Coordinated optical and ultrastructural investigation of new synapses

Despite decades of study, the earliest stages in the formation of a connection between two neurons, known as a “synapse,” remain mysterious. The technical difficulty of precisely capturing the structure and function of a new synapse has precluded a consensus on the mechanisms of synapse genesis. One class of theories emphasizes the importance of molecular targeting – special proteins expressed in cellular membranes that allow pre- and postsynaptic neurons to sample each other and choose to form and stabilize a link. Another group of theories posits the crucial role of neurotransmitter release from would-be pre-synaptic terminals in broadcasting an availability signal and attracting potential synaptic partners. In addition to these distinct proposed genesis mechanisms, the temporal estimates for when a new synapse becomes functional range across orders of magnitude. This Whitehall Foundation Grant proposal seeks to provide an unprecedented, spatio-temporally controlled analysis of new synapse ultrastructure by bridging multi-laser 2-photon technologies for de novo synaptogenesis induction with electron microscopy. Recently, while studying basal ganglia circuits during the remarkably dynamic period, when the opening of the senses sculpts the emerging, increasingly complex goal-directed behaviors, I discovered a new, neuromodulation-dependent form of plasticity that may play a critical role in development. I accomplished this by adapting a technique for de novo induction of dendritic spines for genetically identified neurons in the relatively intact circuitry of the acute slice. This technology hinges on custom-built 2-photon 2-laser systems that combine imaging of dendritic fluorescence signal with glutamate uncaging. Glutamate photorelease near a young dendrite can produce a new dendritic spine, with a time-course of seconds and high success rates, in nearly arbitrary locations on the dendrite. My recent studies defined a critical role for neuromodulation-induced signaling of Protein Kinase A in regulating the probability of and the glutamate threshold for de novo spinogenesis. Importantly, follow-up patching of the parent neuron reveals that stimulating newborn spines evokes glutamatergic currents and fluxes Calcium, although to a lesser extent than driving neighboring, older spines. Some new dendritic spines retract, disappearing within seconds to minutes, while others persist for the life-time of the slice. What distinguishes those that go from those that stay is unknown. Functional evidence supporting synaptic signature on newly induced spines demonstrates that an immature synapse can form within minutes – considerably faster than generally believed. However, most functional techniques lack the spatial resolution to characterize size and shape of the post-synaptic density, determine whether new spines are capable of recruiting presynaptic axonal terminals, or discover how synapses differ as a function of dendritic activity state at the time of their birth. Electron microscopy (EM) is the gold standard technique for this class of questions. With precise spatio-temporal control of dendritic spine induction in a relatively intact circuit, I have the unique opportunity to bridge EM with 2-photon uncaging/imaging, in order to address the following fundamental neurobiological questions: (1) Do newly produced dendritic spines recruit presynaptic terminals? (2) Does the age of dendritic spine, on the order of minutes to hours, determine the ultrastructural qualities of its synapse? (3) How do synapses vary as a functi