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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - SINCHAIS (In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behavior)

Teaser

Summary

Ionotropic receptors and ion channels on neuronal membranes are often composed of multiple subunits, forming ion pores within the heteromeric complexes. Although the subunit compositions of these channels are critical for functional properties and regulation of surface expression, very little is known about subunit composition of single ion channels in situ, because of technical problems. Overall objectives of the project is to develop a high resolution labelling methods for different subunits of glutamate receptors and voltage-dependent calcium channels (VDCCs), using electron microscopy (EM) tags bound to nano-size distinct metal particles, and identify subunit composition at the single channel level. Development of such methods is important and widely applicable for many other protein complexes, which compositions are critical to understand their functions. In case of glutamate receptors and VDCCs, subunit composition of the channels controls neurotransmission in the postsynaptic and presynaptic sites, and regulates synaptic plasticity. We aim to clarify how the subunit composition and its changes are involved in physiological learning and memory formation. We focus on spatial and emotional memory formations in the hippocampus, amygdala, and medial habebula to interpeduncular nucleus pathways. Quantitative measurements of single subunits compositions and their absolute numbers will be also important for constructing realistic simulation of signal integration in individual neurons and information processing in the brain circuits.

Work performed

We have developed a reactive tag system using a newly developed short amino acid sequences for EM tag combined with synthetic chemical probe, which makes covalent link with the tag (Tabata et al., under revision). When we expressed a tagged receptor in HEK cells, 1.4nm gold-conjugated chemical probe specifically labelled surface receptors with much higher sensitivity than FLAG tag located to an adjacent sequence. The resolution of this labelling is a few nanometer, which is more than 4 times higher than conventional immunolabeling using primary and secondary antibodies (20 nm). We have generated knock-in mice with EM tag sequences inserted in AMPA-type and NMDA-type glutamate receptors to enable us to examine the subunit composition of single ionotropic receptors in situ. To visualize multiple subunits in single channels, we have developed second chemical labelling method using a different tag-probe pair (Zenmyo et al., 2018). However, because it showed cross reactivity with the first one, we are now testing the third tag-probe pair. For asking physiological roles of glutamate receptors in hippocampal pathways involved in emotional memory formation, we identified a new glutamatergic ventro-dorsal hippocampal projection mediated by hilar mossy cells (Fredes et al, submitted). We found that activation of ventral mossy cells is necessary and sufficient to activate dorsal granule cells, which otherwise are rarely activated by other glutamatergic pathways. This indicates unexpected ventro-dorsal interaction of the hippocampus critical for linking emotional inputs to context memory formation in the dorsal hippocampus. We will further examine which subunit composition of glutamate receptors are involved in this emotion-facilitated context memory formation, using the newly generated knock-in mice.

Final results

Although we have succeeded to develop one kind of new tagging method with high sensitivity and resolution, we will need at least two orthogonal methods to label two different subunits in single channels. We have a few candidates and expect that we will be able to visualize at least two subunits in single AMPA or NMDA channels until the end of the project. At the same time, we will identify new pathways involved in spatial and emotional memory formation and elucidate how these glutamatergic receptors with defined subunit composition are involved in the memory formation in vivo.