Editing of the serotonin 2C receptor pre-mRNA: Effects of the Morris Water Maze

Abstract

The pre-mRNA encoding the serotonin 2C receptor, HTR2C (official mouse gene symbol, Htr2c), is subject to adenosine deamination that produces inosine at five sites within the coding region. Combinations of this site-specific A-to-I editing can produce 32 different mRNA sequences encoding 24 different protein isoforms with differing biochemical and pharmacological properties. Studies in humans have reported abnormalities in patterns of HTR2C editing in psychiatric disorders, and studies in rodents show altered patterns of editing in response to drug treatments and stressful situations. To further explore the biological significance of editing of the Htr2c mRNA and its regulation, we have examined patterns of Htr2c editing in C57BL/6J mice after exposure to the hidden platform version of the Morris Water Maze, a test of spatial learning that, in mice, is also associated with stress. In brains of both swimming controls and mice trained to find the platform, subtle time dependent changes in editing patterns are seen as soon as 1 h after a probe trial and typically last less than 24 h. Changes in whole brain with cerebellum removed differ from those seen in isolated hippocampus and cortex. Unexpectedly, in hippocampi from subsets of mice, abnormally low levels of editing were seen that were not correlated with behavior or with editing levels in cortex. These data implicate responses to spatial learning and stress, in addition to stochastic processes, in the generation of subtle changes in editing patterns of Htr2c.

Figure 1

Primer extension analysis. a) Schematic of primer extension assays. The genomic sequence spanning the edited region is shown in the center, 5' to 3'. The five A residues that can be edited are named A-E in the order indicated. Primer extension reactions 1-5 are shown with the corresponding dideoxynucleotide chain terminator. Horizontal lines indicate positions at which chain termination may occur. Products obtained in each reaction are named for the edited site combination and the corresponding encoded amino acids (underlined). + indicates the site is edited to inosine; − indicates it remains the genomic adenosine. b) Representative gels for reactions 1-5. Bands are labeled by edited status (site + or − editing) and length in nucleotides. P, primer.

Figure 1

Primer extension analysis. a) Schematic of primer extension assays. The genomic sequence spanning the edited region is shown in the center, 5' to 3'. The five A residues that can be edited are named A-E in the order indicated. Primer extension reactions 1-5 are shown with the corresponding dideoxynucleotide chain terminator. Horizontal lines indicate positions at which chain termination may occur. Products obtained in each reaction are named for the edited site combination and the corresponding encoded amino acids (underlined). + indicates the site is edited to inosine; − indicates it remains the genomic adenosine. b) Representative gels for reactions 1-5. Bands are labeled by edited status (site + or − editing) and length in nucleotides. P, primer.

Figure 2

Results of Morris Water Maze with wild type (WT) C57BL/6J and two lines of ADAR2 transgenic mice, Tg14 and Tg15. a) Latency: learning in the MWM is indicated by the decreased amount of time (in seconds) required to find the hidden platform with increasing number of training sessions. Both wild type and transgenic mice learned to locate the hidden platform in approximately 30 seconds after three blocks of training. b) Results of the probe trial (hidden platform removed) of Morris Water Maze test. After two days of training in the MWM, mice were tested immediately in a single probe trial. Learning is assessed by recording the percentage of time spent in the training quadrant (former location of the platform). There is no significant difference between wild type and transgenic mice in preference for the training quadrant (p=0.84, One Way ANOVA). TQ, training quadrant.

Figure 3

A subset of mice show low levels of editing at the A and B sites in hippocampus. a) Proportion of transcripts edited at the A site in 13 naive (N), 9 swimming control (SC) and 9 mice trained to find the platform (WM). Five mice are edited at <30% while 25 are edited at ≥70%. b) Proportion of transcripts edited at both the A and B sites in the same animals. c) Animals with low levels of editing at the A site in hippocampus (Hp) show high levels of editing at the A site in cortex (Cr). d) Animals with low levels of A+B+ editing in hippocampus show high levels of A+B+ in cortex.

Figure 4

Significant changes in editing patterns in hippocampus. a) Levels of A−B+ were affected by treatment (p<0.01). b) Levels of E+C+D+ were affected by treatment (p<0.05) and time (p<0.01). c) Levels of C+D+ were affected by time (p<0.05). d) Levels of transcripts encoding methionine (M) were affected by treatment (p<0.05). e) Levels of transcripts encoding serine-valine (SV) were affected by time (p<0.05).

Figure 5

Significant changes in editing patterns in cortex. a) Levels of A−B+ were affected by time (p<0.01); time and treatment show an interaction (p<0.05). b) Levels of E+C+D+ are affected by time (p<0.05). c) Levels of D+ were affected by treatment (p<0.05). d) Levels of transcripts encoding methionine were affected by time (p<0.05).