Neuron Activity-Dependent Tool Series: scFLARE2

Release time：2025-01-14 11:07:48

The brain contains a large number of neurons with functional heterogeneity. In response to stimuli, specific neuron groups assemble into imprints, forming functional encoding units at both the cellular and circuit levels. To analyze these functional encoding units, IEG (immediate early gene) expression is widely used as an indirect representation of neuronal activation. However, the transition from neuronal activation to IEG expression involves a series of cellular electrochemical activities, resulting in insufficient temporal resolution and sensitivity to represent the rapid dynamics of the nervous system in real-time.

In 2017, Wang et al. developed the FLARE tool, a novel light- and calcium-dependent neuronal activity labeling system, capable of genetically tagging specifically activated neurons within the acute experimental time window [1]. Compared to systems such as TRAP2 and RAM that rely on drugs as inducers, the scFLARE2 system can rapidly respond to neuronal activity with a time window of 1 to 30 minutes.

1. Principle

The FLARE tool is based on the modular gene expression structure of the Tango protein interaction system, calcium detection by GECIs (genetically encoded calcium indicators), and the light-responsive properties of the light-oxygen-voltage (LOV) domain. It achieves high spatiotemporal resolution for light-controlled, calcium-dependent labeling of active neuronal clusters and target gene expression.

In this system, calmodulin (CaM) and the CaM-binding peptides M13/M2, components of GECIs, replace the GPCR and β-arrestin elements of the Tango system. The transcription factor tTA is linked to one component of the system through a cleavage sequence recognized by tobacco etch virus protease (TEV Protease, TEVp) and anchored to the cell membrane. The other component is fused with TEVp.

When intracellular Ca²⁺ concentration increases, Ca²⁺ binds to CaM, inducing a conformational change that recruits M13/M2, bringing the two separate structural modules into close proximity. At this point, exposure to blue light triggers a conformational change in the LOV domain, exposing the hidden TEVp cleavage site. The subsequent TEVp-mediated cleavage releases tTA from the membrane, allowing it to enter the nucleus and activate the expression of target genes. This process enables light-controlled, calcium-dependent activity labeling.

2. Example Application

Case 1:

On December 15, 2023, the research team led by Sheena A. Josselyn at the University of Toronto published a study titled “Examining memory linking and generalization using scFLARE2, a temporally precise neuronal activity tagging system” in the journal Cell Reports [2].

In this study, researchers injected AAV-scFLARE2 and AAV-TRE-mCherry viruses into the lateral amygdala (LA) and allowed expression for four weeks. They paired auditory conditioned stimuli (CS) with footshock unconditioned stimuli (US) during training to induce fear responses in mice in response to the tone. During the training, mice were exposed to blue light (BL) of varying durations (3 or 10 minutes) and frequencies (0.25 or 20 Hz). Memory tests were conducted 24 hours later, and cFos immunostaining was used to identify neurons active during memory retrieval.

The results demonstrated that even applying BL for as short as 3 minutes during training, scFLARE2 effectively labeled active neurons in the LA. This finding highlights the shorter time window of scFLARE2 compared to IEG-based labeling systems.

Figure 1: Using scFLARE2 to label active lateral amygdala neurons in a single threat memory experiment

The researchers further used scFLARE2 and TRE-eNpHR3.0-mCherry to label active neurons during auditory threat training by applying blue light (BL). During the test phase, they applied red light (RL) to inhibit scFLARE2-labeled neurons expressing the RL-sensitive inhibitory opsin eNpHR3.0. Neurons active during the test phase were identified using cFos immunohistochemistry.

The results showed that RL inhibition of scFLARE2-labeled neurons reduced cFos expression and disrupted memory retrieval during the test phase.

Figure 2: Functional validation of scFLARE2's effective labeling in a single threat memory experiment using optogenetics

Case 2

On November 15, 2024, the research team led by C. K. Kim from the Department of Neuroscience at the University of California published an article titled “Isolation of psychedelic-responsive neurons underlying anxiolytic behavioral states” in the journal Science [3].

Using GRIN lens-based calcium imaging, the researchers observed that the injection of the psychedelic compound 2,5-dimethoxy-4-iodoamphetamine (DOI) immediately increased the activity of approximately 54% of neurons in the medial prefrontal cortex (mPFC). To further investigate the transcriptomic characteristics of DOI-activated neuronal populations, the team injected AAV-scFLARE2 and AAV-TRE-GFP into the mPFC of mice and implanted optical fiber cannulas for blue light delivery to label DOI-activated mPFC neurons.

Mice were treated with DOI or saline, followed by 15 minutes of blue light stimulation to drive scFLARE2 labeling of activated neurons. The mice were sacrificed 24 hours later for histological analysis. Results showed that mice injected with DOI expressed more GFP+ cells compared to saline-treated control mice, with approximately 40% of scFLARE2-expressing cells activated in DOI-treated mice.

Figure 3: Labeling DOI-activated neurons using scFLARE2

To investigate whether reactivation of DOI-labeled neurons has anxiolytic effects, the researchers used scFLARE2 to label DOI-activated neurons and injected the excitatory optogenetic virus AAV-TRE-bReaChES into the mPFC region. The results showed that, compared to the saline group, DOI-treated mice exhibited reduced burying behavior 30 minutes after treatment, though this effect did not persist into the next day. However, on the second day, optogenetic activation of DOI-labeled neurons significantly reduced burying behavior, and the time spent by mice in the open arms of the EPM (elevated plus maze) was significantly increased. These results indicate that light stimulation of DOI-activated neurons in the mPFC 24 hours after drug administration is sufficient to reproduce the acute anxiolytic effect of the drug.

Figure 4: Reactivation of DOI-labeled neurons exerts anxiolytic effects without inducing a psychedelic state.

References:
[1] Wang W, Wildes CP, Pattarabanjird T, Sanchez MI, Glober GF, Matthews GA, Tye KM, Ting AY. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons. Nat Biotechnol. 2017 Sep;35(9):864-871. doi: 10.1038/nbt.3909. Epub 2017 Jun 26. PMID: 28650461; PMCID: PMC5595644.
[2] Jung, J. H., Wang, Y., Rashid, A. J., Zhang, T., Frankland, P. W., & Josselyn, S. A. (2023). Examining memory linking and generalization using scFLARE2, a temporally precise neuronal activity tagging system. Cell reports, 42(12), 113592. https://doi.org/10.1016/j.celrep.2023.113592
[3] Muir J, Lin S, Aarrestad IK, Daniels HR, Ma J, Tian L, Olson DE, Kim CK. Isolation of psychedelic-responsive neurons underlying anxiolytic behavioral states. Science. 2024 Nov 15;386(6723):802-810. doi: 10.1126/science.adl0666. Epub 2024 Nov 14. PMID: 39541450.

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