Dynamic mapping of micro-viscosity in the intact brain

Physiological activity of the brain relies on the rapid movement of signalling molecules, outside and inside nerve cells. Recent studies have found that eukariot cells can modulate their cytosol viscosity in response to temperature and energy demands, by regulating the synthesis of glycogen and trehalose, and thus maintain constant cytoplasm diffusivity (hence invariant reaction rates) across a 20C.

It has also been discovered that cytoplasm viscosity regulates the assembly dynamics of microtubules and thus cell morphology. Therefore, the knowledge of molecular diffusivity, which is defined by local microviscosity, on the small scale in the brain is crucial. Firstly, it should reveal critical constraints for the dynamics of molecular reactions and ionic currents that shape neural activity.

Secondly, it could provide a potentially important readout of local physiological and pathological changes in brain cells, which cannot be detected by other means.

Our program builds on a set of our in-house established methods, which include two-photon excitation fluorescence lifetime imaging (FLIM) and time-resolved fluorescence anisotropy imaging (TR-FAIM) implemented in vitro and in organised brain tissue. The main methodological objective of the proposed project is to expand and implement such methods in the intact animal brain at microscopic resolution, the approach that has not been attainable previously.

This implementation will enable the mapping and monitoring of extracellular and intracellular microviscosity in live animals (rodents), using FLIM-enabled protocols for either water-soluble small-molecule fluorophores or fluorescent indicators or molecular motors that could be genetically encoded for cell-targeted expression.

The ultimate scientific objective of our research will be to establish, where feasible, the causal associations between micro-viscosity of the specific extracellular or intracellular environments and the physiological or pathological traits of the target cells and their compartments, which in turn affect the functioning of the local neural microcircuitry. This will be achieved by employing the in-house established sensory stimulation paradigm (physiological aspect) and a chronic animal epilepsy model (pathological aspect).

We will use high-resolution time-resolved, multiplexed where required, imaging of the identified brain cells and their environments, including principal neurons, interneurons, astrocytes, and blood vessels, in the sensor cortex and barrel cortex areas of the rodent brain. The fluorescent probes will be applied either through direct application using patch-clamp techniques (relevant for small synthetic dyes), or through genetic targeting of specific cells (relevant for BODIPY-type molecular motors).

The experiments will comprise control and test conditions, the latter involving either physiological tests or a pathology/ disease model, respectively.

The current host group comprises experts in single-cell electrophysiology, time-resolved and multiplexed fluorescence imaging, two-photon microscopy, optics, cellular and neural-networks biophysics, molecular biology. We are equipped with several cutting-edge two-photon excitation-uncaging imaging systems coupled with patch-clamp physiology, including a system dedicated to brain imaging in live rodents, and all the related infrastructure support. The host department provides world-leading academic environment.