BACKGROUND:

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Neurons in the brain have extensive dendritic arbours that receive synaptic inputs throughout. The transformation of all these inputs to an output occurs through the integration of synaptic events and the generation and transmission of an action potential. Therefore, a neuron's input integration is crucial for determining how information is processed in the brain. Neurons receive two types of input: excitatory signals that promote output, and inhibitory signals that prevent it. The behaviour of a neuron is therefore precisely regulated by how these two opposing forces interact with one another in space and time. Developing and maintaining a correct balance between excitation (E) and inhibition (I) is thought to be fundamental for appropriate circuit wiring and healthy function. Correspondingly, disruption of this E/I balance is hypothesised to underly many neurological conditions, including epilepsy, schizophrenia and autism spectrum disorder (ASD).

 

ASD describes a family of neurodevelopmental conditions with heterogenous etiologies converging into common symptoms. Clinical diagnosis is based on two core criteria present early in development: (1) deficits in social communication and interaction, and (2) stereotyped, repetitive behaviour and restricted interest. Despite considerable research into E/I balance and its disturbance, we know little about how excitatory and inhibitory synapses are distributed across neurons at the subcellular level, or how the interaction between them influences neuronal output. My project builds on recent work carried out in the Burrone lab where the dendritic distribution of excitatory and inhibitory synapses was mapped during early neurodevelopment in healthy mice. Working at the intersection of mouse and human models, I am aiming to characterize subcellular E/I balance in murine ASD models and eventually extend this approach to patient iPSC derived cortical neuron cultures.

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METHODOLOGY:

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My project follows two main avenues – animal in vivo work and human in vitro work. To label excitatory and inhibitory synapses in the mouse hippocampus, I will introduce so called intrabodies to the embryonic brain via in utero electroporation. These fluorescent markers bind to the excitatory postsynaptic protein PSD95 and the inhibitory postsynaptic protein Gephyrin and thereby allow us to trace and quantify both synapse types.

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Currently, I am trying to establish a way to perform the same synapse labelling in human iPSC derived neuronal cultures. I am testing the same intrabodies while simultaneously considering alternative labelling methods such as endogenous tagging of PSD95 and Gephyrin. Since we are interested in both excitatory and inhibitory connections, I am using a co-culture of induced glutamatergic and GABAergic neurons.

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Once the synapses are labelled, they can be visualized by confocal microscopy. For image analysis, the java based program ImageJ/FIJI is commonly used. Here, I can set parameters and optimise my synapse counting on a few images to then write a script that can be applied to all other images, automating the analysis.

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RESULTS:

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As I have just started my project, I am still optimizing my methods and haven’t acquired many results yet, but here is the experimental setup (top) and a confocal image (bottom) of forward programmed human cortical neuron co-culture with induced glutamatergic neurons in green (iGlut) and GABAergic neurons in red (iGABA). 

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FUTURE WORK:

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Eventually, we want to test if whatever E/I correlation we find on a structural level is reflected functionally as well. For this, I will perform electrophysiology where the amplitude of excitatory and inhibitory postsynaptic currents can be measured by whole-cell patch clamping.

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To analyse electrophysiological data, ready-made software such as pClamp or PatchMaster are commonly used. Alternatively, custom made analysis scripts can be written in Matlab or Python. Analysis includes extracting traces and processing signals – such as removing noise – as well as statistics.

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FUNDED BY:

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CONTACT: