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A. Collaborating Investigator(s): Kristen M. Harris,1 Terry Sejnowski,2 Tom Bartol, 2 J. Faeder3

 

 

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Fig VII.6. Adult (A) and P15 (B) dendrites in 3DEM show synapse enlargement at the expense of small spines in adults, and new spines at P15 under TBS-LTP relative to control conditions. (C) Single EM through two spines (yellow) with red outlined synapses that were connected to one dendrite.

B. Institutions: 2University of Texas a t Austin, 2Salk, 3Pitt

C. Funding Status: G1: R01MH104319 (Harris) 9/14-07/19; G2: R01MH095980 (Harris) 7/12-06/17

D. Driving relationship between TR&D2 and DBP7: Neuronal dendrites, axons and synapses are structurally distorted in individuals with mental retardation and other neurological disorders. We would like to interpret this distortion, but dendrites and spines differ widely in their appearance and composition in normal brains. Our overall goal is to characterize this structural variation towards understanding how neurons regulate, sustain, and alter synaptic connectivity as brain function develops and changes with learning, memory, and pathology. We have made significant progress using reconstruction from serial section transmission EM (3DEM) and in developing a new approach of transmission EM on the scanning electron microscope (tSEM) that greatly increases throughput of high quality images.105,106 We discovered that the summed synaptic surface area is balanced along hippocampal dendrites, either having many small spines with small synapses or few large spines with large synapses.107 Even following substantial structural synaptic plasticity during LTP induced by theta-burst stimulation (TBS-LTP), a rebalancing occurs to achieve equal summed synaptic surface area along control dendrites and LTP dendrites with enlarged synapses. These findings lead to the hypothesis that heterosynaptic competition for intrinsic resources regulates synapse number and size along adult neurons (Fig VII.6 A).

 

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Fig VII.7. Reconstructed dendritic segments were divided into spiny clusters and aspiny segments (>75 nm with no spine origins). SER and SA (green) were identified. The decrease in spine density was found only along those spiny clusters that had no SER in any of the spines.

In our funded research we are determining mechanisms underlying the developmental onset of the late phase LTP (L-LTP) lasting more than 3 hours.108 We have shown that when L-LTP is induced in developing animals at postnatal day (P)15, instead of enlarging existing synapses, more dendritic spines form. P15 is an age of rapid spinogenesis, hence there was substantial resources for adding new spines, that resulted in a greater summed synaptic input than under control conditions (Fig VII.6 B). We are also investigating the role of silent synaptic growth109 as a basis for the augmentation of LTP that could form the basis for the enhanced efficacy of spaced over massed learning.110 Critical to these studies is the improvement of alignment and reconstruction tools which will be developed by TR&D2 team members, driven by our data.

Two example findings illustrate how axonal and dendritic structure and composition support synaptic plasticity in the hippocampus, and provide high motivation for the proposed collaborative efforts with TR&D2 to enhance our data collection and analyses. We discovered that only ~20% of the axons that pass next to dendrites actually form synaptic contacts.111 We are investigating what intrinsic rules govern the number and size of synapses supported along axons. Recently we showed that spines arising from the same dendrite synapsing on the same presynaptic axon have the same synaptic surface areas, head volumes, vesicle numbers and neck diameters.112 This finding suggests a powerful structure-function relationship. Axons in CA1 stratum radiatum were evaluated with 3DEM after TBS-LTP.113 The frequency of axonal boutons with a single postsynaptic partner was decreased by 33% at 2 hours, corresponding perfectly to the 33% loss of small dendritic spines (head diameters <0.45 µm). Presynaptic vesicles as well as transport packets between boutons were reduced for at least 2 hours after TBS-LTP. These findings show that specific presynaptic ultrastructural changes complement postsynaptic ultrastructural plasticity during LTP. Smooth ER (SER) forms a membranous network that extends throughout neurons. SER regulates intracellular calcium and the posttranslational modification and trafficking of membrane and proteins. Greater SER volume, folding, or branching reduces the movement of membrane cargo and local delivery of resources increases in the vicinity of complex SER.114 Recently we have found that TBS-LTP initiates SER remodeling in adult hippocampal dendrites. In spines, SER volume increased and more spines contained a spine apparatus (SA), which is composed of highly folded SER. Synaptic growth was greatest at these spines. In parallel, dendritic shaft SER was less branched after TBS-LTP. Dendritic segments with no SER-containing spines had fewer neighboring spines whereas those surrounding a spine apparatus had spine densities equal to controls (Fig VII.7). Thus, dendritic spines with an apparatus collaborate with neighboring, but compete with more distant, spines for critical resources.

E. Innovations: Our past collaborative work has involved truly heroic studies, requiring a brute-force manual approach to obtain the original 3DEM reconstructions.112,115 The editing was done section by section with iterative 3D reconstructions to confirm the edits were in the correct locations. The improved alignment, segmentation, and model-editing tools of this proposal will support the specific aims of two funded grants in the Harris laboratory. They will make these in-depth analysis more routine and allow realistic MCell models to simulate biochemical signaling in the 3D subcellular structure of dendrites, axons, spines and synapses. Surface meshes used to represent cell membranes and subcellular structures must meet very strict geometric standards (e.g. water-tight, non-intersecting, manifold) like those we have created in our recent publications. We will add to these models the effects of synaptic plasticity.

 

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Fig VII.8. Three virtual sections (S1-S3) from tomography (3 nm thick) show no vesicles at nascent zones (NZ); versus many at active zones (AZ).(AZ, blue docked, yellow pores).

 

 

F. Methods and Procedures: We will provide high resolution images and 3DEM reconstructions to test the alignment, segmentation, and editing routines proposed in this grant. Our tissue processing and 3DEM methods have been rigorously tested and published.105,106 Example series have been posted to the OPENCONNECTOME.116 We will share data and images while accomplishing the Aims of our grants G1 and G2 (paraphrased here): Aim 1 (G1): To test the hypothesis that the abrupt onset of L-LTP at P12 is associated with first occurrence of dendritic spines using our rat model systems of in vivo perfusion-fixed hippocampus, TBS-LTP in hippocampal slices, and quantitative analyses from 3DEM. Aim 2 (G1): To test whether dendritic spines are induced by the first bout of TBS at P10, after which a second bout of TBS can produce L-LTP, but not at P8 when multiple bouts do not.

Aims 1-4 (G2): Recently, we have shown that initially saturated LTP can be subsequently augmented once a couple hours elapse between episodes of LTP induction.117 We have identified, nascent zones, which are dynamic regions with a postsynaptic density (PSD) that lack presynaptic vesicles (Fig VII.8).118 Immediately following induction, vesicles accumulate at prior nascent zones converting them to active zones. By 2 hours, nascent zones have returned. We will test the roles of protein synthesis, SER, and candidate molecules in building or stabilizing synapses during saturation and augmentation of LTP.

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