1. Electrochemical Methodology Elucidates Changes in Synaptic 5-HT Caused by Intermediate Reductions in SERT Expression A.M. Andrews, T.A. Mathews, E.L. Unger, X.A. Perez * Department of Chemistry. Pennsylvania State University, University Park, Pa What can HETEROZYGOTE SERT knockout mice tell us about the neurobiology of human personality? Radiometric Serotonin uptake SERT Expression Extracellular Levels of Serotonin Serotonin Uptake Rates Anxiety-related Behavior Neuroadaptation in Other Neurotransmitter Systems ?? Number of entries and time spent in the closed arms of the plus-maze for (-/-) (n=8), (+/-) (n=8) and (+/+) (n=8, * p<0.02). SERT KO mice are more anxious than wildtype littermates. These data demonstrate that moderate chenges in expression lead to changes in anxiety-related behavior similar to what has been found in human populations. Introduction * The serotonin (5-HT) neurotransmitter system is thought to play a key role in the regulation of mood, anxiety states and motor activity. The presynaptic serotonin transporter (SERT) is the primary means by which the concentration of 5-HT in the extracellular space is modulated and it is the molecular target for the largest class of clinically relevant psychiatric drugs (serotonin reuptake inhibitors; SRIs). * It is known that there is a polymorphism in the SERT gene in the human population. This polymorphism affects SERT mRNA expression and protein expression, serotonin neurotransmission and behavior in humans (Greenberg et al. 1999;Lesch et al. 1996). * To better understand how intermediate changes in serotonin transporter expression affect behavior, genetically altered mice with reduced sertonin transporter expression have been engineered (SERT (+/-) and SERT (-/-) mice). Quantitative autoradiography shows a gene dose-dependent decrease in SERT expression in SERT KO mice. In all brains regions examined, SERT protein was reduced by 50% in SERT (+/-) mice. * Previous [ 3 H]-serotonin uptake data did not show changes in uptake rates in heterozygote vs wiildtype SERT KO mice (Bengel et al. 1998). * Our chronoamperometry data shows: 1. A 50% decrease in 5-HT uptake rates in heterozygote SERT knockout mice (146 ± 8.01 pmole/mg protein/min) as compared to wildtype SERT knockout mice (62.7 ± 3.34 pmole/mg protein/min) was observed in synaptosomes from frontal cortex. 2. Similarly, a decrease in 5-HT uptake rates of 162.2 ± 11 vs. 61.53 ±5.52 pmole/mg protein/min for wildtype and heterozygote, respectively was observed in striatum from SERT knockout mice. * Average DA uptake rates in SERT (+/-) mice (446.45 ± 44.2 pmole/mg protein/min)were decreased by ~40% compared to wildtype mice (317.99 ± 32.1pmole/mg protein/min). * Uptake rates in SERT (-/-) mice (305.43 ±25.8 pmole/mg protein/min) decrease by ~46% compared to wildtype mice * Incubation with 1.0  M paroxetine decreased DA uptake rates in wildtype and SERT (+/-) mice to about the same level as that observed in SERT (-/-) mice (210.60 ± 24 and 224.99 ± 13.3 pmole/mg protein/min, respectively). Paroxetine had no effect on the rate of DA uptake in SERT (- /-) mice ( 306.5 ± 25.7 pmole/mg protein/min) * In striatum, basal (uncorrected) extracellular 5-HT levels were 0.32  0.05 nM in SERT +/+ mice, 0.36  0.05 nM in SERT +/- and 1.4  0.2 nM in SERT -/- mice. * High Potassium stimulation shows extracellular 5-HT levels were elevated across the genotypes. Extracellular 5-HT levels were 2.0  0.3 nM in SERT +/+ mice, 2.1  0.5 nM in SERT +/- and 10  2.2 nM in SERT -/- mice. * No net flux showed a six-fold increase in extracellular 5- HT in SERT (-/-) mice (18.0  2.2 nM) versus wildtype mice (3.4  1.1 nM). * No net flux analysis also demonstrated that SERT (+/-) mice had a three-fold increase in extracellular levels of 5-HT (10.2  1.0 nM) versus wildtype mice. K + Stim. * DA levels were not statistically different in wildtype, SERT(+/-) and SERT (-/-), respectively (3.5±0.5 (n=22), 2.9±0.4 (n=17) and 3.7±0.5 nM (n=19). * DA zero net flux was employed in SERT mice to determine if there were neuroadaptive changes in this system unable to be detected using conventional microdialysis techniques. * DA zero net flux showed no change in “true” extracellular DA levels (6.61 vs. 6.09 nM, WT and (-/-) respectively). To measure small but biologically significant changes in serotonin neurotransmission in SERT (+/-) mice, our research has focused on the use of techniques with high temporal resolution, sensitivity and selectivity; such as no-net flux microdialysis and voltammetry Conclusions. Gene dose-dependent decreases in 5-HT uptake are accompanied by related increases in extracellular 5-HT in SERT (+/-) mice, as well as in SERT (-/-) mice. However, the former changes can only be detected using more sophisticated bioanalytical techniques Acknowledgements This project was supported by a grant from NIH (RO1 MH64756-01). Frequency (Mean) Mean Time (Sec) Bengel, D.; Murphy, D. L.; Andrews, A. M.; Wichems, C. H.; Feltner, D.; Heils, A.; Mossner, R.; Westphal, H.; Lesch, K. P. Molecular Pharmacology 1998, 53, 649-55. Wildtype (+/+) Heterozygote (+/-) Homozygote (-/-) 749.12 Alcian blue staining of sections shown in A-C