Magnetic resonance microscopy of Aplysia neurons : studying neurotransmitter-modulated transport and response to stress
Recent progress in magnetic resonance imaging (MRI) has opened the way for micron-scale resolution, and thus for imaging biological cells. In this thesis work, we performed magnetic resonance microscopy (MRM) on the nervous system of Aplysia californica, a model particularly suited due to its simplicity and to its very large neuronal cell bodies, in the aim of studying cellular-scale processes with various MR contrasts. Experiments were performed on a 17.2 Tesla horizontal magnet, at resolutions down to 25 µm isotropic. Initial work consisted in conceiving and building radiofrequency microcoils adapted to the size of single neurons and ganglia. The first major part of the project consisted in using the manganese ion (Mn2+) as neural tract tracer in the buccal ganglia of Aplysia. Manganese is an MR contrast agent that enters neurons via voltage-gated calcium channels. We performed the mapping of axonal projections from motor neurons into the peripheral nerves of the buccal ganglia. We also confirmed the existence of active Mn2+ transport inside the neural network upon activation with the neurotransmitter dopamine. In the second major part of the project, we tested the potential of two diffusion MRI sequences for microscopy. On the one hand, we explored a very original mechanism for diffusion weighting, DESIRE (Diffusion Enhancement of SIgnal and REsolution), particularly suited for small samples. The two-dimensional DESIRE sequence was implemented and successfully tested on phantoms. The measured enhancement was consistent with theoretical predictions. Using this sequence to produce diffusion weighted images with an unprecedented contrast in biological tissue remains a challenge. On the other hand, a more “standard” sequence was implemented to measure the apparent diffusion coefficient (ADC) in nervous tissue with MRM. This sequence was a three-dimensional DP-FISP (Diffusion Prepared Fast Imaging with Steady-state free Precession), which met criteria for high resolution in a short acquisition time, with minimal artifacts. Using this sequence, we studied the changes in water ADC at different scales in the nervous system, triggered by cellular challenges. The challenges were hypotonic shock or exposure to ouabain. ADC measurements were performed on single isolated neuronal bodies and on ganglia tissue, before and after challenge. Both types of stress produced an ADC increase inside the cell and an ADC decrease at tissue level. The results favor the hypothesis that the increase in membrane surface area associated with cell swelling is responsible for the decrease of water ADC in tissue, typically measured in ischemia or other conditions associated with cell swelling.