Two-photon imaging and analysis of neural network dynamics

The glow of a starry night sky, the smell of a freshly brewed cup of coffee or the sound of ocean waves breaking on the beach are representations of the physical world that have been created by the dynamic interactions of thousands of neurons in our brains. How the brain mediates perceptions, creates thoughts, stores memories and initiates actions remains one of the most profound puzzles in biology, if not all of science. A key to a mechanistic understanding of how the nervous system works is the ability to analyze the dynamics of neuronal networks in the living organism in the context of sensory stimulation and behaviour. Dynamic brain properties have been fairly well characterized on the microscopic level of individual neurons and on the macroscopic level of whole brain areas largely with the help of various electrophysiological techniques. However, our understanding of the mesoscopic level comprising local populations of hundreds to thousands of neurons (so called 'microcircuits') remains comparably poor. In large parts, this has been due to the technical difficulties involved in recording from large networks of neurons with single-cell spatial resolution and near- millisecond temporal resolution in the brain of living animals. In recent years, two-photon microscopy has emerged as a technique which meets many of these requirements and thus has become the method of choice for the interrogation of local neural circuits. Here, we review the state-of-research in the field of two-photon imaging of neuronal populations, covering the topics of microscope technology, suitable fluorescent indicator dyes, staining techniques, and in particular analysis techniques for extracting relevant information from the fluorescence data. We expect that functional analysis of neural networks using two-photon imaging will help to decipher fundamental operational principles of neural microcircuits.Comment: 36 pages, 4 figures, accepted for publication in Reports on Progress in Physic

Microstimulation and multicellular analysis: A neural interfacing system for spatiotemporal stimulation

Willfully controlling the focus of an extracellular stimulus remains a significant challenge in the development of neural prosthetics and therapeutic devices. In part, this challenge is due to the vast set of complex interactions between the electric fields induced by the microelectrodes and the complex morphologies and dynamics of the neural tissue. Overcoming such issues to produce methodologies for targeted neural stimulation requires a system that is capable of (1) delivering precise, localized stimuli a function of the stimulating electrodes and (2) recording the locations and magnitudes of the resulting evoked responses a function of the cell geometry and membrane dynamics. In order to improve stimulus delivery, we developed microfabrication technologies that could specify the electrode geometry and electrical properties. Specifically, we developed a closed-loop electroplating strategy to monitor and control the morphology of surface coatings during deposition, and we implemented pulse-plating techniques as a means to produce robust, resilient microelectrodes that could withstand rigorous handling and harsh environments. In order to evaluate the responses evoked by these stimulating electrodes, we developed microscopy techniques and signal processing algorithms that could automatically identify and evaluate the electrical response of each individual neuron. Finally, by applying this simultaneous stimulation and optical recording system to the study of dissociated cortical cultures in multielectode arrays, we could evaluate the efficacy of excitatory and inhibitory waveforms. Although we found that the proximity of the electrode is a poor predictor of individual neural excitation thresholds, we have shown that it is possible to use inhibitory waveforms to globally reduce excitability in the vicinity of the electrode. Thus, the developed system was able to provide very high resolution insight into the complex set of interactions between the stimulating electrodes and populations of individual neurons.Ph.D.Committee Chair: Stephen P. DeWeerth; Committee Member: Bruce Wheeler; Committee Member: Michelle LaPlaca; Committee Member: Robert Lee; Committee Member: Steve Potte

Quantifying network properties in multi-electrode recordings: spatiotemporal characterization and inter-trial variation of evoked gamma oscillations in mouse somatosensory cortex in vitro.

Linking the structural connectivity of brain circuits to their cooperative dynamics and emergent functions is a central aim of neuroscience research. Graph theory has recently been applied to study the structure-function relationship of networks, where dynamical similarity of different nodes has been turned into a "static" functional connection. However, the capability of the brain to adapt, learn and process external stimuli requires a constant dynamical functional rewiring between circuitries and cell assemblies. Hence, we must capture the changes of network functional connectivity over time. Multi-electrode array data present a unique challenge within this framework. We study the dynamics of gamma oscillations in acute slices of the somatosensory cortex from juvenile mice recorded by planar multi-electrode arrays. Bursts of gamma oscillatory activity lasting a few hundred milliseconds could be initiated only by brief trains of electrical stimulations applied at the deepest cortical layers and simultaneously delivered at multiple locations. Local field potentials were used to study the spatio-temporal properties and the instantaneous synchronization profile of the gamma oscillatory activity, combined with current source density (CSD) analysis. Pair-wise differences in the oscillation phase were used to determine the presence of instantaneous synchronization between the different sites of the circuitry during the oscillatory period. Despite variation in the duration of the oscillatory response over successive trials, they showed a constant average power, suggesting that the rate of expenditure of energy during the gamma bursts is consistent across repeated stimulations. Within each gamma burst, the functional connectivity map reflected the columnar organization of the neocortex. Over successive trials, an apparently random rearrangement of the functional connectivity was observed, with a more stable columnar than horizontal organization. This work reveals new features of evoked gamma oscillations in developing cortex

Bayesian multi-modal model comparison: a case study on the generators of the spike and the wave in generalized spike–wave complexes

We present a novel approach to assess the networks involved in the generation of spontaneous pathological brain activity based on multi-modal imaging data. We propose to use probabilistic fMRI-constrained EEG source reconstruction as a complement to EEG-correlated fMRI analysis to disambiguate between networks that co-occur at the fMRI time resolution. The method is based on Bayesian model comparison, where the different models correspond to different combinations of fMRI-activated (or deactivated) cortical clusters. By computing the model evidence (or marginal likelihood) of each and every candidate source space partition, we can infer the most probable set of fMRI regions that has generated a given EEG scalp data window. We illustrate the method using EEG-correlated fMRI data acquired in a patient with ictal generalized spike–wave (GSW) discharges, to examine whether different networks are involved in the generation of the spike and the wave components, respectively. To this effect, we compared a family of 128 EEG source models, based on the combinations of seven regions haemodynamically involved (deactivated) during a prolonged ictal GSW discharge, namely: bilateral precuneus, bilateral medial frontal gyrus, bilateral middle temporal gyrus, and right cuneus. Bayesian model comparison has revealed the most likely model associated with the spike component to consist of a prefrontal region and bilateral temporal–parietal regions and the most likely model associated with the wave component to comprise the same temporal–parietal regions only. The result supports the hypothesis of different neurophysiological mechanisms underlying the generation of the spike versus wave components of GSW discharges

Widefield, Spatiotemporal Mapping of Spontaneous Activity of Mouse Cultured Neuronal Networks Using Quantum Diamond Sensors

Quantum diamond sensors containing Nitrogen Vacancy (NV) centers were integrated into the culture of spontaneously electrically active cultures of live mouse primary cortical neurons. Two diamond formats were used enabling extracellular studies of cells cultured directly on a planar diamond plate and intracellular studies via endocytosis mediated integration of nanoscale diamond particles within cells. Over a 14 days culture period functional neuronal networks formed as confirmed by endogenous calcium transients detected via fluorescent imaging of a calcium probe (Fluo-5 AM). Spatially correlated images of photoluminescence from NV centers were also recorded demonstrating successful acquisition of optically detected magnetic resonance from regions of endogenously firing neuronal cultures. Further, a fast and simple measurement protocol based on acquisition of NV photoluminescence without and with the application of near resonant microwaves is presented. This protocol was applied to live signaling cells and dead cells. The relative photoluminescence from NVs with and without microwaves varied for the case of live as compared to dead cells warranting future investigation. Overall this work presents a protocol for integration of NV diamond sensors in a spontaneously active network of neuronal cells of relevance to the field of neuroscience. Photoluminescence from NV centers was detected with high spatiotemporal resolution using measurement protocols potentially capable of single shot detection of neuronal activity. It is anticipated that the methodologies introduced in this work will underpin the establishment of NV diamond sensors as a radically new measurement platform capable of rapid, non-destructive functional studies of cells

Dynamic reconfiguration of human brain networks during learning

Human learning is a complex phenomenon requiring flexibility to adapt existing brain function and precision in selecting new neurophysiological activities to drive desired behavior. These two attributes -- flexibility and selection -- must operate over multiple temporal scales as performance of a skill changes from being slow and challenging to being fast and automatic. Such selective adaptability is naturally provided by modular structure, which plays a critical role in evolution, development, and optimal network function. Using functional connectivity measurements of brain activity acquired from initial training through mastery of a simple motor skill, we explore the role of modularity in human learning by identifying dynamic changes of modular organization spanning multiple temporal scales. Our results indicate that flexibility, which we measure by the allegiance of nodes to modules, in one experimental session predicts the relative amount of learning in a future session. We also develop a general statistical framework for the identification of modular architectures in evolving systems, which is broadly applicable to disciplines where network adaptability is crucial to the understanding of system performance.Comment: Main Text: 19 pages, 4 figures Supplementary Materials: 34 pages, 4 figures, 3 table

Does higher sampling rate (multiband + SENSE) improve group statistics - An example from social neuroscience block design at 3T

Multiband (MB) or Simultaneous multi-slice (SMS) acquisition schemes allow the acquisition of MRI signals from more than one spatial coordinate at a time. Commercial availability has brought this technique within the reach of many neuroscientists and psychologists. Most early evaluation of the performance of MB acquisition employed resting state fMRI or the most basic tasks. In this study, we tested whether the advantages of using MB acquisition schemes generalize to group analyses using a cognitive task more representative of typical cognitive neuroscience applications. Twenty-three subjects were scanned on a Philips 3 ​T scanner using five sequences, up to eight-fold acceleration with MB-factors 1 to 4, SENSE factors up to 2 and corresponding TRs of 2.45s down to 0.63s, while they viewed (i) movie blocks showing complex actions with hand object interactions and (ii) control movie blocks without hand object interaction. Data were processed using a widely used analysis pipeline implemented in SPM12 including the unified segmentation and canonical HRF modelling. Using random effects group-level, voxel-wise analysis we found that all sequences were able to detect the basic action observation network known to be recruited by our task. The highest t-values were found for sequences with MB4 acceleration. For the MB1 sequence, a 50% bigger voxel volume was needed to reach comparable t-statistics. The group-level t-values for resting state networks (RSNs) were also highest for MB4 sequences. Here the MB1 sequence with larger voxel size did not perform comparable to the MB4 sequence. Altogether, we can thus recommend the use of MB4 (and SENSE 1.5 or 2) on a Philips scanner when aiming to perform group-level analyses using cognitive block design fMRI tasks and voxel sizes in the range of cortical thickness (e.g. 2.7 ​mm isotropic). While results will not be dramatically changed by the use of multiband, our results suggest that MB will bring a moderate but significant benefit

Quantitative diffusion MRI with application to multiple sclerosis

Diffusion MRI (dMRI) is a uniquely non-invasive probe of biological tissue properties, increasingly able to provide access to ever more intricate structural and microstructural tissue information. Imaging biomarkers that reveal pathological alterations can help advance our knowledge of complex neurological disorders such as multiple sclerosis (MS), but depend on both high quality image data and robust post-processing pipelines. The overarching aim of this thesis was to develop methods to improve the characterisation of brain tissue structure and microstructure using dMRI. Two distinct avenues were explored. In the first approach, network science and graph theory were used to identify core human brain networks with improved sensitivity to subtle pathological damage. A novel consensus subnetwork was derived using graph partitioning techniques to select nodes based on independent measures of centrality, and was better able to explain cognitive impairment in relapsing-remitting MS patients than either full brain or default mode networks. The influence of edge weighting scheme on graph characteristics was explored in a separate study, which contributes to the connectomics field by demonstrating how study outcomes can be affected by an aspect of network design often overlooked. The second avenue investigated the influence of image artefacts and noise on the accuracy and precision of microstructural tissue parameters. Correction methods for the echo planar imaging (EPI) Nyquist ghost artefact were systematically evaluated for the first time in high b-value dMRI, and the outcomes were used to develop a new 2D phase-corrected reconstruction framework with simultaneous channel-wise noise reduction appropriate for dMRI. The technique was demonstrated to alleviate biases associated with Nyquist ghosting and image noise in dMRI biomarkers, but has broader applications in other imaging protocols that utilise the EPI readout. I truly hope the research in this thesis will influence and inspire future work in the wider MR community

Optimization of proprioceptive stimulation for echo-planar and inverse magnetic resonance imaging

In echo-planar imaging (EPI), the optimal passive movement parameters (rate and duration) for studying proprioceptive brain responses are unknown. The aim of this thesis was to test the effect of stimulus rate on brain responses evoked by proprioceptive stimulation in EPI. In addition, we attempted to develop a measurement protocol for experiments focused on proprioception in ultrafast inverse magnetic resonance imaging (InI) and investigate the amplitude of blood oxygen level-dependent (BOLD) signal at varying stimulus duration. This experimental setup was supposed to be applied in future connectivity studies of the proprioceptive brain network. We found that the optimum rate for right index finger proprioceptive stimulation in EPI varies from 3 to 6 Hz. While we managed to sample the BOLD responses every 100 ms (a 20-fold increase in temporal resolution compared to EPI), the experimental design in InI is challenging due to methodological constraints. Thus, the appropriate stimulation parameters for InI still remain a topic for further research