Optogenetic Investigation of Gliotransmission – OptoGlia

Brain tissue includes two main cell types, neurons and glia. Neurons are viewed to be the information-processing cells. Astrocytes that form a heterogeneous cell population were thought to exert a supportive role and occupy 20-50% of the grey matter. OptoGlia will focus its attention on protoplasmic linear astrocytes, in culture and in slices of the primary sensory cortex and hippocampus.
Ten years ago, it was suggested that in addition to their supportive role astrocytes contribute actively to information processing by regulating synaptic activity, leading to the concept of the tripartite synapse, which includes the neuronal pre- and post-synaptic elements and an astrocyte process, all interacting in a reciprocal manner. Astrocytes respond to neuronal activity with cytosolic calcium elevations that, in turn, induce the release of neuroactive gliotransmitters from astrocytes. These gliotransmitters can stimulate other astrocytes and modify the excitability of neighboring pre- and post-synaptic elements.
The concept that astrocytes may exert an active and rapid feedback control of the neuronal activity is of great functional importance for understanding the working of the brain under both normal and pathological conditions. However, it is still far from being admitted.
First, although astrocytes can release glutamate and other gliotransmitters in a calcium-dependent manner, the mechanisms of release are not yet clarified and it is not clear if the release occurs under physiological conditions. It has been argued that stimuli used to evoke gliotransmitter release could rather correspond to a pharmacological stimulation because gliotransmission was not detected in transgenic animal models designed to recapitulate natural stimuli. OptoGlia will develop and use a new optogenetic toolbox to allow a more physiological and graded stimulation of astrocytes in culture and in brain tissue.
Second, the mechanisms of gliotransmitter release are highly debated and several calcium-dependent gliotransmitter release pathways have been suggested and may co-exist. Fast neuron-like exocytosis was proposed, however, the presence of major molecular components of the fast vesicular release machinery, such as the vesicular glutamate transporters (VGluts), and components of the SNARE protein complex (like VAMP2), has not been convincingly demonstrated. Using optogenetic activation of astrocytes, super-resolution imaging techniques, transgenic knockin and knockout mice, OptoGlia will address these issues.
Third, the release of vesicular organelles from astrocytes has been shown, but no consensus has been reached as to nature of the organelles, nor to what is released. OptoGlia will study single-organelle exocytosis using optogenetics, TIRF microscopy, and organelle-specific pHluorin-based fluorescent proteins to monitor the exocytosis of VAMP2- and VAMP3-positive vesicles, as well as CD63-expressing lysosomal organelles.
Fourth, in brain slices, neuronal slow inward currents (SICs) and slow outward currents (SOCs) can be detected in response to astrocytic glutamate and GABA release. The mechanisms are unclear: both exocytosis and volume-regulated anion channels (VRACs) were suggested, among other pathways. In a single neuron, both SICs and SOCs can be recorded but never occur simultaneously, suggesting a diversity of glutamate- and GABA-containing astrocytes, or local barriers of amino acid diffusion in a single astrocyte. Finally, SICs are potentially important, due to their ability to synchronize neuronal activity. SICs could have an important epileptogenic potential which is largely unexplored in the context of treating epilepsy. Using the optogenetic tools developed and validated by OptoGlia, we will examine the mechanisms of glutamate and GABA release which are responsible for SICs and SOCs. We will also investigate the impact of reactive astrocytes on neuronal activity after status eplipticus.