31). Six of seven stents were determined correctly as dislocated by ultrasonography. Here, specificity was 85.7%. A positive predictive value of 96.7% (29/30) was examined. The negative predictive value was 75.0% (6/8). CONCLUSION: Transabdominal ultrasonography detects the majority of prophylactic pancreatic stents. Thereby, it helps to identify patients with an indication for endoscopy sufficiently. X-ray imaging could subsequently be omitted in about 70% of examinations, reducing the radiation exposure for the patient and the endoscopy staff.

Spontaneous Ca2+ Fluctuations Arise in Thin Astrocytic Processes With Real 3D Geometry.

Fluctuations of cytosolic Ca2+ concentration in astrocytes are regarded as a critical non-neuronal signal to regulate neuronal functions. Although such fluctuations can be evoked by neuronal activity, rhythmic astrocytic Ca2+ oscillations may also spontaneously arise. Experimental studies hint that these spontaneous astrocytic Ca2+ oscillations may lie behind different kinds of emerging neuronal synchronized activities, like epileptogenic bursts or slow-wave rhythms. Despite the potential importance of spontaneous Ca2+ oscillations in astrocytes, the mechanism by which they develop is poorly understood. Using simple 3D synapse models and kinetic data of astrocytic Glu transporters (EAATs) and the Na+/Ca2+ exchanger (NCX), we have previously shown that NCX activity alone can generate markedly stable, spontaneous Ca2+ oscillation in the astrocytic leaflet microdomain. Here, we extend that model by incorporating experimentally determined real 3D geometries of 208 excitatory synapses reconstructed from publicly available ultra-resolution electron microscopy datasets. Our simulations predict that the surface/volume ratio (SVR) of peri-synaptic astrocytic processes prominently dictates whether NCX-mediated spontaneous Ca2+ oscillations emerge. We also show that increased levels of intracellular astrocytic Na+ concentration facilitate the appearance of Ca2+ fluctuations. These results further support the principal role of the dynamical reshaping of astrocyte processes in the generation of intrinsic Ca2+ oscillations and their spreading over larger astrocytic compartments.

Investigations on spreading of PRRSV among swine herds by improved minimum spanning network analysis.

In Hungary, the economic losses caused by porcine reproductive and respiratory syndrome virus (PRRSV) led to the launching of a national PRRSV Eradication Program. An important element of the program was investigating the spread of PRRSV among swine herds and the possible ways of introduction by sequencing of the open reading frame 5 (ORF5) gene. However, the classical phylogenetic tree presentation cannot explain several genetic relationships clearly, while more precise visualization can be represented by network tree diagram. In this paper, we describe a practical and easy-to-follow enriched minimum spanning similarity network application for improved representation of phylogenetic relations among viral strains. This method eliminated the necessity of applying a predefined, arbitrary cut-off or computationally extensive algorithms. The network-based visualization allowed processing and visualizing large amount of data equally for the laboratory, private and official veterinarians, and helped identify the potential connections between different viral sequences that support data-driven decisions in the eradication program. By applying network analysis, previously unknown epidemiological connections between infected herds were identified, and virus spreading was analyzed within short period of time. In our study, we successfully built and applied network analysis tools in the course of the Hungarian PRRSV Eradication Program.

Connexin 43 Differentially Regulates Epileptiform Activity in Models of Convulsive and Non-convulsive Epilepsies.

The influence of astrocytic cell networks on neuronal network activity is an emerging issue in epilepsy. Among the various mechanisms by which astrocytes modulate neuronal function, synchronization of astrocytes via gap junction channels is widely considered to be a crucial mechanism in epileptic conditions, contributing to the synchronization of the neuronal cell networks, possibly inducing recurrent epileptiform activity. Here, we explored whether modulation of astrocytic gap junctions could alter epileptic seizures in different types of epilepsy. Opening of gap junctions by trimethylamine intensifies seizure-like events (SLEs) in the low-[Mg2+] in vitro model of temporal lobe epilepsy, while alleviates seizures in the in vivo WAG/Rij rat model of absence epilepsy. In contrast, application of the gap junction blocker carbenoxolone prevents the appearance of SLEs in the low-[Mg2+] epilepsy model, but aggravates seizures in non-convulsive absence epilepsy, in vivo. Pharmacological dissection of neuronal vs. astrocytic connexins shows that astrocytic Cx43 contribute to seizure formation to a significantly higher extent than neuronal Cx36. We conclude that astrocytic gap junctions are key players in the formation of epileptiform activity and we provide a scheme for the different mode of action in the convulsive and non-convulsive epilepsy types.

Cell cycle dependent RRM2 may serve as proliferation marker and pharmaceutical target in adrenocortical cancer.

Adrenocortical cancer (ACC) is a rare, but agressive malignancy with poor prognosis. Histopathological diagnosis is challenging and pharmacological options for treatment are limited. By the comparative reanalysis of the transcriptional malignancy signature with the cell cycle dependent transcriptional program of ACC, we aimed to identify novel biomarkers which may be used in the histopathological diagnosis and for the prediction of therapeutical response of ACC. Comparative reanalysis of publicly available microarray datasets included three earlier studies comparing transcriptional differences between ACC and benign adrenocortical adenoma (ACA) and one study presenting the cell cycle dependent gene expressional program of human ACC cell line NCI-H295R. Immunohistochemical analysis was performed on ACC samples. In vitro effects of antineoplastic drugs including gemcitabine, mitotane and 9-cis-retinoic acid alone and in combination were tested in the NCI-H295R adrenocortical cell line. Upon the comparative reanalysis, ribonucleotide reductase subunit 2 (RRM2), responsible for the ribonucleotide dezoxyribonucleotide conversion during the S phase of the cell cycle has been validated as cell cycle dependently expressed. Moreover, its expression was associated with the malignancy signature, as well. Immunohistochemical analysis of RRM2 revealed a strong correlation with Ki67 index in ACC. Among the antiproliferative effects of the investigated compounds, gemcitabine showed a strong inhibition of proliferation and an increase of apoptotic events. Additionally, RRM2 has been upregulated upon gemcitabine treatment. Upon our results, RRM2 might be used as a proliferation marker in ACC. RRM2 upregulation upon gemcitabine treatment might contribute to an emerging chemoresistance against gemcitabine, which is in line with its limited therapeutical efficacy in ACC, and which should be overcome for successful clinical applications.

The role of the p53 protein in nitrosative stress-induced apoptosis of PC12 rat pheochromocytoma cells.

PC12 rat pheochromocytoma cells are widely used to investigate signaling pathways. The p143p53PC12 cell line expresses a Val143Ala mutant p53 protein that is less capable of binding to the p53 consensus site in DNA than its wild-type counterpart. Nitric oxide (NO), depending on its concentration, is able to activate several signal transduction pathways. We used sodium nitroprusside (SNP), an NO donor compound, to analyze NO-induced cellular stress in order to clarify the mechanism and role of nitrosative stress in pathological processes, including inflammation and cancer. SNP caused cell death when applied at a concentration of 400 µM, p143p53PC12 cells showing higher sensitivity than wild-type PC12 cells. The mechanisms leading to the increased SNP-sensitivity of p143p53PC12 cells were then investigated. The 400-µM SNP treatment caused stress kinase activation, phosphorylation of the eukaryotic initiation factor eIF2α and p53 protein, proteolytic activation of protein kinase R, caspase-9, and caspase-3, p53 stabilization, CHOP induction, cytochrome c release from mitochondria, and a decline in the level of the Bcl-2 protein in both cell lines. All these SNP-induced changes were more robust and/or permanent in cells with the mutant p53 protein. We thus conclude that (1) the main cause of the SNP-induced apoptosis of PC12 cells is the repression of the bcl-2 gene, evoked through p53 stabilization, stress kinase activation, and CHOP induction; (2) the higher SNP sensitivity of p143p53PC12 cells is the consequence of the stronger and earlier activation of the intrinsic apoptotic pathway.