Biology session

Photoactive cage molecules can be bound to both excitatory and inhibitory neurotransmitters and their analogues, and used as carrier molecules, thus creating a caged neurotransmitter that is excited when exposed to light, releasing its payload. By combining this technique with fast two-photon microscopy, the fast and localised synaptic transmission patterns between individual neurons can be precisely reproduced both in vitro and in vivo, allowing us to investigate the mechanisms of dendritic integration and signal processing. However, the currently available and widely used caged excitatory neurotransmitter, MNI-Glu, derived from mononitroindolin, is just at the limit of its usability and have several drawbacks. For example, its photochemical efficiency is low in order to control the rate of spontaneous hydrolysis, as caged neurotransmitters with an increased rate of photochemical release also suffer from high spontaneous hydrolysis rates. Using quantum chemical modeling we have understood the mechanisms of hydrolysis and two-photon activation, and synthetised photochemically more effective caged compounds. These novel compounds have higher photochemical release efficiency, therefore less excitation energy is required to release the same amount of neurotransmitter. However, the resulting higher spontaneous hydrolysis rates increased the free glutamate concentration in the extracellular space, which is kept in balance by the cells’ own glutamate uptake mechanisms. The increased free glutamate concentration indirectly affected neuronal activity and dendritic signal integration through NMDA receptors. To overcome this problem, we used a new enzymatic elimination method, in which administration of the enzyme glutamate-dehydrogenase and its coenzyme, NADP+ decreased the free glutamate concentration to control levels. Through this method the use of the new, photochemically more effective caged glutamate compounds in neurophysiological experiments is possible. Here I report, that three novel caged glutamate compounds were successfully synthetised and tested in biological experiments in in vitro acute mouse hippocampal slices. Among the three new glutamate compounds, the dinitroindolin-derived DNI-Glu•TFA has a 7.2 times greater photochemical efficiency and a significantly lower GABAA receptor blocking effect, compared to MNI-Glu•TFA.