Summary Neurotransmitter release probability (Pr) largely determines the dynamic properties of synapses. While much is known on the role of presynaptic proteins in transmitter release, their specific contribution to synaptic plasticity is unclear. One such protein, tomosyn, is believed to reduce Pr by interfering with the SNARE complex formation. Tomosyn is enriched at hippocampal mossy fiber-to-CA3 pyramidal cell synapses (MF-CA3), which characteristically exhibit low Pr, strong synaptic facilitation and pre-synaptic PKA-dependent LTP. To evaluate tomosyn's role in MF-CA3 function, we used a combined knockdown (KD)-optogenetic strategy whereby presynaptic neurons with reduced tomosyn levels were selectively activated by light. Using this approach in mouse hippocampal slices we found that facilitation, LTP, and PKA-induced potentiation were significantly impaired at tomosyn-deficient synapses. These findings not only indicate that tomosyn is a key regulator of MF-CA3 plasticity, but also highlight the power of a combined KD-optogenetic approach to determine the role of presynaptic proteins.

Neural networks engaged in high-frequency activity rely on sustained synaptic vesicle recycling and coordinated recruitment from functionally distinct synaptic vesicle (SV) pools. However, the molecular pathways matching neural activity to SV dynamics and release requirements remain unclear. Here we identify unique roles of SNARE-binding Tomosyn1 (Tomo1) proteins as activity-dependent substrates that regulate dynamics of SV pool partitioning at rat hippocampal synapses. Our analysis is based on monitoring changes in distinct functionally defined SV pools via V-Glut1-pHluorin fluorescence in cultured hippocampal neurons in response to alterations in presynaptic protein expression. Specifically, we find knockdown of Tomo1 facilitates release efficacy from the Readily Releasable Pool (RRP), and regulates SV distribution to the Total Recycling Pool (TRP), which is matched by a decrease in the SV Resting Pool. Notably, these effects were reversed by Tomo1 rescue and overexpression. Further, we identify that these actions of Tomo1 are regulated via activity-dependent phosphorylation by cyclin-dependent kinase 5 (Cdk5). Assessment of molecular interactions that may contribute to these actions identified Tomo1 interaction with the GTP-bound state of Rab3A, an SV GTPase involved in SV targeting and presynaptic membrane tethering. In addition, Tomo1 via Rab3A-GTP was also observed to interact with Synapsin 1a/b cytoskeletal interacting proteins. Finally, our data indicate that Tomo1 regulation of SV pool sizes serves to adapt presynaptic neurotransmitter release to chronic silencing of network activity. Overall, the results establish Tomo1 proteins as central mediators in neural activity-dependent changes in SV distribution among SV pools.

Restoration of the communication between brain circuitry is a crucial step in the recovery of brain damage induced by traumatic injuries or neurological insults. In this work we present a study of real-time unidirectional communication between a spiking neuronal network (SNN) implemented on digital platform and an in-vitro biological neuronal network (BNN), generating similar spontaneous patterns of activity both spatial and temporal. The communication between the networks was established using patterned optogenetic stimulation via a modified digital light projector (DLP) receiving real-time input dictated by the spiking neurons’ state. Each stimulation consisted of a binary image composed of 8 × 8 squares, representing the state of 64 excitatory neurons. The spontaneous and evoked activity of the biological neuronal network was recorded using a multi-electrode array in conjunction with calcium imaging. The image was projected in a sub-portion of the cultured network covered by a subset of the all electrodes. The unidirectional information transmission (SNN to BNN) is estimated using the similarity matrix of the input stimuli and output firing. Information transmission was studied in relation to the distribution of stimulus frequency and stimulus intensity, both regulated by the spontaneous dynamics of the SNN, and to the entrainment of the biological networks. We demonstrate that high information transfer from SNN to BNN is possible and identify a set of conditions under which such transfer can occur, namely when the spiking network synchronizations drive the biological synchronizations (entrainment) and in a linear regime response to the stimuli. This research provides further evidence of possible application of miniaturized SNN in future neuro-prosthetic devices for local replacement of injured micro-circuitries capable to communicate within larger brain networks.

The mammalian hippocampal formation (HF) plays a key role in several higher brain functions, such as spatial coding, learning and memory. Its simple circuit architecture is often viewed as a trisynaptic loop, processing input originating from the superficial layers of the entorhinal cortex (EC) and sending it back to its deeper layers. Here, we show that excitatory neurons in layer 6b of the mouse EC project to all sub-regions comprising the HF and receive input from the CA1, thalamus and claustrum. Furthermore, their output is characterized by unique slow-decaying excitatory postsynaptic currents capable of driving plateau-like potentials in their postsynaptic targets. Optogenetic inhibition of the EC-6b pathway affects spatial coding in CA1 pyramidal neurons, while cell ablation impairs not only acquisition of new spatial memories, but also degradation of previously acquired ones. Our results provide evidence of a functional role for cortical layer 6b neurons in the adult brain.