The specific achievements of the GIANTSYN project were as follows: first, we elucidated the biophysical mechanisms of synaptic transmission. To achieve this, we extended our subcellular patch-clamp recording technology, and developed a method to non-invasively stimulate hippocampal mossy fiber terminals in the cell-attached configuration. We determined key biophysical parameters of transmission (Vandael et al., 2020). Using freeze fracture replica labeling electron microscopy, we localized the presynaptic Ca2+ channels in the mossy fiber terminals in relation to the active zones. Using “flash and freeze” electron microscopy, we found that the number of docked vesicles at active zones was reduced after stimulation, providing evidence for full fusion of synaptic vesicles as a main mode of exocytosis at this synapse (Borges-Merjane et al., 2020; Vandael et al., 2020). Our results revealed, for the first time, the biophysical mechanisms of synaptic transmission at a cortical synapse.
Second, we unraveled the mechanisms of synaptic plasticity. We focused on posttetanic potentiation (PTP), the most prominent form of plasticity at the hippocampal mossy fiber synapse, a potential correlate of hippocampal short-term memory. Biophysical analysis revealed that PTP was, to a large extent, generated by an increase in the size of the readily releasable vesicle pool. “Flash and freeze” electron microscopy analysis indicated that after tetanic stimulation followed by a recovery period, the docked vesicle pool was increased beyond the control value, implying “overfilling” of the pool (Vandael et al., 2020). Our results reveal the mechanisms of a major form of synaptic plasticity at the hippocampal mossy fiber synapse, and suggest that changes in the configuration of presynaptic vesicle pools play a key role. Our experiments identified, for the first time, a structural pool engram that potentially underlies short-term memory.
Third, we determined the structural and functional connectivity in the dentate gyrus–CA3 network. To address this issue, we established a technique for rabies virus-mediated transsynaptic labeling, based on a new N2c rabies virus. Using CA3 pyramidal neurons as starter cells, rabies virus-mediated transsynaptic labeling from CA3 pyramidal neurons revealed the classical synaptic inputs to CA3 from the granule cells via the hippocampal mossy fibers and from the entorhinal cortex layer 2 via the perforant path. In addition, we discovered a new powerful input from the deep entorhinal layers (layer 6b or “subplate”; Ben-Simon et al., 2022). Our results challenge the classical view of the trisynaptic circuit of the hippocampus. Our findings also falsify the widely held hypothesis that deep cortical layers and subplate neurons play an exclusive role in development.
Fourth, we wanted to analyze the network function of hippocampal mossy fiber synapses. To achieve this, we developed a method to obtain in vivo whole-cell patch-clamp recordings from dentate gyrus granule cells, the presynaptic cells that give rise to the hippocampal mossy fiber pathway. We discovered that morphologically identified granule cells fired sparsely, but when they fired, they frequently generated bursts or “superbursts” of action potentials, suggesting a new type of coding in the dentate gyrus (Zhang et al., 2020). In parallel, we established behavioral paradigms, including spatial coding, learning, and contextual fear conditioning. We found that in Prox1-CreERT2 mice, sparse optogenetic granule cell stimulation resulted in increased motor activity and impairment of fear memory recall, consistent with the proposed detonator function of the synapse. Finally, we developed network models of the dentate gyrus–CA3 region. We found that the mossy fiber synapse regulates the balance between pattern separation and pattern completion (Guzman et al., 2021). The results shed new light on the function of the hippocampal mossy fiber synapse in the network.
In conclusion, the major goals of the original application were achieved as proposed. Thus, the hippocampal mossy fiber synapse has become the first synapse in the history of neuroscience where we reach deep insights into both synaptic biophysics and contribution to higher network computations. In the long run, the results may open new perspectives for the diagnosis and treatment of brain diseases in which mossy fiber transmission, plasticity, or connectivity are impaired.
