Comment on "Multiple repressive mechanisms in the hippocampus during memory formation"

Abstract

Cho et al. (Reports, 2 October 2015, p. 82) report that gene repression after contextual fear conditioning regulates hippocampal memory formation. We observe low levels of expression for many of the top candidate genes in the hippocampus and robust expression in the choroid plexus, as well as repression at 4 hours after contextual fear conditioning, suggesting the inclusion of choroid plexus messenger RNAs in Cho et al. hippocampal samples.

Figure 1. Gene expression analyses in choroid plexus and hippocampus reveal tissue-specific responses to contextual fear conditioning

(A) The ChP of the lateral ventricle (blue) is proximate to the hippocampus (brown), requiring specialized dissection procedures to isolate and separate the tissues. (B–F) C57BL/6N 9-week old male mice, treated in accordance with the protocol approved by the IACUC of Boston Children’s Hospital, were individually housed in a 12h light/dark cycle (7:00/19:00) and handled daily for 3 minutes each, for four consecutive days. Following handling on day 4 (at 10:00), mice were either returned to their home cage (naïve mice), or exposed to foot shock (148 seconds in chamber, 0.6 mA shock for 2 seconds, 30 seconds in chamber, returned to home cage at 180 seconds). Four hours later (at 14:00), ChP and hippocampus from pairs of naïve and trained mice were concurrently isolated, ensuring that each trained mouse had an exact time-matched naïve control. Tissue RNA was purified, analyzed by qPCR (TaqMan, Applied Biosystems, n=3, with each gene-specific measurement and standard performed in triplicate), and the data were used to generate panels B–F. (B) The expression levels of 10 selected mRNAs out of the top 15 differentially-expressed genes reported by Cho et al. (1) are 100–20,000 fold higher in the ChP than in the hippocampus (n=12). (C) High expression of ChP-specific transcripts in the hippocampal samples used by Cho et al. (1). The expression levels of the selected genes in the hippocampal samples were normalized to Neurod6 using material generated in this study (n=12) and by analysis of the Cho et al. (1) data in the GEO Dataset GSE72064 (n=15) [Ttr levels are not plotted; the values are 12% in this study and 3170% in Cho et al. (1)]. (D–F) mRNA levels 4h after foot shock in the lateral and fourth ventricle ChP and in the hippocampus. Data are represented as mean ± S.E.M.; n=3; *p<0.05, one-way ANOVA with repeated measures for ChP samples, t-test for hippocampal samples. Ttr expression was not affected by foot shock in any of the tissues. Of note, since both the ChP (13) and hippocampus (14) demonstrate circadian gene expression patterns, the magnitude of gene expression changes could vary as a function of time of day used for the naïve control, as well as the time of day that the experiment was performed.