Generation And Phenotype Analysis Of Dopamine Receptor D5 And D5b Transgenic Rats

We have cloned a new subtype of human dopamine D5 receptor(D5b).The sequence of human D5B receptor is 98%identical to human D5 receptor.It contains a long open reading frame that encodes a functional protein,which is one amino acid longer than human D5 receptor.The pharmacological study showed that the D5b receptor had distinct pharmacological properties from D5 receptor.In order to study the function of D5 and D5b receptor,we have generated forebrain-specific transgenic rats overexpressing human D5b receptors driven by CamKⅡαpromoter.The receptors were specifically expressed in the forebrain of transgenic rats at both mRNA and protein levels.We used molecular biology,biochemistry and behavioral techniques to systematically analyze the effects of D5/D5b gene overexpression restricted to the forebrain on learning and memory abilities of rats.The main objectives are as follows:1 The expression of human D5 or D5b receptor in the forebrain of transgenic ratsReversed transcription-PCR,in situ hybridization strategies and western blot were applied to identify the expression of human D5/D5b gene.Data showed that the D5/D5b gene was overexpressed in cortex and hippocampus in transgenic rats.2 Phenotyping analysis of D5 and D5b transgenic ratsHE staining was performed on the brain slices from WT,D5 and D5b transgenic rats.The results revealed that,the morphologies of forebrain regions of D5 and D5b transgenic rats were similar to that of wild type control.Numerous studies have demonstrated that dopamine D5 receptor is associated with learning and memory.To investigate the effects of D5 and D5b gene overexpression to learning and memory,we used behavioral studies,including novel object recognition,water maze and fear conditioning to analyze the two transgenic rats.Our results showed that both D5 and D5b rats exhibited impairment in memory. This indicated that D5 and D5b subtypies overexpressed in the forebrain negatively regulate memory. 3 Gene expression changes in the hippocampus of D5 and D5b transgenic ratsIn comparison to wild type control,65 genes and 215 genes were found to change their expresstion levels respectively in the hippocampus of D5 and D5b transgenic rats.There are 202 genes whose expressions show alternations between D5b and D5 transgenic rats.Functional analysis revealed that these genes were related to metabolism,signal transduction,transcription,transport,cytoskeleton and others. Several critical hormone encoding genes such as growth hormone 1(Gh1),prolactin (Pr1),Proopiomelanocortin(POMC),and synaptic transmission related gene synaptotagmin XIV(Syt14),were down-regulated.Real-time PCR was performed and obtained consistent results.Taken together,these results suggested that the impairment of memory in D5 and D5b transgenic rats may,at least partially,due to the changed expression levels of the hormone encoding genes.On the other hand, tryptophan hydroxylase(Tph1) gene expression level was higher in the hippocampus of D5 rats rather than in D5b rats,which plays an important role in serotonin biosynthesis and leave the possibility that the differences between D5 and D5b transgenic rats may lie in the Tph1-associated function such as emotion,sleep and psychology.