In Vivo Spike-Timing-Dependent Plasticity in the Optic Tectum of Xenopus Laevis

Spike-timing-dependent plasticity (STDP) is found in vivo in a variety of systems and species, but the first demonstrations of in vivo STDP were carried out in the optic tectum of Xenopus laevis embryos. Since then, the optic tectum has served as an excellent experimental model for studying STDP in sensory systems, allowing researchers to probe the developmental consequences of this form of synaptic plasticity during early development. In this review, we will describe what is known about the role of STDP in shaping feed-forward and recurrent circuits in the optic tectum with a focus on the functional implications for vision. We will discuss both the similarities and differences between the optic tectum and mammalian sensory systems that are relevant to STDP. Finally, we will highlight the unique properties of the embryonic tectum that make it an important system for researchers who are interested in how STDP contributes to activity-dependent development of sensory computations

Genetically encoded proton sensors reveal activity-dependent pH changes in neurons

The regulation of hydrogen ion concentration (pH) is fundamental to cell viability, metabolism, and enzymatic function. Within the nervous system, the control of pH is also involved in diverse and dynamic processes including development, synaptic transmission, and the control of network excitability. As pH affects neuronal activity, and can also itself be altered by neuronal activity, the existence of tools to accurately measure hydrogen ion fluctuations is important for understanding the role pH plays under physiological and pathological conditions. Outside of their use as a marker of synaptic release, genetically encoded pH sensors have not been utilized to study hydrogen ion fluxes associated with network activity. By combining whole-cell patch clamp with simultaneous two-photon or confocal imaging, we quantified the amplitude and time course of neuronal, intracellular, acidic transients evoked by epileptiform activity in two separate in vitro models of temporal lobe epilepsy. In doing so, we demonstrate the suitability of three genetically encoded pH sensors: deGFP4, E2GFP, and Cl-sensor for investigating activity-dependent pH changes at the level of single neurons

Pro-maturational effects of human iPSC-derived cortical astrocytes upon iPSC-derived cortical neurons

Astrocytes influence neuronal maturation and function by providing trophic support, regulating the extracellular environment, andmodulating signaling at synapses. The emergence of induced pluripotent stem cell (iPSC) technology offers a human system with whichto validate and re-evaluate insights from animal studies. Here, we set out to examine interactions between human astrocytes and neuronsderived from a common cortical progenitor pool, thereby recapitulating aspects ofin vivocortical development. We show that the corticaliPSC-derived astrocytesexhibit many of the molecular and functional hallmarks of astrocytes. Furthermore, optogenetic and electrophys-iological co-culture experiments reveal that the iPSC-astrocytes can actively modulate ongoing synaptic transmission and exertpro-maturational effects upon developing networks of iPSC-derived cortical neurons. Finally, transcriptomic analyses implicate syn-apse-associated extracellular signaling in the astrocytes’ pro-maturational effects upon the iPSC-derived neurons. This work helps laythe foundation for future investigations into astrocyte-to-neuron interactions in human health and disease

Calcium-dependent neuroepithelial contractions expel damaged cells from the developing brain

Both developing and adult organisms need efficient strategies for wound repair. In adult mammals, wounding triggers an inflammatory response that can exacerbate tissue injury and lead to scarring. In contrast, embryonic wounds heal quickly and with minimal inflammation, but how this is achieved remains incompletely understood. Using in vivo imaging in the developing brain of Xenopus laevis, we show that ATP release from damaged cells and subsequent activation of purinergic receptors induce long-range calcium waves in neural progenitor cells. Cytoskeletal reorganization and activation of the actomyosin contractile machinery in a Rho kinase-dependent manner then lead to rapid and pronounced apical-basal contractions of the neuroepithelium. These contractions drive the expulsion of damaged cells into the brain ventricle within seconds. Successful cell expulsion prevents the death of nearby cells and an exacerbation of the injury. Cell expulsion through neuroepithelial contraction represents a mechanism for rapid wound healing in the developing brain

Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.Initiative Joint Undertaking under grant agreement no. 115439, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and EFPIA companies' in kind contribution. A.H., S.C., and M.Z.C. were also funded by the NIHR (Oxford BRC). K.M. and A.B. were also supported by the NIHR GOSH BRC

Calcium and chloride dynamics in immature neurons and their role in dendritic growth

Activity-dependent dendritic development is an important process in the maturation of neuronal circuits. The precise morphology of a neuron’s dendritic tree dictates which other cells it is able to interact with and how it will receive and process synaptic information. The aim of this Thesis was to investigate the mechanisms by which dendrites grow and, in particular, how changes in intracellular ion concentrations contribute to these mechanisms. One important activity-dependent signal is calcium as it can translate neuronal activity into morphological changes. Despite this, very little is known about calcium signalling during the period of dendritic development. Using single-cell electroporation of immature CA1 hippocampal pyramidal neurons, I characterised the spatial and temporal properties of local calcium transients in growing dendrites. This revealed a high frequency of transients at shaft filopodia and stable branchpoints, but an almost complete absence from the tips of dendritic branches. Another important factor during development is the intracellular chloride concentration because this regulates neuronal excitability. Prematurely lowering intracellular chloride by expressing the chloride co-transporter KCC2 led to less stable dendritic filopodia and stunted dendritic growth. These effects were independent of local calcium signalling and suggested that chloride regulation itself may be fundamental to normal dendritic growth. To examine this further I developed imaging techniques to measure the spatial and temporal dynamics of chloride in growing dendrites. This work revealed a somatodendritic gradient of increasing intracellular chloride, whereby the highest concentrations were found at sites of growth. Further analysis suggested a close link between local chloride regulation and morphological changes. The dendritic tips that exhibited high intracellular chloride levels and the potential to rapidly modulate these levels, also exhibited the greatest morphological dynamics. These findings have important implications for understanding the mechanisms of dendritic growth and establish the spatiotemporal regulation of chloride as a key parameter.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Calcium and chloride dynamics in immature neurons and their role in dendritic growth

Activity-dependent dendritic development is an important process in the maturation of neuronal circuits. The precise morphology of a neuron’s dendritic tree dictates which other cells it is able to interact with and how it will receive and process synaptic information. The aim of this Thesis was to investigate the mechanisms by which dendrites grow and, in particular, how changes in intracellular ion concentrations contribute to these mechanisms. One important activity-dependent signal is calcium as it can translate neuronal activity into morphological changes. Despite this, very little is known about calcium signalling during the period of dendritic development. Using single-cell electroporation of immature CA1 hippocampal pyramidal neurons, I characterised the spatial and temporal properties of local calcium transients in growing dendrites. This revealed a high frequency of transients at shaft filopodia and stable branchpoints, but an almost complete absence from the tips of dendritic branches. Another important factor during development is the intracellular chloride concentration because this regulates neuronal excitability. Prematurely lowering intracellular chloride by expressing the chloride co-transporter KCC2 led to less stable dendritic filopodia and stunted dendritic growth. These effects were independent of local calcium signalling and suggested that chloride regulation itself may be fundamental to normal dendritic growth. To examine this further I developed imaging techniques to measure the spatial and temporal dynamics of chloride in growing dendrites. This work revealed a somatodendritic gradient of increasing intracellular chloride, whereby the highest concentrations were found at sites of growth. Further analysis suggested a close link between local chloride regulation and morphological changes. The dendritic tips that exhibited high intracellular chloride levels and the potential to rapidly modulate these levels, also exhibited the greatest morphological dynamics. These findings have important implications for understanding the mechanisms of dendritic growth and establish the spatiotemporal regulation of chloride as a key parameter.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Short-term ionic plasticity at GABAergic synapses

Fast synaptic inhibition in the brain is mediated by the pre-synaptic release of the neurotransmitter γ-Aminobutyric acid (GABA) and the post-synaptic activation of GABA-sensitive ionotropic receptors. As with excitatory synapses, it is being increasinly appreciated that a variety of plastic processes occur at inhibitory synapses, which operate over a range of timescales. Here we examine a form of activity-dependent plasticity that is somewhat unique to GABAergic transmission. This involves short-lasting changes to the ionic driving force for the postsynaptic receptors, a process referred to as short-term ionic plasticity. These changes are directly related to the history of activity at inhibitory synapses and are influenced by a variety of factors including the location of the synapse and the post-synaptic cell’s ion regulation mechanisms. We explore the processes underlying this form of plasticity, when and where it can occur, and how it is likely to impact network activity