Periodic Reporting for period 4 - SINCHAIS (In situ analysis of single channel subunit composition in neurons: physiological implication in synaptic plasticity and behavior)
Periodo di rendicontazione: 2021-01-01 al 2022-06-30
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.
We have developed a reactive tag system using newly developed short amino acid sequences for the EM tag combined with the synthetic chemical probe, which makes the covalent link with the tag (Tabata et al., 2019). When we expressed a tagged receptor in HEK cells, a 1.4nm gold-conjugated chemical probe specifically labeled surface receptors with much higher sensitivity than the FLAG tag located in an adjacent sequence. The resolution of this labeling is a few nanometers, 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 a second chemical labeling method using a different tag-probe pair (Zenmyo et al., 2018). However, because it showed cross-reactivity with the first one, we tested the third tag-probe pair, which makes a covalent linkage between the His tag and a newly synthesized modified NTA probe. 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, 2020). 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. Using the newly generated knock-in mice, we further examine which subunit composition of glutamate receptors is involved in this emotion-facilitated context memory formation. In naive mice, we found that GluA1 and GluA2 subunits of the AMPA receptors have relatively homogeneous distribution patterns in the hippocampal pyramidal cell synapses and high extrasynaptic density, whereas GluA3 has a different pattern showing less density in the extrasynaptic sites and concentration in the center compared with the periphery of the postsynaptic density (Jevtic et al, under preparation). However, contextual fear learning has induced a shift of GluA1 to a more centered position with a significant increase of density specific to the CA1 area close to the CA3 area. This implies a compartment-specific change in the subunit composition of AMPA receptors by physiological learning for the first time. We are examining if this is the case using the newly developed chemical probe labeling for AMPA receptors (Islam et al., in preparation).
We have succeeded in developing two kinds of new tagging methods with chemical probes with high sensitivity and resolution, one with the hD2 tag (Tabata et al., 2019) and the other with a newly developed modified His-tag (Suetsugu et al., in preparation). We also tagged AMPA receptor subunits with spot and ALFA tags in knock-mice lines (Islam et al., in preparation). We are testing if the combination of these tagging methods enables us to label two different subunits in single channels of AMPA and NMDA receptors. At the same time, we examine if the subunit composition changes in the identified hippocampal 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.