Egr1-EGFP transgenic mouse allows in vivo recording of Egr1 expression and neural activity
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
References (40)
- et al.
Motor learning consolidates arc-expressing neuronal ensembles in secondary motor cortex
Neuron
(2015) - et al.
Limitations of the GENSAT Egr1-EGFP transgenic mouse strain for neural circuit activity mapping
Neurosci. Lett.
(2020) - et al.
Why neurons mix: high dimensionality for higher cognition
Curr. Opin. Neurobiol.
(2016) - et al.
GENSAT BAC cre-recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits
Neuron
(2013) - et al.
Imaging calcium in neurons
Neuron
(2012) - et al.
Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations
Neuron
(2013) - et al.
Inverse synaptic tagging of inactive synapses via dynamic interaction of Arc/Arg3.1 with CaMKIIbeta
Cell
(2012) - et al.
Upregulation of the immediate early gene arc in the brains of rats exposed to environmental enrichment: implications for molecular plasticity
Brain Res. Mol. Brain Res.
(2001) - et al.
Simultaneous denoising, deconvolution, and demixing of calcium imaging data
Neuron
(2016) - et al.
Increased expression of the immediate-early gene arc/arg3.1 reduces AMPA receptor-mediated synaptic transmission
Neuron
(2006)
Npas4: linking neuronal activity to memory
Trends Neurosci.
Memory engram cells have come of age
Neuron
Alteration of neuronal firing properties after in vivo experience in a FosGFP transgenic mouse
J. Neurosci.
Sensitive red protein calcium indicators for imaging neural activity
Elife
The memory function of sleep
Nat. Rev. Neurosci.
Activity-induced histone modifications govern Neurexin-1 mRNA splicing and memory preservation
Nat. Neurosci.
Impaired long-term memory and NR2A-type NMDA receptor-dependent synaptic plasticity in mice lacking c-Fos in the CNS
J. Neurosci.
Reverse replay of behavioural sequences in hippocampal place cells during the awake state
Nature
A gene expression atlas of the central nervous system based on bacterial artificial chromosomes
Nature
Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene
Nature
Cited by (3)
Approaches and considerations of studying neuronal ensembles: a brief review
2023, Frontiers in Cellular NeuroscienceNeural mechanism of long-term memory storage and modulation
2023, Kexue Tongbao/Chinese Science BulletinSuv39h1 regulates memory stability by inhibiting the expression of Shank1 in hippocampal newborn neurons
2022, European Journal of Neuroscience