Generation and analysis of transgenic mice over-expressing the 5-hydroxytryptamine (5-HT) transporter

Abstract

To explore the regulation and function of the serotonin transporter (5-HTT), transgenic (Tg) mice that over express the human 5-HTT were made. A 500Kb modified yeast artificial chromosome (YAC35D8) containing the human 5-HTT gene and its 5' regulatory sequence was injected into fertilized mouse oocytes, and Tg offspring were identified by PCR. Analysis by in situ hybridisation showed that h5-HTT mRNA is over-expressed in the cortex, hippocampus and mesencephalic raphe nuclei of these animals. RNase protection and RT-PCR showed that the human 5-HTT is expressed in these regions at levels comparable to, or greater than, that of the endogenous 5-HTT whilst the mouse 5-HTT RNA remained unaltered.[³H]-Citalopram, a high affinity 5-HTT ligand, was used to measure the protein expression. [³H]-citalopram binding showed a higher Kd value (/3<0.02) and a 2.3 - 3.5 fold increase in binding site density (R<0.01) in cortical membranes from Tg mice compared to controls. In addition, the Hill coefficient was less than 1 (consistent with the presence of two binding sites corresponding to the human and mouse 5-HTT).Using HPLC, tissue extracts from the brainstem, hippocampus, midbrain, hypothalamus cerebellum and basal ganglia were analysed for 5-HT, DOPAC and 5-HIAA. The transgenic mice show a drop in 5-HT levels in all areas except the cerebellum (-17 to -40%) and a slight increase in 5-HIAA levels (6-17%); although this was only significant in the cortex (42% p<0.05). Thus the 5-HIAA to 5-HT ratio was increased by 27-76% in transgenic animals (p<0.005).Irwin screening for behavioural abnormalities discovered no overt phenotype in transgenic mice. Mice were then treated with MDMA, a psychoactive chemical that acts at the 5-HTT, in a ramped dosing regime (1,3,10,30 mg/kg) one dose per day and horizontal, locomotor activity and body temperature recorded using telemetry equipment. Drug naive transgenic mice were less active during the dark period of the diurnal cycle. There was a shift in the response curve for MDMA suggesting that transgenic mice have an increased sensitivity to MDMA. Transgenic mice showed an altered thermoregulatory response, with a pronounced hypothermia (reaching -8°C in one case) occurring within 7 hrs of administration of the 10 mg/kg dose of MDMA in contrast to a slight hyperthermia in wild-type animals.In conclusion these mice show a moderate over-expression of the human 5-HTT, which results in significant alterations in the metabolism of 5-HT. This may provide a useful animal model of the neurochemical disturbances occurring in affective disorder in man.