Imaging Synapses - Karel Svoboda (HHMI)

Introduce foundational imaging methods including Golgi staining techniques used by Ramón y Cajal and standard fluorescence microscopy. Demonstrate how fluorescent markers label neural structures with high selectivity, such as red markers filling neuronal dendrites and green fluorophores marking synaptic proteins.

Describe two-photon excitation microscopy as a laser scanning technique that overcomes scattering limitations in deep brain tissue imaging. Highlight its advantages including high resolution, non-invasive long-term imaging capability, and specificity for targeting labeled structures in intact mouse brains.

Show in vivo imaging of a neuron labeled with red cytoplasmic marker and photoactivatable green fluorescent protein tagged to PSD95. Activate the green fluorophore at time zero and track fluorescence decay to study synaptic protein trafficking in the intact brain.

Label a neuron with red calcium-insensitive and green calcium-sensitive fluorophores, then deliver synaptic stimulation coincident with a blue square stimulus. Record calcium flashes in dendritic spines and shaft synapses to visualize NMDA receptor-mediated synaptic activation.

Image dendrites and axons in the barrel cortex of living mice every four days over an extended timeline. Document spine stability patterns, identifying that most spines persist for days to months while thin hair-like filopodia appear and disappear on shorter timescales.

Alter the animal's experience by trimming whiskers or training the animal in a perceptual learning task with remaining whiskers. Monitor how thin dendritic protrusions stabilize and develop large mushroom-shaped spine heads in response to novel experience, compared to baseline conditions.

Use serial section electron microscopy to create 3D reconstructions of newly grown spines observed in two-photon imaging. Confirm that stabilized thin protrusions and large spine heads form bona fide synapses with presynaptic structures in the surrounding neuropil.

Establish the relationship between structural spine changes and functional synaptic strength by demonstrating that spine size positively correlates with synaptic efficacy. Show that conversion from thin to large mushroom spines is associated with long-term potentiation and coupled changes in synaptic strength.