Anal. Chem. 2005, 77, 818-826
Chronoamperometry To Determine Differential Reductions in Uptake in Brain Synaptosomes from Serotonin Transporter Knockout Mice Xiomara A. Perez† and Anne M. Andrews*,†,‡
Department of Chemistry and Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802-4615
The serotonin transporter (SERT) is a neuronal plasma membrane protein whose primary function is to take up the neurotransmitter serotonin from the extracellular space, thereby controlling the spatial and temporal aspects of serotonergic signaling in the brain. In humans, a commonly expressed genetic variant of the serotonin transporter gene results in 40% reductions in SERT expression that have been linked to increases in anxietyrelated personality traits and susceptibility to stressassociated depression. Mice have been engineered to express similar reductions in SERT expression to investigate transporter-mediated control of serotonin neurotransmission and behavior. We employed carbon fiber microelectrode voltammetry (chronoamperometry) to examine serotonin clearance rates in brain liposomes (synaptosomes) prepared from mice with 50% (SERT+/-) or complete (SERT-/-) loss of SERT expression. Initial characterization of uptake showed that transport of serotonin was enhanced in the presence of oxygen and abolished when synaptosomes were stirred. Additionally, uptake was prevented by inclusion of the serotoninselective reuptake inhibiting drug paroxetine in the incubation medium. Most notably, unlike prior studies using established radiochemical methods in synaptosomes, we determined 60% reductions in serotonin uptake rates in SERT+/- mice in two different brain regionssstriatum and frontal cortex. Serotonin uptake was not detected in either brain region in SERT-/- mice. Thus, electroanalytical methods offer distinct advantages stemming from excellent temporal resolution for determining transporter kinetics. Moreover, these appear necessary for delineating moderate but biologically important changes in neurotransmitter transporter function. Advances in genetic engineering techniques over the last 30 years have spawned numerous genetically modified animal strains that are important for agriculture and medicine.1-5 Genetically * To whom correspondence should be addressed. Phone: (814) 865-2970. Fax: (814) 863-5319. E-mail:
[email protected]. † Department of Chemistry. ‡ Huck Institutes of the Life Sciences. (1) Tecott, L. H.; Wehner, J. M. Arch. Gen. Psychiatry 2001, 58, 995-1004. (2) Wheeler, M. B.; Walters, E. M.; Clark, S. G. Anim. Reprod. Sci. 2003, 79, 265-289.
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modified mice, in particular, have become indispensable tools in medical research for increasing our understanding of the contributions of various genes to disease states and their treatment. In one such model, mice have been engineered to express reduced amounts of the serotonin transporter.6 The serotonin (5-hydroxytryptamine, 5-HT) neurotransmitter system is known to modulate many critical functions including mood, anxiety states, motor activity, and cognitive function.7-10 The serotonin transporter (SERT) is a presynaptic 68-kDa transmembrane protein that regulates the concentration of serotonin in the extraneuronal space by taking serotonin back up into presynaptic neurons after it has been released.11 SERT is also the molecular target for many therapeutic drugs used in the treatment of depression and anxiety disorders (i.e., Prozac, Paxil, Luvox, Celexa, and Lexapro),12-15 as well as recreationally abused substances including cocaine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy).6,7,16,17 The former, which are collectively referred to as serotonin-selective reuptake inhibitors (SRIs), constitute the most widely prescribed class of antidepressant/antianxiety drugs. These drugs prevent serotonin inward transport from the extracellular space by blocking the transporter.18-20 Long-term treatment with (3) Shashikant, C. S.; Ruddle, F. H. Curr. Issues Mol. Biol. 2003, 5, 75-98. (4) Petters, R. M.; Sommer, J. R. Transgenic Res. 2000, 9, 347-351; discussion 345-346. (5) Di Berardino, M. A. Differentiation 2001, 68, 67-83. (6) Bengel, D.; Murphy, D. L.; Andrews, A. M.; Wichems, C. H.; Feltner, D.; Heils, A.; Mossner, R.; Westphal, H.; Lesch, K. P. Mol. Pharmacol. 1998, 53, 649-655. (7) Sora, I.; Hall, F. S.; Andrews, A. M.; Itokawa, M.; Li, X. F.; Wei, H. B.; Wichems, C.; Lesch, K. P.; Murphy, D. L.; Uhl, G. R. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5300-5305. (8) Murphy, D. L. Neuropsychopharmacology 1990, 3, 457-471. (9) Hornykiewicz, O. Neurology 1998, 51, S2-9. (10) Coccaro, E. F.; Murphy, D. L. Serotonin in Major Psychiatric Disorders; American Psychiatric Press: Washington, DC, 1990. (11) Blakely, R. D.; De Felice, L. J.; Hartzell, H. C. J. Exp. Biol. 1994, 196, 263281. (12) Sheehan, D. V. J. Clin. Psychiatry 1999, 60 (Suppl 18), 16-21. (13) Liebowitz, M. R. J. Clin. Psychiatry 1999, 60 (Suppl 18), 22-26. (14) Goodman, W. K. J. Clin. Psychiatry 1999, 60 (Suppl 18), 27-32. (15) Keller, M. B. J. Clin. Psychiatry 1999, 60 (Suppl 17), 41-45; discussion 46-48. (16) Gough, B.; Imam, S. Z.; Blough, B.; Slikker, W., Jr.; Ali, S. F. Ann. N. Y. Acad. Sci. 2002, 965, 410-420. (17) Callaway, C. W.; Wing, L. L.; Geyer, M. A. J. Pharmacol. Exp. Ther. 1990, 254, 456-464. (18) Frazer, A. J. Clin. Psychiatry 1997, 58 (Suppl 6), 9-25. (19) Frazer, A. J. Clin. Psychiatry 2001, 62 (Suppl 12), 16-23. 10.1021/ac049103g CCC: $30.25
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SRIs (weeks-months) causes sustained reductions in serotonin uptake that most likely lead to prolonged increases in extraneuronal serotonin concentrations.18,21,22 This fundamental homeostatic alteration in serotonin neurotransmission is thought to lead to additional adaptive responses in the brain that ultimately underlie the therapeutic efficacy of the SRIs.23-25 In addition to pharmacologic modulation of the serotonin transporter, genetically controlled alterations in SERT occur in humans. A polymorphic element (alternate DNA base sequence) was discovered in the promoter region of the human SERT gene26 that results in a 40% decrease in SERT expression in ∼70% of the normal population.27 This decrease in SERT has been correlated with an increase in anxiety-related personality traits, particularly neuroticism in men and women.27-31 An increase in anxiety-like behavior also occurs in mice expressing reduced amounts of SERT.32-34 Thus, SERT knockout mice, particularly those with 50% reductions in SERT expression, appear to model closely the genetic variability in SERT that occurs in humans.35 The use of electroanalytical methods to investigate brain chemistry originated in the laboratory of Adams in 1967;36,37 however, reports on the specific use of chronoamperometry for neurochemical applications did not begin to appear until the early 1980s.38-41 Today, the voltammetry techniques most widely utilized to investigate various aspects of neuronal intercellular signaling, in addition to chronoamperometry, include constant potential amperometry and fast-scan cyclic voltammetry (for reviews, see refs 42-44). Together, these methods most often have been used to study the catecholamine neurotransmitter dopamine. Serotonin, (20) Benmansour, S.; Owens, W. A.; Cecchi, M.; Morilak, D. A.; Frazer, A. J. Neurosci. 2002, 22, 6766-6772. (21) Fuller, R. W. Life Sci. 1994, 55, 163-167. (22) Nestler, E. J. Biol. Psychiatry 1998, 44, 526-533. (23) Nibuya, M.; Nestler, E. J.; Duman, R. S. J. Neurosci. 1996, 16, 2365-2372. (24) Murphy, D. L.; Andrews, A. M.; Wichems, C. H.; Li, Q.; Tohda, M.; Greenberg, B. J. Clin. Psychiatry 1998, 59 (Suppl 15), 4-12. (25) Blier, P.; de Montigny, C. Trends Pharmacol. Sci. 1994, 15, 220-226. (26) Heils, A.; Teufel, A.; Petri, S.; Stober, G.; Riederer, P.; Bengel, D.; Lesch, K. P. J. Neurochem. 1996, 66, 2621-2624. (27) Lesch, K. P.; Bengel, D.; Heils, A.; Sabol, S. Z.; Greenberg, B. D.; Petri, S.; Benjamin, J.; Muller, C. R.; Hamer, D. H.; Murphy, D. L. Science 1996, 274, 1527-1531. (28) Greenberg, B. D.; Li, Q.; Lucas, F. R.; Hu, S.; Sirota, L. A.; Benjamin, J.; Lesch, K. P.; Hamer, D.; Murphy, D. L. Am. J. Med. Genet. 2000, 96, 202216. (29) Schinka, J. A.; Busch, R. M.; Robichaux-Keene, N. Mol. Psychiatry 2004, 9, 197-202. (30) Osher, Y.; Hamer, D.; Benjamin, J. Mol. Psychiatry 2000, 5, 216-219. (31) Sen, S.; Burmeister, M.; Ghosh, D. Am. J. Med. Genet. 2004, 127B, 8589. (32) Holmes, A.; Lit, Q.; Murphy, D. L.; Gold, E.; Crawley, J. N. Genes Brain Behav. 2003, 2, 365-380. (33) Holmes, A.; Hariri, A. R. Genes Brain Behav. 2003, 2, 332-335. (34) Holmes, A.; Murphy, D. L.; Crawley, J. N. Biol. Psychiatry 2003, 54, 953959. (35) Murphy, D. L.; Uhl, G. R.; Holmes, A.; Ren-Patterson, R.; Hall, F. S.; Sora, I.; Detera-Wadleigh, S.; Lesch, K. P. Genes Brain Behav. 2003, 2, 350364. (36) Hawley, M. D.; Tatawawadi, S. V.; Piekarski, S.; Adams, R. N. J. Am. Chem. Soc. 1967, 89, 447-450. (37) Adams, R. N. Anal. Chem. 1976, 48, 1126A-1138A. (38) Salamone, J. D.; Lindsay, W. S.; Neill, D. B.; Justice, J. B. Pharmacol. Biochem. Behav. 1982, 17, 445-450. (39) Schenk, J. O.; Miller, E.; Rice, M. E.; Adams, R. N. Brain Res. 1983, 277, 1-8. (40) Blakely, R. D.; Duvarney, R. C. Brain Res. Bull. 1983, 10, 315-320. (41) Hefti, F.; Felix, D. J. Neurosci. Methods 1983, 7, 151-156. (42) Michael, D. J.; Wightman, R. M. J. Pharm. Biomed. Anal. 1999, 19, 3346.
like dopamine, is oxidized at potentials of 0.99) and cation selectivity ratios greater than 700:1 were used in subsequent experiments. Synaptosome Preparation and Uptake Experiments. Synaptosomes were prepared using a modification of the technique previously described by Hyde and Bennett56 to replicate the conditions used for the original radiometric uptake studies in SERT knockout mice.6 For each day’s experiments, bilateral striata and frontal cortex from 6 mice (2 per genotype) were quickly dissected by hand over ice. Tissues were pooled by brain region and genotype and homogenized in 10 volumes of Tris-sucrose buffer (0.5 mM Tris-HCl, 0.32 mM sucrose, pH 7.4) with a Teflon pestle. Homogenates were centrifuged at 2000g for 10 min. The resulting supernatants were removed carefully and centrifuged at 16000g for 10 min. The pellets then were resuspended in 40 volumes of assay buffer (124 mM NaCl, 1.80 mM KCl, 1.24 mM KH2PO4, 1.40 mM MgSO4, 2.50 mM CaCl2, 26.0 mM NaHCO3, 10.0 mM glucose, saturated with 95% O2/5% CO2, pH 7.4 with phosphoric acid). A 50-µL sample of each homogenate was reserved for protein analysis according to the method of Lowry et al.57 Precalibrated microelectrodes and reference electrodes were placed in synaptosomes in a final volume of 2 mL. A +0.55-V pulsed potential was applied, and the current was recorded until a stable baseline was obtained. Serotonin (100 µL) was added to each synaptosomal solution to yield a final concentration of 1.0 µM, and the change in current with respect to time was recorded and compared for each of the three genotypes of SERT knockout mice. Where indicated, synaptosomal preparations were preincu(54) Hoffman, A. F.; Gerhardt, G. A. J. Neurochem. 1998, 70, 179-189. (55) Gerhardt, G. A. In Neuromethods; Boulton, A., Baker, G., Adams, R. N., Eds.; Humana Press: Totowa, NJ, 1995; Vol. 27, pp 117-151. (56) Hyde, C. E.; Bennett, B. A. Brain Res. 1994, 646, 118-123. (57) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275.
bated with paroxetine for 30 min (1.0 µM final concentration). Prior to the addition of serotonin, synaptosomes were centrifuged briefly at 16000g and diluted in fresh oxygenated assay buffer. Uptake rates are expressed as picomoles of 5-HT taken up per milligram of protein per minute. Uptake rates are reported as “not detectable” when TC (defined below) was
