Spatial quantification of the synaptic activity phenotype across large populations of neurons with Markov random fields

The collective and coordinated synaptic activity of large neuronal populations is relevant to neuronal development as well as a range of neurological diseases. Quantification of synaptically-mediated neuronal signalling permits further downstream analysis as well as potential application in target validation and in vitro screening assays. Our aim is to develop a phenotypic quantification for neuronal activity imaging data of large populations of neurons, in particular relating to the spatial component of the activity. We extend the use of Markov random field (MRF) models to achieve this aim. In particular, we consider Bayesian posterior densities of model parameters in Gaussian MRFs to directly model changes in calcium fluorescence intensity rather than using spike trains. The basis of our model is defining neuron 'neighbours' by the relative spatial positions of the neuronal somata as obtained from the image data whereas previously this has been limited to defining an artificial square grid across the field of view and spike binning. We demonstrate that our spatial phenotypic quantification is applicable for both in vitro and in vivo data consisting of thousands of neurons over hundreds of time points. We show how our approach provides insight beyond that attained by conventional spike counting and discuss how it could be used to facilitate screening assays for modifiers of disease-associated defects of communication between cells. We supply the MATLAB code and data to obtain all of the results in the paper. [email protected] and [email protected]. Supplementary data are available at Bioinformatics online. Motivation Results Availability Contact Supplementary Informatio

Computational aspects of parvalbumin-positive interneuron function

The activity of neurons is dependent on the manner in which they process synaptic inputs from other cells. In the event of clustered synaptic input, neurons can respond in a nonlinear manner through synaptic and dendritic mechanisms. Such mechanisms are well established in principal excitatory neurons throughout the brain, where they increase neuronal computational ability and information storage capacity. In contrast for parvalbumin-positive (PV+) interneurons, the most common cortical class of in- hibitory interneuron, synaptic integration is thought to be either linear or sub-linear in nature, facilitating their role as mediators of precise and fast inhibition. This thesis addresses situations in which PV+ interneurons integrate synaptic inputs in a nonlinear manner, and explores the functions of this synaptic processing. First, I describe a form of cooperative supralinear synaptic integration by local excitatory inputs onto PV+ interneurons, and I extend these results to show how this augments the computational capability of PV+ cells within spiking neuron networks. I also explore the importance of polyamine-modulation of synaptic receptors in mediating sublinear synaptic integration, and discuss how this expands the array of mechanisms known to perform similar functions in PV+ cells. Finally, I present work manipulating PV+ cells experimentally during epilepsy. I consider these findings together with recent scientific advances and suggest how they account for a number of open questions and previously contradictory theories of PV+ interneuron function

Micro-, Meso- and Macro-Connectomics of the Brain

Neurosciences, Neurolog

Model and Appearance Based Analysis of Neuronal Morphology from Different Microscopy Imaging Modalities

The neuronal morphology analysis is key for understanding how a brain works. This process requires the neuron imaging system with single-cell resolution; however, there is no feasible system for the human brain. Fortunately, the knowledge can be inferred from the model organism, Drosophila melanogaster, to the human system. This dissertation explores the morphology analysis of Drosophila larvae at single-cell resolution in static images and image sequences, as well as multiple microscopy imaging modalities. Our contributions are on both computational methods for morphology quantification and analysis of the influence of the anatomical aspect. We develop novel model-and-appearance-based methods for morphology quantification and illustrate their significance in three neuroscience studies. Modeling of the structure and dynamics of neuronal circuits creates understanding about how connectivity patterns are formed within a motor circuit and determining whether the connectivity map of neurons can be deduced by estimations of neuronal morphology. To address this problem, we study both boundary-based and centerline-based approaches for neuron reconstruction in static volumes. Neuronal mechanisms are related to the morphology dynamics; so the patterns of neuronal morphology changes are analyzed along with other aspects. In this case, the relationship between neuronal activity and morphology dynamics is explored to analyze locomotion procedures. Our tracking method models the morphology dynamics in the calcium image sequence designed for detecting neuronal activity. It follows the local-to-global design to handle calcium imaging issues and neuronal movement characteristics. Lastly, modeling the link between structural and functional development depicts the correlation between neuron growth and protein interactions. This requires the morphology analysis of different imaging modalities. It can be solved using the part-wise volume segmentation with artificial templates, the standardized representation of neurons. Our method follows the global-to-local approach to solve both part-wise segmentation and registration across modalities. Our methods address common issues in automated morphology analysis from extracting morphological features to tracking neurons, as well as mapping neurons across imaging modalities. The quantitative analysis delivered by our techniques enables a number of new applications and visualizations for advancing the investigation of phenomena in the nervous system

Development of variable and robust brain wiring patterns in the fly visual system

Precise generation of synapse-specific neuronal connections are crucial for establishing a robust and functional brain. Neuronal wiring patterns emerge from proper spatiotemporal regulation of axon branching and synapse formation during development. Several neuropsychiatric and neurodevelopmental disorders exhibit defects in neuronal wiring owing to synapse loss and/or dys-regulated axon branching. Despite decades of research, how the two inter-dependent cellular processes: axon branching and synaptogenesis are coupled locally in the presynaptic arborizations is still unclear. In my doctoral work, I investigated the possible role of EGF receptor (EGFR) activity in coregulating axon branching and synapse formation in a spatiotemporally restricted fashion, locally in the medulla innervating Dorsal Cluster Neuron (M- DCN)/LC14 axon terminals. In this work I have explored how genetically encoded EGFR randomly recycles in the axon branch terminals, thus creating an asymmetric, non-deterministic distribution pattern. Asymmetric EGFR activity in the branches acts as a permissive signal for axon branch pruning. I observed that the M-DCN branches which stochastically becomes EGFR ‘+’ during development are synaptogenic, which means they can recruit synaptic machineries like Syd1 and Bruchpilot (Brp). My work showed that EGFR activity has a dual role in establishing proper M-DCN wiring; first in regulating primary branch consolidation possibly via actin regulation prior to synaptogenesis. Later in maintaining/protecting the levels of late Active Zone (AZ) protein Brp in the presynaptic branches by suppressing basal autophagy level during synaptogenesis. When M-DCNs lack optimal EGFR activity, the basal autophagy level increases resulting in loss of Brp marked synapses which is causal to increased exploratory branches and post-synaptic target loss. Lack of EGFR activity affects the M-DCN wiring pattern that makes adult flies more active and behave like obsessive compulsive in object fixation assay. In the second part of my doctoral work, I have asked how non-genetic factors like developmental temperature affects adult brain wiring. To test that, I increased or decreased rearing temperature which is known to inversely affect pupal developmental rate. We asked if all the noisy cellular processes of neuronal assembly: filopodial dynamics, axon branching, synapse formation and postsynaptic connections scale up or down accordingly. I observed that indeed all the cellular processes slow down at lower developmental temperature and vice versa, which changes the DCN wiring pattern accordingly. Interestingly, behavior of flies adapts to their developmental temperature, performing best at the temperature they have been raised at. This shows that optimal brain function is an adaptation of robust brain wiring patterns which are specified by noisy developmental processes. In conclusion, my doctoral work helps us better understand the developmental regulation of axon branching and synapse formation for establishing precise brain wiring pattern. We need all the cell intrinsic developmental processes to be highly regulated in space and time. It is infact a combinatorial effect of such stochastic processes and external factors that contribute to the final outcome, a functional and robust adult brain

Efferent Modulation of Spontaneous Activity in Developing Sensory Systems

Patterned spontaneous activity plays an instructive role in developing sensory systems. Before hearing onset, inner support cells release ATP and induce spontaneous firing of neighboring inner hair cells. This periphery-initiated spontaneous activity propagates throughout the auditory hierarchy via the afferent pathway, coordinating neural activity in distinct tonotopic zones in the central auditory system. Similarly, spontaneous retinal waves initiated in the retina by starburst amacrine cells (stage II) or bipolar cells (stage III) were observed throughout the visual system via the retinotopic visual afferent circuits. Deciphering the underlying mechanisms of patterned spontaneous activity is critical to elucidate its instructive role in priming the developing nervous system prior to sensory experience. On the other hand, anatomical and functional evidence suggests that centrifugal efferent systems may contribute to neural dynamics before sensory inputs. In the first half of this study, we profiled spatiotemporal and correlational features of auditory spontaneous activity over the entire pre-hearing period. We discovered that the olivocochlear efferent system controlled the coupling strength of bilateral auditory spontaneous activity and demonstrated the profound impact of such modulation on the development of auditory functions. In the second half of this work, we introduced a novel experimental technique that enabled access to in situ retinal calcium dynamics in awake animals. We demonstrated in situ recordings of spontaneous retinal waves from distinct neuronal populations in the retina. Moreover, our result indicated that retinal activity was directly modulated by locomotion. Our approach is well suited to study retinopetal projections in vivo and whether they contributed to locomotion-related modulation on retinal dynamics. Together, these findings provide new perspectives on the functional roles of efferent modulations in shaping spontaneous activity and promoting the development of auditory and visual systems

Identifying regulators of synaptic stability during normal healthy ageing

The loss and dysfunction of selected populations of synapses is characteristic of mammalian brain ageing and alterations in these receptive compartments are considered to underpin age-related cognitive decline. Discrete neuro-anatomical regions of the cortical architecture harbour disparate populations of synapses that demonstrate significant heterogeneity with regards to advancing age. Of particular interest is the hippocampus, which is selectively vulnerable during ageing. The hippocampal synaptic architecture exhibits subtle structural and biophysical alterations, which are considered to promote the manifestation of cognitive symptoms in aged patients. This notion of “selective synaptic vulnerability” has been the focal point of a multitude of morphological studies investigating age-related cognitive decline, which have often provided tentative conclusions as to how this phenomenon may be regulated. The molecular correlates bolstering the reported age-dependent morphological and functional shift remain elusive and studies are only now beginning to unravel how discrete organelles, proteins and signalling cascades may hierarchically or synergistically attenuate synaptic function. Until there is considerable comprehension of how functional mediators drive the biochemical substrates regulating age-related cognitive decline, there are limited strategic avenues for the development of efficacious therapeutic interventions that promote successful ageing. To address the phenomenon of selective synaptic vulnerability, we have utilised an unbiased combinatorial approach, including quantitative proteomic analyses coupled with in vivo candidate assessments in lower order animals (Drosophila), to temporally profile regional synapse and synaptic mitochondrial biochemistry during normal healthy ageing. We begin by demonstrating that cortical mitochondria located at the synaptic terminal are morphologically distinct from non-synaptic mitochondria in adult rodents and human patients. Biochemical isolation and purification of discrete mitochondrial subpopulations from control adult rat fore-brain enabled generation of synaptic and non-synaptic mitochondrial molecular fingerprints using quantitative proteomics, which revealed that expression of the mitochondrial proteome is highly dependent on subcellular localisation. We subsequently demonstrate that the molecular differences observed between mitochondrial sub-populations are capable of selectively influencing synaptic morphology in-vivo. Next, we sought to examine how the synaptic mitochondrial proteome was dynamically and temporally regulated throughout ageing to determine whether protein expression changes within the mitochondrial milieu are actively regulating the age-dependent vulnerability of the synaptic compartment. Proteomic profiling of wild-type mouse cortical synaptic and non-synaptic mitochondria across the lifespan revealed significant age-dependent heterogeneity between mitochondrial subpopulations, with aged organelles exhibiting unique protein expression profiles. Recapitulation of aged synaptic mitochondrial protein expression at the Drosophila neuromuscular junction has the propensity to perturb the synaptic architecture, demonstrating that temporal regulation of the mitochondrial proteome may directly modulate the stability of the synapse in vivo. Although we had comprehensively characterised the temporal regulation of rodent cortical mitochondrial subpopulations, providing a number of novel candidates that may be mediating synaptic vulnerability during ageing, we sought to establish whether similar alterations were occurring in the primate brain. Using synaptic isolates from neuroanatomically distinct age-resistant (occipital cortex) and age-vulnerable (hippocampus) regions, we demonstrate that synaptic ageing is brainregion dependent and that discrete populations of synapses significantly differ at a biochemical level in the healthy human and non-human primate brain. Recapitulation of aged hippocampal protein expression with genetic manipulation in vivo revealed numerous novel candidates that have the propensity to significantly modulate multiple morphological parameters at the synapse. Furthermore, we demonstrate that several of these candidates sit downstream of TGFβ1 and activation of the TGFβ1 signalling cascade in hippocampal synaptic populations drives the aberrant expression of selected candidates during ageing. Finally, we show that selective pharmacological inhibition of this pathway rescues synaptic phenotypes in multiple candidate lines. The data affirmed that activation of the TGFβ1 transduction pathway modulates synaptic stability and thus may contribute to the selective vulnerability of hippocampal synapses during ageing