Exploring the memory: existing activity-dependent tools to tag and manipulate engram cells

Abstract

The theory of engrams, proposed several years ago, is highly crucial to understanding the progress of memory. Although it significantly contributes to identifying new treatments for cognitive disorders, it is limited by a lack of technology. Several scientists have attempted to validate this theory but failed. With the increasing availability of activity-dependent tools, several researchers have found traces of engram cells. Activity-dependent tools are based on the mechanisms underlying neuronal activity and use a combination of emerging molecular biological and genetic technology. Scientists have used these tools to tag and manipulate engram neurons and identified numerous internal connections between engram neurons and memory. In this review, we provide the background, principles, and selected examples of applications of existing activity-dependent tools. Using a combination of traditional definitions and concepts of engram cells, we discuss the applications and limitations of these tools and propose certain developmental directions to further explore the functions of engram cells.

Keywords: activity-dependent tools; engram cells; genetic strategy; memory; neuronal activity.

Figure 1

Design of activity-dependent tools based on Ca²⁺. (A) CaMPARI and CaMPARI2. They are photoconvertible fluorescent proteins that can be switched from green to red when illuminated with violet light and when intracellular calcium levels are elevated due to a stimulus. CaM and its binding peptide sense calcium levels through their interaction. (B) Cal-Light and ST-Cal-Light. They are tools that consist of two components and a reporter or optogenetics tool. The cytoplasmic component is M13 fused to the TEV-C, while the transmembrane component is a fusion of the transmembrane domain with CaM, the TEV-N, TEVcs caged within AsLOV2 domain, and tTA. To trigger the expression of a reporter or OT, it is necessary to elevate Ca²⁺ in the cytosol through a stimulus and illuminate blue light to cause a conformational change in the AsLOV2 to release TEVcs. Then, M13 and CaM bind to each other, TEV-C and TEV-N regain proteolytic functions to reconstitute TEVp, and the uncaged TEVcs are cleaved to drive the expression of tTA and further trigger the expression of a reporter or OT. (C) FLARE and FLiCRE. They contain two components and a reporter or OT. The cytoplasmic component is a fusion of CaM with TEVp, while the membrane component is a fusion of the transmembrane domain with the CaM-binding peptide M13, TEVcs caged within the eLOV domain, tTA, and a reporter/OT. To trigger the expression of a reporter or OT, it is necessary to elevate Ca²⁺ in the cytosol through a stimulus and illuminate blue light to cause a conformational change in the eLOV to release TEVcs. Then, M13 and CaM bind to each other, TEVp cleaves the uncaged TEVcs, and tTA is released to drive the expression of a reporter or OT. FLiCRE is an optimized version of FLARE, which contains TEVp with a faster turnover rate and a version of LOV domain with tighter caged TEVcs named hLOV1. (D) scFLARE. It consists of a membrane component and a reporter. The membrane component consists of a transmembrane domain, engineered CaTEV, TEVcs caged within the engineered hLOV1, and tTA. To trigger the expression of a reporter or OT, it is necessary to elevate Ca²⁺ in the cytosol through a stimulus to activate CaTEV and illuminate blue light to cause a conformational change in the hLOV1 to release TEVcs. Then, CaTEV cleaves the TEVcs, releases tTA, and drives the expression of a reporter or OT. All FLARE-derived systems contain soma-targeting signals in their membrane components.

Figure 2

Design of activity-dependent tools based on chemicals and IEGs. (A) TetTag. To induce reporter gene expression in target neurons, doxycycline must be removed from the food of Fos-tTA mice, and a specific stimulus should be provided concurrently to initiate Fos expression, resulting in tTA expression. Controlled by tetracycline-responsive elements under the regulation of the TetO, tTA drives the expression of the reporter genes. RAM. The RAM synthetic promoter comprises four tandem repeats of synthetic EM located upstream of the minimal Fos promoter. Commonly employed in conjunction with TegTag, the RAM promoter effectively drives the expression of destabilized tTA to initiate the production of a reporter. (B) TRAP and TRAP2. For the expression of reporter genes in activated neurons, it is essential to administer TM or 4-OHT to Fos-CreERT2 mice, while concurrently providing a specific stimulus to initiate Fos expression. The expression of CreERT2, in turn, drives the expression of Cre-dependent reporter genes. E-SARE. In this synthetic promoter, the SARE enhancer is fused to the minimal Arc promoter in five tandem repeats. It is usually used with the TRAP tools and drives the Cre recombinase effectively to trigger the expression of reporter genes. (C) LacZ-Daun02. To selectively inactivate neurons activated by stimulus, c-fos-lacZ rats were designed to label activated neurons expressing both c-Fos and the β-gal encoded by the lacZ gene. After providing a specific stimulus, precursor drug Daun02 is injected into specific brain regions where β-gal catalyzes the conversion of Daun02 to daunorubicin. This drug could inactivate neurons activity. So, neurons expressing β-gal and c-Fos can be selectively inactivated in this way. (D) CANE. To induce reporter gene expression in activated neurons, it is essential to provide a specific stimulus to initiate Fos expression and inject lentiviruses coated with an EnvA into FosTVA mice. Subsequently, the Fos promoter will stimulate the expression of dsTVA, which can bind a coat protein of EnvA. The EnvA-RV-infected neurons will then deliver the Cre recombinase that drives the expression of reporter genes. (E) vGATE. Prior to initiating the experiment, it is required to inject three components (tPFos-rtTA + Ptetpi-YC3.60/Cre + POT-flex-effector) packaged by AAV into WT mice. To induce reporter gene expression in activated neurons, a specific stimulus must be provided to initiate Fos expression, and Dox should be added to the food of WT mice to activate rtTA. The Fos promoter drives the expression of Dox-activated rtTA, which is further autoregulated in a loop manner. Both reporter genes and Cre recombinase are expressed by rtTA, which subsequently induces the expression of the effector. (F) TRACE. The TRAP method is combined with the expression of a reporter gene from the AAV2 retrograde virus. This approach labels activated neurons in the target region and afferent neurons from other regions with the reporter gene. Yellow neurons in (F) indicate successful activation and labeling, while gray neurons in (F) denote inactivation. In brain slices, neurons with yellow nuclei and purple cytoplasm signify successful activation and labeling, whereas neurons with yellow nuclei and gray cytoplasm represent activation without successful labeling. Neurons in brain slices that are gray indicate inactivation.

Figure 3

Schematic diagram of activity-dependent tools for longitudinal records of cellular events. (A) Experimental diagram of iPAK4-based systems to record cFos-driven transcription events. IPAK4, as a protein scaffold, can safely grow in mammalian cells over time. The HaloTag-iPAK4 can stably bind to the HT dye and achieve fiducial timestamps to label fibers without affecting the physiological stage of the cell. The cFos promoter can drive the expression of EGFP-iPAK4. The red and yellow areas on the protein filament mean fiducial timestamps and the green areas mean records of cFos-driven transcription events. IPAK4, a fusion of the catalytic domain of the Pak4 kinase and the 38 amino acid iBox domain of its inhibitor Inka1. EGFP, enhanced green fluorescent protein. CMV, cytomegalovirus. HaloTag, a protein tag that can combine with multiple fluorescent dyes. (B) Schematic diagram of XRI strategy. The XRI is a genetically encoded self-assembling protein system and mainly consists of three parts. The 1POK (E239Y), as a filament-forming subunit, plays a role in self-assembling, and the MBP tag effects in blocking the unwanted lateral binding and growth of the protein assembly as an insulator. The epitope tag connects the two parts together. When cells are alive, self-assembling tagged proteins are continuously added to the growing chain, allowing for continuous recording of the presence of different tagged proteins available carrying specific promoter and reporter genes. In 2 weeks, the result of the growing chain can be obtained through immunofluorescence imaging. The blue areas on the growing protein chain represent components on the self-assembled protein that change expression over time, as the background. The pink and purple areas represent components of the self-assembled protein that is driven by cellular events of interest. 1POK (E239Y), self-assembling protein variant. MBP, maltose-binding protein.