Egr1-EGFP transgenic mouse allows in vivo recording of Egr1 expression and neural activity

https://doi.org/10.1016/j.jneumeth.2021.109350Get rights and content

Highlights

  • Egr1-EGFP expression is correlated with native Egr1 in Egr1-EGFP mice.

  • Egr1-EGFP signals are correlated with task-induced neural activities.

  • Egr1-EGFP signals continuously accumulate during persistent neuronal activation.

Abstract

Background

Immediate-early genes (IEGs) have been serving as markers of active neurons for their rapid responses to stimulation. With the development of IEG-EGFP reporters by the GENSAT project, application of the IEGs have been greatly expanded. However, detailed validations for these systems are still lacking, causing trouble in the interpretation of the fluorescence signals.

New method

In this work, taken Egr1-EGFP transgenic mice as an example, we proposed an improvement for the usage of the Egr1-EGFP reporter system based on detailed validation of its fluorescence signals.

Results

Firstly, the exogenous EGFP mRNA levels were linearly correlated with the endogenous Egr1 mRNA levels in neurons. Secondly, the 3-hr-changes of the Egr1-EGFP signals before and after the stimulus were positively correlated with the stimulus-induced neuronal activities. Interestingly, persistent neuronal activity patterns in the post-stimulus phase also showed correlation with the stimulus-induced Egr1-EGFP signal changes. Furthermore, enriched environments engaged dramatic neuronal activations, allowing detailed characterization of Egr1-EGFP expression dynamics.

Comparison with existing method(s)

People used to infer the neuronal activities based on the raw fluorescence signals of IEG-EGFP reporter system, which was strongly obstructed by distinct protein regulation or dynamic properties between the EGFP and the IEGs. We demonstrated a better way for data analysis and experimental design.

Conclusions

Taken together, this work proves that Egr1-EGFP signal is weakly but significantly correlated to task-induced neural activity and gives detailed characterization of the signal dynamics. It not only provides basis for the understanding of the IEG-EGFP fluorescence signals but also offers instructions for proper experimental design with IEG-EGFP reporter systems.

Introduction

Neuronal activities in brain are rather complex and high-dimensional (Fusi et al., 2016, Rigotti et al., 2013), which requires joint efforts of diverse activity recording systems with multiple temporal and spatial scales. Immediate-early genes (IEGs) are genes that rapidly respond to neuronal activity (Greenberg and Ziff, 1984) and have been using as important markers of active neurons (Guzowski et al., 1999, Minatohara et al., 2015, Reijmers et al., 2007). Traditionally, these IEGs have been intensively studied for their physiological function (Jones et al., 2001, Fleischmann et al., 2003, Rial Verde et al., 2006, Sun and Lin, 2016). With the development of IEG fluorescence reporter systems by the GENSAT project (Gong et al., 2003, Heintz, 2004), the application of IEGs has been extended into in vivo studies in rodents, regarding various behavior tasks (Xie et al., 2014, Wang et al., 2019, Cao et al., 2015, Kim et al., 2016, Okuno et al., 2012, Barth et al., 2004). Despite the wide and thorough usage of tools with high temporal resolution, such as calcium imaging (Grienberger and Konnerth, 2012, Pnevmatikakis et al., 2016), the applications of techniques with low temporal resolution but large spatial scales, such as IEG-based reporter systems, are relatively limited in in vivo studies.

Recently, memory engrams have been identified in multiple brain regions by the IEG-based labeling and optogenetic manipulation of task-activated neuronal ensembles (Liu et al., 2012, Tonegawa et al., 2015, Guenthner et al., 2013). Researchers have been refocusing on the potential usage of IEGs and reassessing the GENSAT IEG-reporter systems (Demchuk et al., 2020, He et al., 2019, Li et al., 2019a, Sun et al., 2020, Mahringer et al., 2020). However, issues concerning the relationship between neuronal firing and IEG expression still remain, especially in the IEG-EGFP reporting system. While cfos-GFP (Barth et al., 2004), EGFP-Arc (Okuno et al., 2012) and Egr1-EGFP (Xie et al., 2014) systems were designed to visualize the in vivo neuronal activity level by the endogenous fluorescence signals, a recent study reported that the Egr1-EGFP strain did not show activity-dependent EGFP expression (Demchuk et al., 2020). In contrast, another study reported that the Egr1-EGFP signal was correlated positively with neuronal activity (Mahringer et al., 2020). Such conflicts in evidences suggest that more detailed investigation on the relationship between Egr1-EGFP signal and neural activity is needed.

In this study, we validated the correlation between Egr1-EGFP expression and neuronal activities, and further characterized the dynamics of the Egr1-EGFP expression. Firstly, we proved that both the mRNA and protein levels of EGFP driving by the Egr1 promoter is positively correlated with those of the Egr1 expression in each neuron of the Dentate Gyrus (DG), thereby, establishing basis for further validation. Then, combining in vivo imaging of calcium signal and Egr1-EGFP fluorescence in the same set of neurons, we showed that the stimulus-induced 3-h changes of Egr1-EGFP fluorescence signals (ΔF) were positively correlated with the stimulus-induced neuronal calcium activities. Meanwhile, though partially correlated with the neuronal spontaneous activities, cellular Egr1-EGFP fluorescence signal (F) was noisy, due to the residual signals of activated neuron during the past activation period. Interestingly, we found that the cortical activity patterns of calcium events in post-stimulus phase were also highly correlated with the stimulus-induced Egr1-EGFP signal changes, suggesting the existence of sustained activities in those IEG-expressing neurons during post-stimulus period. Finally, we quantified the dynamics of Egr1-EGFP expression using the enriched environment task and characterized the ascending and descending phases of signal changes. Taken together, this work proves the correlation between Egr1-EGFP signals and neuronal activities and provides fundamental basis for the experimental design and data interpretation of IEG-based fluorescence reporter system.

Section snippets

Animals

Animal care was in accordance with the Institutional guidelines of the animal facility of the National Center for Protein Science Shanghai. Protocols were proved by IACUC in the animal facility of the National Center for Protein Science Shanghai. All animals used were male and were housed in groups of 3–5 on a standard 12-h light/12-h dark cycle with food and water ad alibitum. BAC-EGR-1-EGFP (Tg(Egr1-EGFP)GO90Gsat) mouse was originated from the GENSAT project and distributed from Jackson

The mRNA and protein levels of exogenous EGFP is highly correlated with the endogenous Egr1 in the Egr1-EGFP reporter system

A recent study reported the expression differences between Egr1-EGFP protein and the EGR1 protein (Demchuk et al., 2020), which could be caused by their different protein properties or the potential loss of proper activity-dependent EGFP mRNA expression in Egr1-EGFP mouse. To confirm the proper expression of EGFP mRNA driven by Egr1 promoters in Egr1-EGFP transgenic mice (Fig. 1A), we first tested the expression of EGFP and Egr1 at transcriptional level. As the EGFP+ and the EGFP- neurons in

Discussion

Different from the other genes targeted by GENSAT reporter lines (Morales and Hatten, 2006, Gerfen et al., 2013), the IEGs showed fast temporal dynamics which may not be fully achieved when mimicked by the EGFP gene (Demchuk et al., 2020). In the present study, we provided detailed validations of the Egr1-EGFP reporter line. From mRNA level, the Egr1 promoter-driven EGFP mRNA expression was highly correlated with the intrinsic Egr1 level in the DG. From protein level, the induced Egr1-EGFP

Conclusion

In summary, our work proves that the GENSAT Egr1-EGFP transgenic mouse strain is viable and suitable for detecting neuronal activity, which requires proper extraction of activity-induced signal changes through detailed data analysis process. Similar to other activity-reporter systems, the underlying neuronal correlates and detailed dynamic properties of the IEG-based reporter systems are of great importance for accurate interpretation and analysis. Proper understanding of the advantages and

CRediT authorship contribution statement

Guangyu Wang: Investigation, Methodology, Data collection and analysis, Visualization, Writing – original draft. Hong Xie: Methodology, Supervision. Yi Hu: Data collection and analysis. Qinan Chen: Data collection and analysis. Chenhui Liu: Data collection and analysis. Kaiyuan Liu: Data collection and analysis. Yuze Yan: Data collection and analysis. Ji-Song Guan: Supervision, Conceptualization, Funding acquisition, Writing – review & editing.

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgements

This work was financially supported by the National Key Research and Development Program of China (No. 2017YFA0505500), by the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB38040400), by the National Natural Science Foundation of China (NSFC) (Nos. 31671104, 31970903, 31771476, 31930022), by Shanghai ministry of science and technology (No. 19ZR1477400), and by Shanghai Municipal Science and Technology Major Project (No. 2017SHZDZX01). The work was partially

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