Two-Photon Fluorescence Microscopy and Uncaging.
Two-photon fluorescence microscopy allows structure and activity to be observed at the level of single synapses both in vitro and in vivo.
Caged compounds are molecules rendered biologically inactive by the covalent addition of a light-sensitive caging group that blocks the action of the molecule with its receptor. A flash of light removes the group, thereby producing a biologically active molecule. Two-photon uncaging allows neurotransmitters such as glutamate to be produced in femtoliter volumes (1 µm3) within a millisecond. With more widespread activation, action potentials can be evoked in the target neuron.
Two-photon microscopy is being used by Professor Sam Wang to look for coincidence detection in the cerebellum, a likely site for motor learning. This learning has been suggested to be mediated in part by long-term depression (LTD), a calcium- dependent process in which coincident activity of parallel fiber (PF) and climbing fiber (CF) synapses causes a long-lasting decrease in PF synaptic strength onto Purkinje cells. In the Marr/Albus/Ito hypothesis for motor learning, the effectiveness of conditional stimulus information arriving over the PF pathway undergoes LTD in response to instruction by unconditional stimulus information arriving over the CF pathway.
When CF activation is paired with bursts of PF activity, they together can cause larger calcium signals in individual PF dendritic spines than from either stimulation alone. These synergistic signals are the result of calcium release from intracellular stores and constitute a form of coincidence detection not seen before in the nervous system. We are now testing the hypothesis that these signals represent a functional link between cellular phenomena and motor learning.
Contact:
Sam Wang