Wong, Evelyn; (2023) Characterizing single-cell computation using all-optical interrogation of cortical dendrites in vivo. Masters thesis (M.Phil), UCL (University College London).

Preview

Text MPhil Thesis - FINAL VERSION-compressed.pdf - Other Download (5MB) | Preview

Abstract

Pyramidal neurons in the mouse neocortex develop elaborate dendritic compartments that integrate signals to generate stimulus-specific responses. Parameters such as the origin, strength, and location of inputs on the dendritic arbor define computations performed by dendrites to modulate neuronal activity. Although the role of dendrites in synaptic integration has been studied in brain slices in vitro, little is known about how their integrative properties functionally relate to dendritic computations in vivo. The small size of dendrites and mechanical instability of the brain have precluded the use of direct methods such as in vivo patch clamp recording for making functional measurements of dendritic activity during behavior. In this thesis, I have optimized existing optical methods to selectively target and monitor responses in single dendrites in the mouse neocortex, providing a proof-of-principle for all-optical interrogation of dendritic computation. I used an ultra-sparse expression strategy to express the powerful channelrhodopsin ChRmine and the highly sensitive calcium indicator GCaMP8s in Layer 2/3 pyramidal neurons in primary visual cortex (V1) for simultaneous optical stimulation and recording of basal dendrites. Previously, our lab developed a protocol for all-optical interrogation experiments (Russell et al. 2022), using a spatial light modulator (SLM) to activate cellular targets in an awake, head-fixed mouse. We re-configured this approach to optically stimulate single dendritic segments, or combinations of dendritic segments from different dendrites of the same neuron. Challenges included optimizing the relative expression of ChRmine and GCaMP8s in the soma and dendrites to both allow efficient activation and avoid overexpression, as well as calibration of the SLM to reliably target single dendrites (~1-2 µm diameter) while accounting for movement caused by respiration or running. Two-photon imaging of GCaMP8s responses revealed intensity-dependent calcium signals in dendrites with increasing laser power and number of targets. Analysis of somatic activation driven by dendritic optogenetic stimulation revealed supra- or sub-linear summation of multiple dendritic targets depending on the spatial pattern of stimulation. Applying this technique to probe dendritic integration during sensory stimulus processing or animal behavior could provide us with one of the tools needed to understand the role of single-neuron processing in neural computations in the brain.

Download activity - last month

Export as XMLJSONCSV

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as XMLJSONCSV

Archive Staff Only

View Item