Study of the modulation of GABAA receptors by using RuBi-GABA uncaging with linear and non-linear photoactivation in rat cerebellar granule cells in vitro

The neuronal transmission regulates our emotions, thoughts, relationships with the external world, and our life, integrating and transforming the information from our neuronal circuit brain's electric signals. These signals come from the passage of ions through channels in the cellular plasma membrane; an imbalance is enough to cause pathologies involving the Central Nervous System, e.g., epilepsy and other neuropsychiatric conditions. This research concerned the study of a receptor, GABAA receptor, which modulates the neuronal transmission in the Central Nervous System through its activation by the inhibitory neurotransmitter GABA. It is possible to precisely follow the GABAA receptor activation in a limited cell portion coupling electrophysiology and optical microscopy. This method enables selecting a region of interest on the neuron for verifying differences in the GABAA receptor modulation after the activation by its neurotransmitter and pharmacological molecules. The purpose can be achieved thanks to the caged compounds: inert or inactive molecules until the light activates them where and when needed. The release of photoactivable compounds is suitable to control interactions in living cells. In this project, the uncaging of a caged molecule, RuBi-GABA, was investigated and characterized. The method was studied as a useful technique for activating the receptor in localized areas of interest, changing various physical parameters. After investigating all the physiological variations due to these parameters' changes in 4 dimensions (X, Y, Z, t), the focus was moved to the use of the pharmacological molecule Antisecretory Factor. This compound acts in vivo by inhibiting intestinal hypersecretion and various inflammation forms, although its mechanism of action is mostly unknown. The Antisecretory Factor has a facilitating action on the GABAA receptor in the presence of the neurotransmitter GABA. This pharmacological molecule action mechanism was explored on a limited number of receptors and in different parts of the neuron, such as soma, growth cones, and neurites. The study of the receptor responses in a confined cell area may open the door to future studies that can be used for the topical application of drugs on a specific target without activating the non-specific response of cells not of interest, thanks to the photoactivation of selective caged compounds.