The spatiotemporal organization of cerebellar network activity resolved by two-photon imaging of multiple single neurons

In order to investigate the spatiotemporal organization of neuronal activity in local microcircuits, techniques allowing the simultaneous recording from multiple single neurons are required. To this end, we implemented an advanced spatial-light modulator two-photon microscope (SLM-2PM). A critical issue for cerebellar theory is the organization of granular layer activity in the cerebellum, which has been predicted by single-cell recordings and computational models. With SLM-2PM, calcium signals could be recorded from different network elements in acute cerebellar slices including granule cells (GrCs), Purkinje cells (PCs) and molecular layer interneurons. By combining WCRs with SLM-2PM, the spike/calcium relationship in GrCs and PCs could be extrapolated toward the detection of single spikes. The SLM-2PM technique made it possible to monitor activity of over tens to hundreds neurons simultaneously. GrC activity depended on the number of spikes in the input mossy fiber bursts. PC and molecular layer interneuron activity paralleled that in the underlying GrC population revealing the spread of activity through the cerebellar cortical network. Moreover, circuit activity was increased by the GABA-A receptor blocker, gabazine, and reduced by the AMPA and NMDA receptor blockers, NBQX and APV. The SLM-2PM analysis of spatiotemporal patterns lent experimental support to the time-window and center-surround organizing principles of the granular layer

High-Pass Filtering and Dynamic Gain Regulation Enhance Vertical Bursts Transmission along the Mossy Fiber Pathway of Cerebellum

Signal elaboration in the cerebellum mossy fiber input pathway presents controversial aspects, especially concerning gain regulation and the spot-like (rather than beam-like) appearance of granular to molecular layer transmission. By using voltage-sensitive dye imaging in rat cerebellar slices (Mapelli et al., 2010), we found that mossy fiber bursts optimally excited the granular layer above ∼50 Hz and the overlaying molecular layer above ∼100 Hz, thus generating a cascade of high-pass filters. NMDA receptors enhanced transmission in the granular, while GABA-A receptors depressed transmission in both the granular and molecular layer. Burst transmission gain was controlled through a dynamic frequency-dependent involvement of these receptors. Moreover, while high-frequency transmission was enhanced along vertical lines connecting the granular to molecular layer, no high-frequency enhancement was observed along the parallel fiber axis in the molecular layer. This was probably due to the stronger effect of Purkinje cell GABA-A receptor-mediated inhibition occurring along the parallel fibers than along the granule cell axon ascending branch. The consequent amplification of burst responses along vertical transmission lines could explain the spot-like activation of Purkinje cells observed following punctuate stimulation in vivo

Fluorescence Imaging of Cortical Calcium Dynamics: A Tool for Visualizing Mouse Brain Functions, Connections, and Networks

Hemodynamic-based markers of cortical activity (e.g. functional magnetic resonance imaging (fMRI) and optical intrinsic signal imaging) are an indirect and relatively slow report of neural activity driven by electrical and metabolic activity through neurovascular coupling, which presents significant limiting factors in deducing underlying brain network dynamics. As application of resting state functional connectivity (FC) measures is extended further into topics such as brain development, aging, and disease, the importance of understanding the fundamental basis for FC will grow. In this dissertation, we extend functional analysis from hemodynamic- to calcium-based imaging. Transgenic mice expressing a fluorescent calcium indicator (GCaMP6) driven by the Thy1 promoter in glutamatergic neurons were imaged transcranially in both anesthetized (using ketaminze/xylazine) and awake states. Sequential LED illumination (λ=470, 530, 590, 625nm) enabled concurrent imaging of both GCaMP6 fluorescence emission (corrected for hemoglobin absorption) and hemodynamics. EEG measurements of the global cortical field potential were also simultaneously acquired. First, we validated the ability of our system to capture GCaMP6 fluorescence emission and hemodynamics by implementing an electrical somatosensory stimulation paradigm. The neural origins of the GCaMP6 fluorescent signal were further confirmed by histology and by comparing the spectral content of imaged GCaMP6 activity to concurrently-acquired EEG. We then constructed seed-based FC and coherence network maps for low (0.009-0.08Hz) and high, delta-band (0.4-4.0Hz) frequency bands using GCaMP6 and hemodynamic contrasts. Homotopic GCaMP6 FC maps using delta-band data in the anesthetized states show a striking correlated and anti-correlated structure along the anterior to posterior axis. We next used whole-brain delay analysis to characterize this correlative feature. This structure is potentially explained by the observed propagation of delta-band activity from frontal somatomotor regions to visuoparietal areas, likely corresponding to propagating delta waves associated with slow wave sleep. During wakefulness, this spatio-temporal structure is largely absent, and a more complex and detailed FC structure is observed. Collectively, functional neuroimaging of calcium dynamics in mice provides evidence that spatiotemporal coherence in cortical activity is not exclusive to hemodynamics and exists over a larger range of frequencies than hemoglobin-based contrasts. Concurrent calcium and hemodynamic imaging enables direct temporal and functional comparison of spontaneous calcium and hemoglobin activity, effectively spanning neurovascular coupling and functional hyperemia. The combined calcium/hemoglobin imaging technique described here will enable the dissociation of changes in ionic and hemodynamic functional structure and provide a framework for subsequent studies of sleep disorders and neurological disease

Human brain slices for epilepsy research:pitfalls, solutions and future challenges

Increasingly, neuroscientists are taking the opportunity to use live human tissue obtained from elective neurosurgical procedures for electrophysiological studies in vitro. Access to this valuable resource permits unique studies into the network dynamics that contribute to the generation of pathological electrical activity in the human epileptic brain. Whilst this approach has provided insights into the mechanistic features of electrophysiological patterns associated with human epilepsy, it is not without technical and methodological challenges. This review outlines the main difficulties associated with working with epileptic human brain slices from the point of collection, through the stages of preparation, storage and recording. Moreover, it outlines the limitations, in terms of the nature of epileptic activity that can be observed in such tissue, in particular, the rarity of spontaneous ictal discharges, we discuss manipulations that can be utilised to induce such activity. In addition to discussing conventional electrophysiological techniques that are routinely employed in epileptic human brain slices, we review how imaging and multielectrode array recordings could provide novel insights into the network dynamics of human epileptogenesis. Acute studies in human brain slices are ultimately limited by the lifetime of the tissue so overcoming this issue provides increased opportunity for information gain. We review the literature with respect to organotypic culture techniques that may hold the key to prolonging the viability of this material. A combination of long-term culture techniques, viral transduction approaches and electrophysiology in human brain slices promotes the possibility of large scale monitoring and manipulation of neuronal activity in epileptic microcircuits

Quantifying activity in nascent neuronal networks derived from embryonic stem cells

PhD ThesisThe relationship between spatiotemporal patterns of spontaneous activity and functional specialisation in developing neuronal networks is complex and its study is crucial to our understanding of how network communication is initiated. This project quantifies transitions between structural and functional states in embryonic stem cell cultures during differentiation. The work also focussed on the role of γ-aminobutyric acid (GABA), known to be vital for neuronal network development. The work used many techniques, including carbon nanotube (CNT) -patterned substrates to manipulate network architecture, multi-electrode arrays (MEAs) and calcium imaging to quantify function. An embryonic stem cell line (CC9) was used to generate ‘de novo’ neuronal networks and these were monitored over 13 – 22 days in vitro (DIV), while network structure forms and stabilizes. On CNT-patterned arrays, differentiating CC9s migrated and sub-clustered on CNT islands showing that network structure could be manipulated. No spontaneous electrophysiological (unit) activity was found in these cultures. However, intracellular calcium responses were readily induced and seen spontaneously at 13-20 DIV. Activity rate, kinetics and number of active cells increased between 16-18 DIV, correlating with changes in network clustering. Post 17 DIV, activity transformed from near-random to periodic and synchronous. Many events were initiated by ‘hubs’ and degrees of critical behaviour were observed, moving towards more efficient information processing states with development. Blockade of GABAA receptors lead to elevated spontaneous activity and supercritical behaviour, depending on developmental stage. Application of exogenous GABA induced large, slow calcium transients in a developmental stage-dependent manner, suggestive of a mixed excitatory/inhibitory role. These findings begin to show how activity develops as stem cells differentiate to form neuronal networks. GABA’s role in controlling patterns of activity was more complex that previously reported for neuronal networks in situ, but GABA clearly played a vital role in shaping population behaviour to optimise information processing properties in early, developing networks

Sensory coding in supragranular cells of the vibrissal cortex in anesthetized and awake mice

Sensory perception entails reliable representation of the external stimuli as impulse activity of individual neurons (i.e. spikes) and neuronal populations in the sensory area. An ongoing challenge in neuroscience is to identify and characterize the features of the stimuli which are relevant to a specific sensory modality and neuronal strategies to effectively and efficiently encode those features. It is widely hypothesized that the neuronal populations employ “sparse coding” strategies to optimize the stimulus representations with a low energetic cost (i.e. low impulse activity). In the past two decades, a wealth of experimental evidence has supported this hypothesis by showing spatiotemporally sparse activity in sensory area. Despite numerous studies, the extent of sparse coding and its underlying mechanisms are not fully understood, especially in primary vibrissal somatosensory cortex (vS1), which is a key model system in sensory neuroscience. Importantly, it is not clear yet whether sparse activation of supragranular vS1 is due to insufficient synaptic input to the majority of the cells or the absence of effective stimulus features. In this thesis, first we asked how the choice of stimulus could affect the degree of sparseness and/or the overall fraction of the responsive vS1 neurons. We presented whisker deflections spanning a broad range of intensities, including “standard stimuli” and a high-velocity, “sharp” stimulus, which simulated the fast slip events that occur during whisker mediated object palpation. We used whole-cell and cell-attached recording and calcium imaging to characterize the neuronal responses to these stimuli. Consistent with previous literature, whole-cell recording revealed a sparse response to the standard range of velocities: although all recorded cells showed tuning to velocity in their postsynaptic potentials, only a small fraction produced stimulus-evoked spikes. In contrast, the sharp stimulus evoked reliable spiking in a large fraction of regular spiking neurons in the supragranular vS1. Spiking responses to the sharp stimulus were binary and precisely timed, with minimum trial-to-trial variability. Interestingly, we also observed that the sharp stimulus produced a consistent and significant reduction in action potential threshold. In the second step we asked whether the stimulus dependent sparse and dense activations we found in anesthetized condition would generalize to the awake condition. We employed cell-attached recordings in head-fixed awake mice to explore the degree of sparseness in awake cortex. Although, stimuli delivered by a piezo-electric actuator evoked significant response in a small fraction of regular spiking supragranular neurons (16%-29%), we observed that a majority of neurons (84%) were driven by manual probing of whiskers. Our results demonstrate that despite sparse activity, the majority of neurons in the superficial layers of vS1 contribute to coding by representing a specific feature of the tactile stimulus. Thesis outline: Chapter 1 provides a review of the current knowledge on sparse coding and an overview of the whisker-sensory pathway. Chapter 2 represents our published results regarding sparse and dense coding in vS1 of anesthetized mice (Ranjbar-Slamloo and Arabzadeh 2017). Chapter 3 represents our pending manuscript with results obtained with piezo and manual stimulation in awake mice. Finally, in Chapter 4 we discuss and conclude our findings in the context of the literature. The appendix provides unpublished results related to Chapter 2. This section is referenced in the final chapter for further discussion

Early brain activity : Translations between bedside and laboratory

Neural activity is both a driver of brain development and a readout of developmental processes. Changes in neuronal activity are therefore both the cause and consequence of neurodevelopmental compromises. Here, we review the assessment of neuronal activities in both preclinical models and clinical situations. We focus on issues that require urgent translational research, the challenges and bottlenecks preventing translation of biomedical research into new clinical diagnostics or treatments, and possibilities to overcome these barriers. The key questions are (i) what can be measured in clinical settings versus animal experiments, (ii) how do measurements relate to particular stages of development, and (iii) how can we balance practical and ethical realities with methodological compromises in measurements and treatments.Peer reviewe

Investigating hippocampal-neocortical interactions around sharp-wave ripples

Coordinated activity in the hippocampal-neocortical network around hippocampal sharp-wave ripples (SWRs) plays an instrumental role in memory processing in the brain. SWRs occur in both sleep and awake states, though under two significantly different behavioural and chemical circumstances. Previous studies have reported different patterns of peri-SWR neocortical modulations between these states; however, their findings have been limited to one or a few discrete regions of the neocortex. To extend previous findings, we conducted wide-field optical imaging of the mouse neocortical voltage and glutamate activity combined with hippocampal electrophysiological recording. We found topographically- and temporally-organized patterns of neocortical glutamate and voltage activity around sleep and awake SWRs, though with pronounced differences. These findings highlight the state-dependency of the hippocampal-neocortical network’s computations and possibly functions. Moreover, they provide a spatiotemporal map of the neocortex around SWRs that could guide future studies on the role of hippocampal-neocortical interactions in memory consolidation

Analysis and modelling of the PY complex in the pyloric circuit of the crab stomatogastric ganglion

PhD ThesisCentral pattern generators (CPGs) are neural circuits that control rhythmic motor patterns such as walking running and swallowing. Injuries can sever the spinal cord or conditions such as Huntington's disease and Parkinson's disease can damage nerves from the brain that control CPGs. Understanding the connectivity of neural circuits has proved insu cient to understand the dynamics of such circuits. Neuromodulators and neurohormones can di erentially a ect every connection in neural circuits and di erent circuits are a ected in very di erent ways. The resulting complexity of such systems make them very di cult to study but research is greatly facilitated by the use of model organisms and computational models. The crustacean stomatogastric ganglion (STG) has been used as a model system for many years. Its relative simplicity and accessibility to neurons makes it an ideal system for the study of neural interaction, CPGs and the e ect of neuromodulators on neural systems. The e ect of dopamine on the pyloric CPG of the crab STG was recorded using voltage sensitive dye imaging and electrophysiological techniques. To analyse voltage sensitive dye (VSD) imaging data a heuristic method was devised that uses the timing of the activity plateaus of neurons for the estimation of the dynamics of the temporal relationship of the neurons' activities. MATLABR was used to create a Hodgkin-Huxley based model of the pyloric constrictor pyloric dilator neurons (PDs) with parameters that could capture the dynamics of neuromodulation. The MATLABR model includes two compartments, the soma and the axon, for the anterior burster neuron, the lateral pyloric neurons (LPs), two PDs and ve individual pyloric constrictor neurons (PYs). By di erentially changing the values of the model synapses, the model is able to reproduce the de-synchronisation of the pyloric constrictor neurons as was observed experimentally i on the dea erented stomatogastric nervous system. Existing models model PYs and PDs as single neurons. These models are unable to show the desynchronising e ect of dopamine on multiple neurons of the same type. The model created for this research is able to re ect the e ect of neuromodulation on the complete circuit by allowing parameters of synapses between neurons of the same type to be adjusted di erentially, re ecting the biological system more accurately