Photolysis applied to synaptic transmission

In synaptic transmission speed and economy are achieved by sub-micron co-localization of the sources and targets of the ligand – Ca2+ions or neurotransmitter and their target receptors. The Ca dependent presynaptic release machinery is colocalised with Ca2+channels, and the postsynaptic receptors for released neurotransmitter are closely apposed to the presynaptic terminal. In this way a few thousand ligand molecules achieve high concentrations to produce fast efficient binding, and they are quickly eliminated once the signal is transmitted.The experimental problem is to reproduce the time and spatial dimensions of ligand application to study or mimic the physiological process. Photolysis of photolabile precursors can overcome the diffusion barriers that would normally limit the rate of activation of intracellular or extracellular receptors by perfusion or electrochemical techniques: the space adjacent to receptors is flooded with ‘caged’ precursor and the timing and spatial application of the photoreleased ligand is determined by a pulse of light. With laser illumination the timing and spatial resolution come close to those of synaptic transmission, sub-millisecond and sub-micron, permitting kinetic experiments on a physiological scale.
Transmitter release from presynaptic terminals is studied by photolysis of caged Ca2+chelators such as DM-nitrophen. Laser photolysis to release high Ca2+ in typical small terminals with a single release site has been used to probe the release mechanism, particularly the number of vesicles ready for release. To investigate neurotransmitter action at postsynaptic receptors in situ the timing and concentration of neurotransmitter is controlled independently of the presynaptic processes. This can be done with a diffraction limited laser spot and caged excitatory or inhibitory neurotransmitters. There have been several problems in generating hydrolytically stable caged neuroactive amino acids. The most successful uses nitroindoline photochemistry, generating stable, fast and efficient cages in the near UV for excitatory L-glutamate or inhibitory GABA or glycine receptors. Problems with receptor interactions of the cage with GABA and glycine receptors have been largely overcome by modifying cage structure without affecting the properties of the nitroindoline chromophore.
The major deficiency remaining in the use of caged amino acid transmitters is the poor penetration of the near UV photolysis spot into brain and other tissues, limited to 20 micrometresbelow the surface. This is largely due to scattered photons producing delocalised photolysis and ligand release. Two-photon photolysis would greatly improve localisation because scattered IR photons do not produce photolysis. However, two-photon absorption is very inefficient compared with one-photon requiring high light intensities and high cage concentrations. Developments to improve the efficiency of two-photon cages will be discussed.