Editing of serotonin 2C receptor mRNA in the prefrontal cortex characterizes high-novelty locomotor response behavioral trait

Abstract

Serotonin 2C receptor (5-HT(2C)R) exerts a major inhibitory influence on dopamine (DA) neurotransmission within the mesocorticolimbic DA pathway that is implicated in drug reward and goal-directed behaviors. 5-HT(2C)R pre-mRNA undergoes adenosine-to-inosine editing, generating numerous receptor isoforms in brain. As editing influences 5-HT(2C)R activity, individual differences in editing might influence dopaminergic function and, thereby, contribute to interindividual vulnerability to drug addiction. Liability to drug-related behaviors in rats can be predicted by their level of motor activity in response to a novel environment. Rats with a high locomotor response (high responders; HRs) exhibit enhanced acquisition and maintenance of drug self-administration compared to rats with a low response (low responders; LRs). We here examined 5-HT(2C)R mRNA editing and expression in HR and LR phenotypes to investigate the relationship between 5-HT(2C)R function and behavioral traits relevant to drug addiction vulnerability. Three regions of the mesocorticolimbic circuitry (ventral tegmental area (VTA), nucleus accumbens (NuAc) shell, and medial prefrontal cortex (PFC)) were examined. 5-HT(2C)R mRNA expression and editing were significantly higher in the NuAc shell compared with both the PFC and VTA, implying significant differences in function (including constitutive activity) among 5-HT(2C)R neuronal populations within the circuitry. The regional differences in editing could, at least in part, arise from the variations in expression levels of the editing enzyme, ADAR2, and/or from the variations in the ADAR2/ADAR1 ratio observed in the study. No differences in the 5-HT(2C)R expression were detected between the behavioral phenotypes. However, editing was higher in the PFC of HRs vs LRs, implicating this region in the pathophysiology of drug abuse liability.

Fig. 1. Illustration of dissections

Twenty micron thick thionin stained sections before and after dissection of regions of interest. For photography, tissue was covered with 100% alcohol and temporarily cover-slipped. Only one hemisphere is shown before dissection and the mirror image of that hemisphere is shown after dissection. a) Level of the PFC dissection. The PFC dissections contained primarily Cg3 and IL. b) Level of the NuAc dissection. The NuAc dissections were taken from its shell region at the level of the gcc. c) Level the VTA dissection. The VTA was dissected only at levels where the fr could be seen. Abbreviations: a, anterior commissure, Acb, nucleus accumbens; AOP, accessory olfactory nucleus posterior; Cg3, cingulate cortex area 3; DP, dorsal peduncular cortex; IL, infralimbic cortex; fmi, forceps minor corpus callosum; fr, fasciculus retroflexus; gcc, genu corpus callosum; LV, lateral ventricle; ml, medial lemniscus; mp, mammillary peduncle; MT, medial terminal nucleus accessory optic tract; S, septal area; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulate.

Fig. 2. Region-specific frequencies of the two most prevalent 5-HT_2CR mRNA variants (ABDandABCD, panel A) and the two most prevalent 5-HT_2CR protein isoforms (VNV and VSV, panel B) in HRs and LRs

Shown are Means±SEM. ABCD and VSV were significantly increased in the PFC of HRs compared to LRs (*p<0.05)

Fig. 3. Regional differences of 5-HT_2CR editing in the rat brain

Shown are Means±SEM of editing efficiencies at the five editing sites (panel A), frequencies of the mRNA variants (panel B), and frequencies of the predicted protein isoforms (panel C) in three different brain regions. All editing parameters are compared for the entire cohort (HRs and LRs combined). Shown are only the variants and isoforms that were observed at greater than ~ 5% frequencies. (^#p<0.02; *p<0.0001).

Fig. 4. Analysis of the 5-HT_2CR mRNA expression

mRNA expression was measured by qPCR. Shown are Means±SEM. 5-HT_2CRsp2 and 5-HT_2CRsp1 transcripts result from splicing at sp2 and sp1sites and encode functional and truncated non-functional receptor, respectively. 5-HT_2CRsp2 and 5-HT_2CRsp1 expression levels were significantly different between pairs of regions (all p values <0.0001), but did not differ between HRs and LRs in any region.

Fig. 5. Analysis of the ADAR1 and ADAR2 mRNA expression

mRNA expression was measured by qPCR. Shown are Means±SEM. Two different TaqMan assays were employed to distinguish between the ADAR2 transcripts that encode functional (ADAR2f) and truncated non-functional (ADAR2nonf) proteins. ADAR1, ADAR2f, and ADAR2nonf expression levels were significantly different between pairs of regions (all p values <0.0001), but did not differ between HRs and LRs in any region.

Fig. 6. Ratio between ADAR2f and ADAR1 expression levels

Shown are Means±SEM. The ratios were significantly different between the NuAc shell and the two other regions (*p <0.0001), but did not differ between the PFC and VTA or between HRs and LRs in any region.