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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MF-Synapse (Presynaptic calcium channels distribution and impact on coupling at the hippocampal mossy fiber synapse)

Teaser

Summary

The hippocampus is a region of the brain extensively studied due to its fundamental role in declarative memory formation and consolidation, also in spatial memory and formation of cognitive maps for navigation through space. Hippocampal granule cells axons form mossy fibers (MF) and terminate in large boutons that relay this input to the proximal dendrites of CA3 pyramidal neurons. This giant synaptic contact forms a key synapse that has been proposed to have a major role in hippocampal function as a conditional â€œdetonator synapseâ€ that reliably discharges the postsynaptic CA3 target neurons. Despite MF synapses widely accepted involvement in processing, storage and recall of information in hippocampus, due to several technical problems there is still very limited information about this central synapse. The goal of this project was to investigate biophysical mechanisms of calcium dependent exocytosis and how it relates to the unique forms of plasticity identified at the MF- CA3 synapse. A better understanding of synaptic transmission dynamics at this important synapse would give great insight on the mechanisms underlying their role in the hippocampus. This synapse is essential for hippocampal network function and may be involved in distinguishing memory storage and retrieval modes in the hippocampus circuit. And in a general sense, we still have a large gap in the knowledge underlying synaptic transmission in central synapses in the brain.

Work performed

For this study and to address our questions, we optimized a technique to use state-of-the-art methods and directly correlate physiology with ultrastructure of synapses. We performed high-pressure freezing (HPF) of live acute brain slices with optogenetic stimulation immediately prior to freezing, with millisecond precision, to capture presynaptic changes after action potential evoked synaptic transmission under physiological conditions. We were able to: (1) express the light-activated channel channelrhodopsin specifically in granule cells in hippocampus dentate gyrus; (2) characterize light-evoked responses in both granule cells and CA3 pyramidal neurons with electrophysiology, matching those evoked traditionally with electrical stimulation; (3) successfully freeze acute slices with a new HPF system still hardly used by many groups in the world; (4) stimulate mossy fibers in the HPF chamber with light and successfully evoke synaptic transmission; (5) apply mild and strong stimulation paradigms, with evidence of depletion of docked vesicles with strong stimulus, as well as evidence of ultrafast endocytosis.
Such depletion of docked vesicles suggests that docked vesicles represent the releasable vesicle pool and therefore that docked vesicles can be used as a structural correlate for the readily-relesable pool (RRP) of vesicles. We however noticed that after this depleting-protocol, some vesicles remained docked. We interpret this result in two fold. Firstly, this could indicate that not all docked vesicles are part of the RRP, although the majority of them are. However this could also indicate that at some point during the stimulus, rapid recruitment and replenishment of the docked vesicle pool started to happen. Thus, this method now also allows for future studies to investigate kinetics of vesicle recruitment and replenishment at the active zone, by looking at different time points after evoked synaptic transmission, with different levels of activity.

Final results

Understanding how the brain function is a matter of great interest to the general public. In a fundamental level, it is relevant for the comprehension of how our nervous system functions under basal conditions as well as under the development of disease states and neurological disorders that can greatly impair human function. The results of this study, together to the potential projects it enabled with new state-of-the-art techniques, could have great impact on fundamental mechanisms of synaptic transmission that are the cellular basis for learning and memory. The neural network targeted for this study, the hippocampus, has a fundamental role in declarative memory formation and consolidation, also in spatial memory and formation of cognitive maps for navigation through space. It is also affected under neurological imbalanced states such as Alzheimerâ€™s disease and epilepsy. Thus the understanding of this particular neural circuit has the potential to shed light on its basal function in the brain as well as what goes wrong under diseased states.