Cognitive Tagging of Neurons: CREMediated Genetic Labeling and Characterization of the Cells Involved in Learning and Memory

Abstract

In this study, we describe use of Cre-mediated recombination to obtain a permanent genetic labeling of the brain neuronal networks activated during a new experience in animals. This method utilizes bitransgenic Fos-Cre-eGFP mice in which a green fluorescent protein is expressed upon tamoxifen-induced Cre-recombination only in the cells where immediate early gene c-fos expression takes place due to the new experience. We used the classical fear conditioning model to show that ex vivo microscopy of the eGFP protein in Fos-Cre-eGFP mice enables mapping of the neurons of the various brain regions that undergo Cre-recombination during acquisition of a new experience. We exposed the animals to the new environment in brief sessions and demonstrated that double immunohistochemical staining enables a characterization of the types of neocortical and hippocampal neurons that undergo experience-dependent Cre-recombination. Notably, Fos-Cre-eGFP labeled cells appeared to belong to excitatory pyramidal neurons rather than to various types of inhibitory neurons. We also showed that a combination of genetic Cre-eGFP labeling with immunohistochemical staining of the endogenous c-Fos protein allows one to identify and compare the neuronal populations that are activated during two different episodes of new experiences in the same animal. This new approach can be used in a wide spectrum of tasks that require imaging and a comparative analysis of cognitive neuronal networks.

Ex vivo and in vivo imaging of mouse parietal association cortex activity in episodes of cued fear memory formation and retrieval

“…7). This result is consistent with the data about the types of FC-induced Fos-TRAPed neurons in the mouse neocortex (Ivashkina et al, 2018). Also, we visualized dendritic shafts (1-2 μm in diameter) with spines at depth of 5-20 μm under the brain surface ( Fig.…”

“…Besides, the method of targeted recombination in active populations (TRAP) can be used to capture the candidate engram neurons (Guzowski et al, 1999, Josselyn et al, 2015. TRAP is an approach to obtain permanent genetic access to distributed neuronal ensembles that are activated by experiences within a limited time window (Guenthner et al, 2013, Ivashkina et al, 2018. This method uses mice in which the tamoxifen-dependent recombinase Cre ERT2 is expressed in an activity-dependent manner under the Fos promotor control.…”

Imaging of C-fos Activity in Neurons of the Mouse Parietal Association Cortex during Acquisition and Retrieval of Associative Fear Memory

“…Similarly, the method of targeted recombination in active populations (TRAP) was used to capture the candidate engram neurons [ 25 , 32 ]. TRAP is an approach to obtain permanent genetic access to distributed neuronal ensembles that are activated by experiences within a limited time window [ 32 , 33 ]. In Fos-TRAP transgenic mice, the tamoxifen-dependent recombinase CreERT2 is expressed in an activity-dependent manner under the control of the c-fos promoter.…”

“…Nonactive cells do not express CreERT2 and do not undergo recombination, even if TM was injected [ 32 ]. Previously the TRAP approach was used to assess the involvement of neurons genetically captured during context fear conditioning (FC) in subsequent memory retrieval [ 33 , 34 ]. Optogenetic silencing of TRAP-labeled neuronal populations in CA3 or DG prevented the expression of the corresponding memory [ 33 ].…”

“…Previously the TRAP approach was used to assess the involvement of neurons genetically captured during context fear conditioning (FC) in subsequent memory retrieval [ 33 , 34 ]. Optogenetic silencing of TRAP-labeled neuronal populations in CA3 or DG prevented the expression of the corresponding memory [ 33 ]. In the present study, we used c-Fos immunostaining, Fos-EGFP in vivo imaging, and Fos-TRAP to investigate the involvement of PtA cortex in the encoding and retrieval of associative fear memory in mice.…”