Abstract: Markus STAHLBERG

Nanoscale Probing of Synapse Function and Plasticity

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Markus A. Stahlberg (1), AK Willig (2), Karl Deisseroth (3), SW Hell (2), C Dean (1)

(1) Trans-Synaptic Signaling, European Neuroscience Institute Göttingen, Göttingen, Germany
(2) Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
(3) Stanford University/Howard Hughes Med. Inst., Stanford, CA

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Complex neuronal networks form the basis for information processing in the brain, leading to phenomena like perception, behavior and memory. These phenomena rely on the ability to modify synaptic function in response to environmental stimuli and neuronal activity. Brainderived neurotrophic factor (BDNF) is essential for long-term potentiation (LTP) of synapses and its localization may be synapse-specific. To test if BDNF-harboring vesicles are specifically recruited to sites of increased activity, we use optogenetics to achieve focal stimulation of single synaptic sites by using FRAP illumination and a novel STED-inspired technique, in which a center spot is illuminated with light to activate channels, and a surrounding area is illuminated by a different wavelength of light, to close channels. Ideally this should prevent light scatter and diffusion of activated channels, and thus create a highly focal region of activation, approaching the size of a single synapse or even smaller. Switchable channelrhodopsin variants under control of the CaMKII promoter were expressed in dissociated hippocampal neurons by calcium phosphate transfection and AAV1/2 transduction. Using STED microscopy, we found that these channels were homogeneously distributed in the membrane. FRAP experiments indicated that the channels diffuse relatively slowly, at a rate of approximately 0.03 ?m2/sec. The activation and deactivation kinetics of switchable channel variants were tested using whole-cell electrophysiological recordings from expressing neurons. Using this approach we examined if BDNF-harboring vesicles are selectively recruited to specific synapses with increased activity. Such a synapse-specific recruitment would imply that neurons have the ability to specifically control the strength of synaptic transmission at the level of single synapses.