From engram to tomogram: the in-situ structure of memory in the mammalian brain by cryo-electron tomography (cryoET)

Long-term memories are encoded within neuronal circuits called the engram. Structural mechanisms involving Ca2+ ion transients drive essential neuronal functions including synaptic plasticity, and the acquisition of memory. However, the mechanisms by which latent and stable macromolecular complexes retain information within these synaptically connected circuits are unknown.

The Frank group were recently the first in the world to have established methods for obtaining in-situ structures of mouse (and human) brain by cryogenic correlated light and electron microscopy (cryoCLEM)-guided cryo-sectioning and cryo-focused ion beam scanning electron microscopy (cryoFIB-SEM) liftout lamella preparation for cryo-electron tomography (cryoET). These methods reveal the molecular architecture of protein complexes, organelles and cells in situ.

Our goal using these cutting-edge structural approaches together with engram labelling in mice, is to understand how the in situ structures engram synapses mediate synaptic plasticity and the acquisition of memory. Objectives:

i) Use cryoCLEM-guided block-face cryoFIB-SEM imaging to obtain volumetric reconstructions to reveal the cellular architecture and connectivity of engram circuits. architecture of the in situ structure of engram and non-engram circuit synapses.

ii) Use cryoCLEM-guided cryoFIB-SEM liftout lamella preparations from engram mouse brain to determine the in-situ molecular structure of engram and non-engram synapses.

iii) Use synaptically-localised Ca2+ integrators to tag active synapse for in-situ structure determination by cryoCLEM-guided cryo-sectioning and cryoFIB-SEM liftout lamellae preparation for cryoET. Novelty and timeliness:

Cryo-electron tomography (cryoET) is the fastest growing discipline in structural biology, where advancements in instrumentation and computational image analysis are transforming our understanding of molecular mechanisms within cells and tissues. With its investment in cryoEM and cryoFIB-SEM, Leeds is at the forefront of this revolution. We expect that by combining fluorescent markers of calcium signalling with cryoEM to determine in situ molecular structures within cells and tissues will represent a further significant advancement in this field.

Experimental approach:

We are combining fluorescent labelling, including genetically-encoded engram labels, calcium integrators, correlated light-electron microscopy, and cryo-electron tomography to locate engram and active synapses within tissue and cell samples to determine in situ molecular structure. The mechanism of activity-dependent structural change within cells and tissues will be investigated using fluorescent calcium indicators to locate active membranes for structure determination.