Synapses are the basic cellular units modified during learning and memory formation. A major barrier to characterizing synaptic function and plasticity is that the synaptic structures of most interest-presynaptic vesicles, presynaptic active zones, the synaptic cleft, postsynaptic densities (PSDs) and postsynaptic receptors-are mostly below the diffraction-limit of light. While some of these structures are resolvable using electron microscopy (EM), EM cannot assay structures in live tissue or real time, and has significant problems identifying specific molecules and with sample preparation artifacts. These limitations prevent observing how synapses change during plasticity events that underlie memory formation, such as long-term potentiation (LTP) and depression (LTD), as well as synaptic changes occurring during neurodegenerative diseases, such as Alzheimer's Disease or during neurodevelopmental diseases, such as Autism Spectrum Disorders. In this proposal different super-resolution fluorescence microscopy techniques are applied to live excitatory CNS synapses. This approach will improve imaging accuracy and resolution to near that of EM. To apply super-resolution microscopy techniques to synapses, small quantum dots (~7 nm diameter) have been developed that allow access to synaptic clefts, 20-30 nm wide. In contrast, the bigger, commercial quantum dots (~21 nm diameter), which are widely used in the field, are found to be unable to enter synaptic clefts. Using super-resolution techniques, the hypothesis will be tested that the two postsynaptic synaptic neurotransmitter receptors, AMPA-type and NMDA-type glutamate receptors (AMPARs and NMDARs), have unique distributions and mobility within PSDs because of their different interactions with the scaffold proteins, PSD-95 and SAP97 in PSDs. The small quantum dots attached to AMPARs and NMDARs will allow tracking of the movements of the neurotransmitter receptors outside and within synapses. To test the hypothesis, three specific aims are proposed;all of them use super-resolution microscopy and generally rely on our unique small quantum dots (with controls using regular dyes).
The first Aim i s to measure PSD-95 and SAP97 distributions within PSDs and their changes with LTP and LTD.
The second Aim i s to measure AMPAR and NMDAR distribution and kinetics within PSDs and the changes that occur with LTP and LTD.
The third Aim i s to examine the role of PSD-95 palmitoylation cycle in AMPAR and NMDAR dynamics and the effects of LTP and LTD.
Synapses are the connections between two neurons and are involved in fundamental processes of learning and forgetting, and they deteriorate in neurodegenerative diseases such as Alzheimer's disease and neurodevelopmental diseases. We propose to use the recent advances in super-resolution fluorescence microscopy to study their structure and their changes in time.