Improved specificity of hippocampal memory trace labeling

Abstract

Recent studies have focused on the identification and manipulation of memory traces in rodent models. The two main mouse models utilized are either a CreER(T2) /loxP tamoxifen (TAM)- or a tetracycline transactivator/tetracycline-response element doxycycline-inducible system. These systems, however, could be improved to label a more specific population of activated neurons corresponding to behavior. Here, we sought to identify an improved selective estrogen receptor (ER) modulator (SERM) in which we could label an individual memory trace in ArcCreER(T2) mice. We found that 4-hydroxytamoxifen (4-OHT) is a selective SERM in the ArcCreER(T2) × Rosa26-CAG-stop(flox) -channelrhodospin (ChR2)-enhanced yellow fluorescent protein (eYFP) mice. The half-life of 4-OHT is shorter than TAM, allowing for more specificity of memory trace labeling. Furthermore, 4-OHT allowed for context-specific labeling in the dentate gyrus and CA3. In summary, we believe that 4-OHT improves the specificity of memory trace labeling and will allow for refined memory trace studies in the future. © 2015 Wiley Periodicals, Inc.

Keywords: Arc; Hippocampus; c-fos; learning; memory.

Figure 1

Characterization of ArcCreER^T2 x ROSA26-CAG-stop^flox-ChR2-eYFP mice. (A) Breeding strategy. Administration of TAM results in an indelible ChR2-eYFP label in the initially activated Arc⁺ cells. (B) Representative image. A single injection of TAM produces a robust ChR2-eYFP in the HPC. (C) The eYFP label is robust, yet there is a background labeling in this line. (D) eYFP⁺ cells corresponding to CFC encoding. (E) c-fos⁺ DG cells corresponding to memory retrieval. (F) Representative co-labeled eYFP⁺/c-fos⁺ cell. Scale bars represent 100 mm.

Figure 2

Comparison of SERMs for eYFP labeling. (A) Experimental design. (B-E) TAM-injected mice have a dense eYFP label. (F-I) 4-OHT-injected mice have a less intense, yet distinct eYFP signal. (J-M) Ral–injected and (N-Q) Ful-injected mice have an extremely dense eYFP label. n = 3–6 mice per group.

Figure 3

4-OHT produces distinct eYFP labeling. (A) Mice administered TAM or 4-OHT exhibited similar levels of freezing during the retrieval test in the CFC chamber. (B) Mice administered TAM and 4-OHT also had comparable number of eYFP⁺ DG cells. (C and E) Mice administered TAM had a background haze staining when compared with (D and F) mice administered 4-OHT. Arrows indicate areas of background haze staining. (G–H) eYFP labeling in the retrosplenial granular cortex following a 4-OHT injection is noticeably less than following a TAM injection. n = 3–9 mice per group. Error bars represent ± SEM.

Figure 4

4-OHT’s recombination efficiency decreases between 12–18 h post injection. (A) Experimental design. (B) The number of eYFP⁺ cells in the DG was significantly lower at 24 and 36 h when compared to the 5 and 9 h time points. The number of eYFP⁺ cells in the DG was significantly lower at 18 h when compared with the 9 h group. n = 3–7 mice per group. Error bars represent + SEM. * p < 0.05; ** p < 0.01, *** p < 0.001.

Figure 5

4-OHT is effective in labeling memory traces. (A) Experimental design. (B) All mice explored context A in a similar manner before shocks were administered. After the shocks were administered, mice froze significantly more than mice given context exposure. (C) During the retrieval test, mice administered CFC exhibited markedly higher levels of freezing than mice exposed to the context without footshock or mice that were administered CFC but then placed into a novel context B. (D) CFC resulted in an increase in the number of eYFP⁺ cells in the DG when compared to mice injected with 4-OHT in their HC. (E) Mice exposed to a context (A or B) had significantly more Arc⁺ cells in the DG than mice left in their HC. (F) The percent of co-labeled eYFP⁺/Arc⁺ cells in the DG was specific to the context experience. (G) CFC resulted in an increase in the number of eYFP⁺ cells when compared to mice injected with 4-OHT in their HC or to mice injected with 4-OHT and given context A exposure. (H) Mice exposed to a context (A or B) had significantly more c-fos⁺ cells in CA3 than mice left in their HC. (I) The percent of co-labeled eYFP⁺/c-fos⁺ cells in CA3 was specific to the context experience. n = 5 mice per group. Error bars represent ± SEM. * p < 0.05; ** p < 0.01, *** p < 0.001.

Figure 6

4-OHT is effective for eYFP labeling when administered following CFC. (A) Mice were administered a 3-shock CFC paradigm and immediately following the training procedure, were injected with 4-OHT. The retrieval test was administered 5 d later. (B) 4-OHT labeling post-CFC produced distinct eYFP labeling in the DG. (C) The number of eYFP⁺ cells per DG section. (D) The number of Arc⁺ cells per DG section. (E) The percent co-labeled eYFP⁺/Arc⁺ cells per DG section. n = 3 mice per group. Error bars represent ± SEM. Scale bar represents 100 µm.