This paper describes the structure of the porcine HTR2C transcripts as determined using RT-PCR with cDNA prepared from a pool of mRNA isolated from brain tissue. The full-length open reading frame sequence is most analogous to human transcript ENST00000276198 (reference sequence NM_000868). Although exon 3 of human transcript ENST00000371951 is conserved at the genomic level, we assume that this isoform and its orthologues have limited expression in humans and in other mammals, and may be confined to specific neurons in the brain. Two major splice sites define the 3' end of exon 6: both are canonical splice junctions. The first splice junction generates the short form of the protein, which leads to premature truncation of the polypeptide, the loss of transmembrane domains 6 and 7 and the intracellular region thought to be essential for G protein binding. The position of the splice site is analogous to that in human and rodent genes: however, in each species the truncation of the polypeptide occurs at a unique position within the final coding exon. The second splice junction generates the full length transcript, but the complex post-transcriptional editing mechanism generates a range of different isoforms that modify the ability of the receptor-ligand complex to initiate and perpetuate phospholipase dependent intracellular signalling pathways.
Only one of the four polymorphisms in the transcript was identified in the protein coding region in this study (S275N). This amino acid lies in the linker region between transmembrane domains 5 and 6, at a site not previously implicated in functional changes to HTR2C proteins. Any impact of this change on downstream signalling pathways is unknown.
For each of the two breed backgrounds in this study, three of the four pairs show upregulation of HTR2C gene expression in infanticidal animals. This is consistent with previously published results  using array probe data and qRT-PCR primers from the final coding exon, where a trend for increased expression of HTR2C in infancticidal animals was noted. In this paper, we have looked at both the relative quantity of the longer and shorter isoforms, and the potential impact of editing on protein expression between the two groups. Our results support increased mRNA expression in the hypothalamic samples from the infanticidal animals. It is interesting that there is a more significant difference between the two behavioural states for the relative quantity of the longer isoform since this is potentially the most active variant of the receptor, which can subsequently be modified through the action of the editing process. However, there is no statistically significant difference in the qualitative data we have obtained for the degree of mRNA editing.
One obvious limitation to this study is the small number of available samples from animals of the same genetic background. However, the implication of the three-way ANOVA is that breed may also be relevant in determining the overall expression of the receptor, and the observed breed specific differences in the incidence of abnormal maternal behaviour support such a complex, multifactorial model.
Although qualitative changes have been described in a number of other studies relating to both human and rodent populations with presumed genetic or drug-induced behavioural differences, it should also be noted that most of these have analysed different regions of the brain from that described in this paper. The distribution of HTR2C isoforms is remarkably diverse and dependent upon the neuroanatomical region under investigation . In general, the evidence for editing patterns influencing behaviour has been replicated in studies of material collected from the (pre)frontal cortex in both humans  and rat [17, 18, 20]. All of these papers support a role for the gene in anxiety related behaviours, including some forms of aggression. In contrast, using whole brain mRNA from a murine model of Lesch-Nyhan Disease , Bertelli et al. showed that although the level of the HTR2C transcript is elevated, the pattern of editing per site is not affected, which is entirely consistent with our observations here. Again, this raises the issue of whether significant changes are confined to specific sub-sets of neurones within the brain, and that the explicit role of editing can only be fully understood with more precisely dissected samples.
To date, the only positive correlation between behaviour and serotonin receptors within the hypothalamus is with HTR2A. This receptor is de-sensitized preferentially following prolonged glucocorticoid exposure . This implicates the HTR2A receptor in the dampened HPA axis response following prolonged stress, and specifically the action of HTR2A bearing neurones within the paraventrical nucleus (PVN). However, the HTR2C receptor did not seem to be directly involved in this model.
Although HTR2C has been implicated in genetic studies of human behavioural abnormalities, many of these have focussed on using well described polymorphic markers, either singly or as haplotypes. Results are contradictory, and currently the role of this specific receptor remains inconclusive. At the DNA level, the locus is complex, since the intronic regions of the gene also harbour loci for non-coding micro-RNA (miRNA) species. Such RNA molecules are known to be expressed at a high level in the brain, and regulate the expression of downstream targets. In addition HTR2C has itself been identified as a potential target for miRNAs encoded elsewhere in the genome. With such molecular and regulatory complexity, clarification of the evidence will require further experimental work before the HTR2C region of the X-chromosome can be excluded from the list of candidate loci involved in the control of behaviours. Firstly, the contributions of HTR2C gene expression in different neuroanatomical regions of the porcine brain need to be analysed more precisely, and, secondly, we need more understanding of how the associated miRNAs regulate expression patterns of both the HTR2C locus, and other brain expressed genes.