Developing tools to optically map the functional connectivity of neuronal microcircuits in the prefrontal cortex

Abstract

This overarching goal of my PhD was to develop optical microscopy tools to study the functional connectivity of neuronal microcircuits. I first conduct dynamic clamp recordings of putative pyramidal neurons in the Prefrontal Cortex to study the effects of intrinsic conductance and synaptic drive of Action Potential (AP) waveform properties. In the set of measured neurons, I find that an increasing, non-linear Goldman-Hodgkin-Katz Cl- leak conductance or an increasing input AMPA conductance have no effect on AP Full Width, Half Maximum (FWHM). However, I observe that increasing AMPA conductance decreases AP latency and peak voltage. Crucially, the latter has implications on optical functional connectivity assays.

I then develop, characterise, and validate a light patterning microscope for optical functional connectivity assays with single neuron precision. I show reliable generation of photostimulation spots with biologically relevant diameters of 5 ± 0.2 µm and 10 ± 0.2 µm, pulse durations of 2 – 10 ms, errors of 20 ± 10 µs, and latencies of 180 ± 10 µs. I demonstrate lateral spatial confinement of photostimulation spots by photostimulating laterally displaced locations relative to a ChR2 expressing neuron and observing an elimination of photocurrents as the spots move away from the cell soma. I attempt single neuron precision functional mapping between Inter-Telencephalic (IT) and non-IT neurons, finding no evidence of single neuron precision functional connections in the samples tested- but do verify that functional connectivity amongst these populations exists.

Finally, I demonstrate the first reported all-optical, crosstalk-free neurophysiology strategy using Chronos, a blue-light sensitive opsin, and CaSiR-1, a red-light emitting calcium dye. I show red light suitable for CaSiR-1 imaging evokes no photocurrents in CHO cells transfected with Chronos, before demonstrating high signal-to-noise, crosstalk-free imaging of CaSiR-1 red fluorescence whilst photostimulating Chronos in acute brain slices at stimulation frequencies up to 20 Hz.