Mapping the spatiotemporal dynamics of calcium signaling in cellular neural networks using optical flow

An optical flow gradient algorithm was applied to spontaneously forming net- works of neurons and glia in culture imaged by fluorescence optical microscopy in order to map functional calcium signaling with single pixel resolution. Optical flow estimates the direction and speed of motion of objects in an image between subsequent frames in a recorded digital sequence of images (i.e. a movie). Computed vector field outputs by the algorithm were able to track the spatiotemporal dynamics of calcium signaling pat- terns. We begin by briefly reviewing the mathematics of the optical flow algorithm, and then describe how to solve for the displacement vectors and how to measure their reliability. We then compare computed flow vectors with manually estimated vectors for the progression of a calcium signal recorded from representative astrocyte cultures. Finally, we applied the algorithm to preparations of primary astrocytes and hippocampal neurons and to the rMC-1 Muller glial cell line in order to illustrate the capability of the algorithm for capturing different types of spatiotemporal calcium activity. We discuss the imaging requirements, parameter selection and threshold selection for reliable measurements, and offer perspectives on uses of the vector data.Comment: 23 pages, 5 figures. Peer reviewed accepted version in press in Annals of Biomedical Engineerin

Probing the dynamics of identified neurons with a data-driven modeling approach

In controlling animal behavior the nervous system has to perform within the operational limits set by the requirements of each specific behavior. The implications for the corresponding range of suitable network, single neuron, and ion channel properties have remained elusive. In this article we approach the question of how well-constrained properties of neuronal systems may be on the neuronal level. We used large data sets of the activity of isolated invertebrate identified cells and built an accurate conductance-based model for this cell type using customized automated parameter estimation techniques. By direct inspection of the data we found that the variability of the neurons is larger when they are isolated from the circuit than when in the intact system. Furthermore, the responses of the neurons to perturbations appear to be more consistent than their autonomous behavior under stationary conditions. In the developed model, the constraints on different parameters that enforce appropriate model dynamics vary widely from some very tightly controlled parameters to others that are almost arbitrary. The model also allows predictions for the effect of blocking selected ionic currents and to prove that the origin of irregular dynamics in the neuron model is proper chaoticity and that this chaoticity is typical in an appropriate sense. Our results indicate that data driven models are useful tools for the in-depth analysis of neuronal dynamics. The better consistency of responses to perturbations, in the real neurons as well as in the model, suggests a paradigm shift away from measuring autonomous dynamics alone towards protocols of controlled perturbations. Our predictions for the impact of channel blockers on the neuronal dynamics and the proof of chaoticity underscore the wide scope of our approach

Automated detection and analysis of fluorescence changes evoked by molecular signalling

Fluorescent dyes and genetically encoded fluorescence indicators (GEFI) are common tools for visualizing concentration changes of specific ions and messenger molecules during intra- as well as intercellular communication. While fluorescent dyes have to be directly loaded into target cells and function only transiently, the expression of GEFIs can be controlled in a cell and time-specific fashion, even allowing long-term analysis in living organisms. Dye and GEFI based fluorescence fluctuations, recorded using advanced imaging technologies, are the foundation for the analysis of physiological molecular signaling. Analyzing the plethora of complex fluorescence signals is a laborious and time-consuming task. An automated analysis of fluorescent signals circumvents user bias and time constraints. However, it requires to overcome several challenges, including correct estimation of fluorescence fluctuations at basal concentrations of messenger molecules, detection and extraction of events themselves, proper segmentation of neighboring events as well as tracking of propagating events. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. This thesis presents three novel algorithms, PBasE, CoRoDe and KalEve, for the automated analysis of fluorescence events, developed to overcome the aforementioned challenges. The algorithms are integrated into a graphical application called MSparkles, specifically designed for the analysis of fluorescence signals, developed in MATLAB. The capabilities of the algorithms are demonstrated by analyzing astroglial Ca2+ events, recorded in anesthetized and awake mice, visualized using genetically encoded Ca2+ indicators (GECIs) GCaMP3 as well as GCaMP5. The results were compared to those obtained by other software packages. In addition, the analysis of neuronal Na+ events recorded in acute brain slices using SBFI-AM serve to indicate the putatively broad application range of the presented algorithms. Finally, due to increasing evidence of the pivotal role of astrocytes in neurodegenerative diseases such as epilepsy, a metric to assess the synchronous occurrence of fluorescence events is introduced. In a proof-of-principle analysis, this metric is used to correlate astroglial Ca2+ events with EEG measurementsFluoreszenzfarbstoffe und genetisch kodierte Fluoreszenzindikatoren (GEFI) sind gängige Werkzeuge zur Visualisierung von Konzentrationsänderungen bestimmter Ionen und Botenmoleküle der intra- sowie interzellulären Kommunikation. Während Fluoreszenzfarbstoffe direkt in die Zielzellen eingebracht werden müssen und nur über einen begrenzten Zeitraum funktionieren, kann die Expression von GEFIs zell- und zeitspezifisch gesteuert werden, was darüber hinaus Langzeitanalysen in lebenden Organismen ermöglicht. Farbstoff- und GEFI-basierte Fluoreszenzfluktuationen, die mit Hilfe moderner bildgebender Verfahren aufgezeichnet werden, bilden die Grundlage für die Analyse physiologischer molekularer Kommunikation. Die Analyse einer großen Zahl komplexer Fluoreszenzsignale ist jedoch eine schwierige und zeitaufwändige Aufgabe. Eine automatisierte Analyse ist dagegen weniger zeitaufwändig und unabhängig von der Voreingenommenheit des Anwenders. Allerdings müssen hierzu mehrere Herausforderungen bewältigt werden. Unter anderem die korrekte Schätzung von Fluoreszenzschwankungen bei Basalkonzentrationen von Botenmolekülen, die Detektion und Extraktion von Signalen selbst, die korrekte Segmentierung benachbarter Signale sowie die Verfolgung sich ausbreitender Signale. Darüber hinaus müssen die Algorithmen zur Signalerkennung empfindlich genug sein, um lokalisierte Signale mit geringer Amplitude sowie begrenzter räumlicher Ausdehnung genau zu erfassen. In dieser Arbeit werden drei neue Algorithmen, PBasE, CoRoDe und KalEve, für die automatische Extraktion und Analyse von Fluoreszenzsignalen vorgestellt, die entwickelt wurden, um die oben genannten Herausforderungen zu bewältigen. Die Algorithmen sind in eine grafische Anwendung namens MSparkles integriert, die speziell für die Analyse von Fluoreszenzsignalen entwickelt und in MATLAB implementiert wurde. Die Fähigkeiten der Algorithmen werden anhand der Analyse astroglialer Ca2+-Signale demonstriert, die in narkotisierten sowie wachen Mäusen aufgezeichnet und mit den genetisch kodierten Ca2+-Indikatoren (GECIs) GCaMP3 und GCaMP5 visualisiert wurden. Erlangte Ergebnisse werden anschließend mit denen anderer Softwarepakete verglichen. Darüber hinaus dient die Analyse neuronaler Na+-Signale, die in akuten Hirnschnitten mit SBFI-AM aufgezeichnet wurden, dazu, den breiten Anwendungsbereich der Algorithmen aufzuzeigen. Zu guter Letzt wird aufgrund der zunehmenden Indizien auf die zentrale Rolle von Astrozyten bei neurodegenerativen Erkrankungen wie Epilepsie eine Metrik zur Bewertung des synchronen Auftretens fluoreszenter Signale eingeführt. In einer Proof-of-Principle-Analyse wird diese Metrik verwendet, um astrogliale Ca2+-Signale mit EEG-Messungen zu korrelieren

FindSim: a Framework for Integrating Neuronal Data and Signaling Models

Current experiments touch only small but overlapping parts of very complex subcellular signaling networks in neurons. Even with modern optical reporters and pharmacological manipulations, a given experiment can only monitor and control a very small subset of the diverse, multiscale processes of neuronal signaling. We have developed FindSim (Framework for Integrating Neuronal Data and SIgnaling Models) to anchor models to structured experimental datasets. FindSim is a framework for integrating many individual electrophysiological and biochemical experiments with large, multiscale models so as to systematically refine and validate the model. We use a structured format for encoding the conditions of many standard physiological and pharmacological experiments, specifying which parts of the model are involved, and comparing experiment outcomes with model output. A database of such experiments is run against successive generations of composite cellular models to iteratively improve the model against each experiment, while retaining global model validity. We suggest that this toolchain provides a principled and scalable way to tackle model complexity and diversity of data sources

Model-free reconstruction of neuronal network connectivity from calcium imaging signals

A systematic assessment of global neural network connectivity through direct electrophysiological assays has remained technically unfeasible even in dissociated neuronal cultures. We introduce an improved algorithmic approach based on Transfer Entropy to reconstruct approximations to network structural connectivities from network activity monitored through calcium fluorescence imaging. Based on information theory, our method requires no prior assumptions on the statistics of neuronal firing and neuronal connections. The performance of our algorithm is benchmarked on surrogate time-series of calcium fluorescence generated by the simulated dynamics of a network with known ground-truth topology. We find that the effective network topology revealed by Transfer Entropy depends qualitatively on the time-dependent dynamic state of the network (e.g., bursting or non-bursting). We thus demonstrate how conditioning with respect to the global mean activity improves the performance of our method. [...] Compared to other reconstruction strategies such as cross-correlation or Granger Causality methods, our method based on improved Transfer Entropy is remarkably more accurate. In particular, it provides a good reconstruction of the network clustering coefficient, allowing to discriminate between weakly or strongly clustered topologies, whereas on the other hand an approach based on cross-correlations would invariantly detect artificially high levels of clustering. Finally, we present the applicability of our method to real recordings of in vitro cortical cultures. We demonstrate that these networks are characterized by an elevated level of clustering compared to a random graph (although not extreme) and by a markedly non-local connectivity.Comment: 54 pages, 8 figures (+9 supplementary figures), 1 table; submitted for publicatio

Population-scale organization of cerebellar granule neuron signaling during a visuomotor behavior.

Granule cells at the input layer of the cerebellum comprise over half the neurons in the human brain and are thought to be critical for learning. However, little is known about granule neuron signaling at the population scale during behavior. We used calcium imaging in awake zebrafish during optokinetic behavior to record transgenically identified granule neurons throughout a cerebellar population. A significant fraction of the population was responsive at any given time. In contrast to core precerebellar populations, granule neuron responses were relatively heterogeneous, with variation in the degree of rectification and the balance of positive versus negative changes in activity. Functional correlations were strongest for nearby cells, with weak spatial gradients in the degree of rectification and the average sign of response. These data open a new window upon cerebellar function and suggest granule layer signals represent elementary building blocks under-represented in core sensorimotor pathways, thereby enabling the construction of novel patterns of activity for learning