To investigate how the coordinated activity of groups of neurons (‘neuronal ensembles’) drives behavior, two-photon all-optical interrogation is increasingly recognized as a potent approach which provides simultaneous imaging and manipulation of brain circuits with high spatial resolution. Most two-photon all-optical methods couple two-photon microscopy and digital holography to stimulate opsin-expressing neurons with limited photodamage and heating, while recording the activity of the same cells using a fluorescent functional indicator. However, the spectral properties of the most popular opsins and indicators used in two-photon all-optical methods are prone to crosstalk between imaging and stimulation. In this thesis work, we aim to develop a method for high-efficiency stimulation of neurons which decreases crosstalk between imaging and stimulation. To this aim, we generated stCoChR, a soma-tagged variant of the blue light-sensitive opsin CoChR. In anesthetized mice, we show efficient stimulation of cortical neurons expressing stCoChR. For these experiments, we performed holographic two-photon stimulation with both a high repetition rate laser and with a low repetition rate amplified laser tuned at the excitation peak of the opsin (920 nm). We found that stCoChR allowed neuronal stimulation with a 10-fold reduction in average power per cell compared to previously characterized blue light sensitive opsins. Moreover, we combined holographic stimulation of stCoChR with two-photon imaging of the red-shifted indicator jRCaMP1a at 1100 nm. Importantly, we found that this choice of opsins, indicator, and excitation wavelengths significantly limited crosstalk between imaging and photostimulation. Altogether the results presented in this thesis demonstrate that co-expression of stCoChR with red-shifted functional indicators and combined to tuned amplified lasers is a promising tool to perform efficient interrogation of large-scale neuronal networks in the intact brain with limited crosstalk between imaging and photostimulation.
|Titolo della tesi:||Optogenetics strategies for high-efficiency all-optical interrogation using blue-light sensitive opsins|
|Data di discussione:||28-apr-2022|
|Appare nelle tipologie:||Tesi di dottorato|