Electrochemical Methodology Elucidates Changes in Synaptic 5-HT Caused by Intermediate Reductions in SERT Expression

1. Bengel, D.; Murphy, D. L.; Andrews, A. M.; Wichems, C. H.; Feltner, D.; Heils, A.; Mossner, R.; Westphal, H.; Lesch, K. P. Molecular Pharmacology1998,53, 649-55. K+ Stim. Wildtype (+/+) Heterozygote (+/-) Homozygote (-/-) Mean Time (Sec) Frequency (Mean) 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 749.12 What can HETEROZYGOTE SERT knockout mice tell us about the neurobiology of human personality? • 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). * 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. Neuroadaptation in Other Neurotransmitter Systems ?? Anxiety-related Behavior SERT Expression Extracellular Levels of Serotonin Alcian blue staining of sections shown in A-C Serotonin Uptake Rates * 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). Radiometric Serotonin uptake 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. 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 * 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) * Previous [3H]-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. 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. 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).