TY  - JOUR
AU  - Anokhin, Konstantin
AU  - Subach, Fedor
AU  - Subach, Oksana
AU  - Saidov, Khalid
AU  - Tiunova, Anna
PY  - 2019
DA  - 2019/03/04
TI  - New Tools in Cognitive Neurobiology: Biotin-Digoxigenin Detection of Overlapping Active Neuronal Populations by Two-Color <i>c-fos</i> Compartment Analysis of Temporal Activity by Fluorescent <i>in situ</i> Hybridization (catFISH) and c-Fos Immunohistochemistry
JO  - OBM Genetics
SP  - 065
VL  - 03
IS  - 01
AB  - Background: The method of cellular compartment analysis of temporal activity by fluorescent in situ hybridization (catFISH) is widely used in cellular and behavioral neurobiology. This technique exploits stimulus-induced expression of immediate early genes (IEGs) and allows identification of two neuronal populations activated in the brain of the same animal in response to neural or behavioral events separated by 25–30 min. The differential labeling is based on the visualization of nuclear RNA and cytoplasmic mRNA, which accumulates in neurons at different intervals after their activation. However, the drawback of this method is that the two RNA forms are labeled by the same color, which complicates their discrimination during image analysis. Methods: We developed a protocol for a two-color c-fos catFISH that allows visualization of cytoplasmic (stimulus 1) and nuclear (stimulus 2) RNA using the combination of digoxigenin- and biotin-conjugated probes. This approach was used to detect the intracellular localization as well as the spliced and unspliced forms of c-fos RNA by color-coded labeling. We also demonstrated the feasibility of combining this protocol with immunofluorescent detection of c-Fos protein, which allows visualization of two neuronal populations activated by stimuli applied 30 to 60 min apart. Results: We used the developed two-color c-fos catFISH protocol to identify neuronal populations activated in the mouse brain by two cognitive stimuli. We identified two color signals that corresponded to cytoplasmic and nuclear c-fos RNA in different brain regions after mice were exposed to two different stimuli separated by 20 min. The two-color c-fos catFISH protocol was also combined with с-Fos immunohistochemistry, allowing colocalization of c-fos mRNA and с-Fos protein activated by two stimuli applied 60 min apart. Conclusions: The combination of the proposed approaches allows visualization of neuronal populations activated in the brain of the same mouse during two or more cognitive episodes in the time window of 60–120 min.
SN  - 2577-5790
UR  - https://doi.org/10.21926/obm.genet.1901065
DO  - 10.21926/obm.genet.1901065
ID  - Anokhin2019
ER  -