Science | Achieve Cell-Resolution Integrated Stress Response State Whole Brain Imaging

Written | Xiao Yin Responsible Editor | Zyme Integrated Stress Response (ISR) is a highly conserved biochemical pathway that will significantly change the newly synthesized protein components after activation.

The important role of ISR in protein homeostasis, synaptic plasticity, learning and memory makes this pathway a therapeutic target for systemic and brain diseases [1-3].

Preclinical studies have shown that small molecule ISR inhibitors can enhance certain forms of learning and memory.
Although there is more and more evidence in different behavioral paradigms that ISR has an important regulatory role for learning and memory, people are concerned about the effector cells involved in ISR.
But little is known about the time course of the effect.

In order to specifically analyze this mechanism, Science recently published a research paper titled Cholinergic neurons constitutively engage the ISR for dopamine modulation and skill learning in mice, and reported the SPOTlight two-color report developed by the Nicole Calakos group of Duke University Medical Center.
The system can realize the whole brain imaging of the integrated stress response state with cell resolution.

First, briefly introduce the biochemical pathway of ISR and the working principle of SPOTlight (Figure 1).

Under normal physiological conditions, the ISR signal is generally considered to be regulated by the phosphorylation state of eukaryotic initiation factor 2α (eIF2a).

eIF2 is necessary for the initiation of protein synthesis, and its phosphorylation is mediated by four dedicated eIF2a kinases (PERK, HRI, PKR and GCN2, in response to different stressors); dephosphorylation is catalyzed by two phosphatase complexes .

Phosphorylation of eIF2a leads to a significant reduction in protein synthesis.
However, the overall reduction in protein is accompanied by an increase in translation of upstream open reading frame (uORF)-containing mRNA, most of which activate transcription factor 4 (ATF4) (Figure 1b).

Figure 1.
SPOTlight design principle (a) and ISR biochemical regulation pathway (b) SPOTlight design principle is to translate green (ISR off) or red (ISR on) fluorescent protein from a single transcript in response to different ISR activation states (Figure 1a), the cells in the ISR inhibited state will express green fluorescent protein, see the green background in the whole brain map, while the ISR activated cells will express red fluorescent protein (Figure 2).

The author first used drug interference combined with immunohistochemical IHC to verify that the reporting system can effectively reflect the regulatory status of ISR (ie the phosphorylation status of eIF2a).

Combined with cell type markers, the SPOTlight signal indicates that striatal cholinergic interneurons (CIN) are in a stable and high ISR activation state.

Next, the authors used AAV-mediated genetic manipulation to inhibit the firing activity of CIN, and found that the phosphorylation state of eIF2a was significantly reduced, suggesting that the stable ISR activation state in CIN depends on its tonic firing activity.

Figure 2.
ISR whole brain image presented by SPOTlight.
Next, the author used drugs and AAV-mediated genetic manipulation to inhibit the activation of ISR in CIN, and found that it affected the normal type 2 dopamine receptor (D2R) to regulate the discharge response of CIN, so that The CIN discharge process changes from slower to faster.

The mechanism of this process may be that the ISR state regulates the small conductance calcium-activated potassium channel.

Because cholinergic signals in the striatum have been reported to be involved in the regulation of dopamine release, the authors further wanted to investigate whether the ISR status of CIN would affect the release of dopamine.

To this end, the researchers used dLight1.
2 fluorescent reporter molecules to detect the transient dopamine release in the striatum of acute brain slices under electrical stimulation [4].It was found that in normal brain slices, the application of sulpiride, an antagonist of D2R, enhanced the dLight signal induced by electrical stimulation, indicating that the net effect of D2R in the striatum under normal physiological conditions is to inhibit the release of dopamine.

Using genetic manipulation (ChAT Cre / DIO-CReP) to inhibit the ISR state in the striatum, the application of sulpiride reduced the induced dopamine release, indicating that when ISR is inhibited in CIN, the net effect of D2R is to enhance the release of dopamine.

In general, inhibiting ISR will reverse the modulating effect of D2R on dopamine release.

At the end of the article, the researchers explored whether the ISR status of CIN in the striatum affects learning ability.
Although previous studies have shown that inhibiting ISR can enhance learning ability, previous studies have inhibited ISR at the system or whole brain level [5, 6 ], this article specifically targets the inhibition of the ISR state in CIN.
Through the water maze experiment and the pressing lever response experiment, it is found that inhibiting ISR in CIN can enhance the skill learning ability of mice.

All in all: The researchers used the SPOTlight dual-color reporting system to unexpectedly find that cholinergic interneurons (CIN) in the mouse striatum are in a stable ISR activation state.

The use of genetic and pharmacological manipulation revealed that the ISR signaling pathway in CIN is necessary for the regulation of normal type 2 dopamine receptors (D2R).

Inhibition of ISR can reverse the discharge of CIN regulated by D2R, induce dopamine release and improve skills learning.

Original link: https://doi.
org/10.
1126/science.
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029; pmid: 22153080 Reprint Instructions [Original Article] BioArt Original Articles are welcome to be shared by individuals.
Reprinting is prohibited without permission.
The copyrights of all published works are owned by BioArt.
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Cell 147, 1384–1396 (2011).
doi: 10.
1016/ j.
cell.
2011.
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029; pmid: 22153080 Reprint Instructions [Original Article] BioArt Original Articles are welcome to be shared by individuals.
Reprinting is prohibited without permission.
The copyrights of all published works are owned by BioArt. BioArt reserves all statutory rights and offenders must be investigated.