In Vivo Investigation of Escitalopram's Allosteric Site on the Serotonin Transporter Open Access

Murray, Karen E (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/cj82k777k?locale=en
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Abstract

Escitalopram is a commonly prescribed antidepressant of the selective serotonin reuptake inhibitor class. Clinical evidence followed by pre-clinical evidence and mapping on the serotonin transporter (SERT) identified that in addition to inhibiting the SERT via a primary site, escitalopram is capable of binding to the SERT via an allosteric site. In vitro studies suggest that the allosteric site alters the kinetics of escitalopram at the SERT. This dissertation examined the in vivo role of the allosteric site in escitalopram action at the SERT. This was completed by developing a knockin mouse model that had an allosteric-null SERT. Autoradiographic studies indicated that the knockin protein was expressed at a lower amount than endogenous mouse SERT, but the knockin mice were a viable tool to study the allosteric site. It was hypothesized that the absence of the allosteric site would result in the need for a higher dose of escitalopram to achieve the same effect seen in mice with intact SERT. Microdialysis studies in the ventral hippocampus found no measurable decrease in the amount of extracellular serotonin after escitalopram challenge in mice without the allosteric site. In marble burying assays there was a modest effect of the absence of the allosteric site, with a larger dose of escitalopram necessary to see the same effect as in mice with intact SERT. In the tail suspension test there was no effect of the presence or absence of the allosteric site. Together these data suggest that there may be a regional specificity in the role of the allosteric site, explaining the modest marble burying effect without matching tail suspension test and microdialysis effects. The knockin mice could be used to explore this further. However, the lack of a robust effect overall suggests that the role of the allosteric site for escitalopram on the SERT does not produce relevant in vivo effects.

Table of Contents

Table of Contents

Chapter 1: Introduction and Background

Depression and Treatments...1 Allosterism...6 Allosteric Site...10 Purpose of Studies...14 Aims and Hypotheses...16 Figures Figure 1: Hypothesis of escitalopram's activity at the SERT...18

Figure 2: Theoretical dose-response curve of the hypothesized effect of escitalopram's allosteric site on the hSERT...19

Chapter 2: Development of hSERT Mice

Necessity of hSERT Knockin Mice...20 Knockin Gene Construct...21 Production of the hSERT colony...22 Maintenance of the hSERT Colony...23

Chapter 3: Autoradiography

Abstract...25 Introduction...26 Materials and Methods...27 Results...30 Conclusions...31 Figures Figure 3: Measured expression of SERT in the cortex...34 Figure 4: Measured expression of SERT in the hippocampus...35 Figure 5: Measured expression of SERT in the raphe nucleus...36 Table Table 1: Autoradiographic measurements in the mouse brain...37 Chapter 4: Microdialysis Abstract...38 Introduction...39 Materials and Methods...41 Results...49 Conclusions...51 Figures Figure 6: Schematic of microdialysis setup...55 Figure 7: Schematic of 6-port valve...56 Figure 8: Microdialysis timeline...57

Figure 9: Percent change from baseline of extracellular serotonin...58

Figure 10: Baseline extracellular serotonin levels...59 Figure 11: Area under the curve of extracellular serotonin...60 Tables Table 2: Serotonin concentrations...61 Table 3: Percent change from baseline...62 Table 4: Area under the curve...63 Chapter 5: Behavior Abstract...64 Introduction...65 Marble Burying...67 Materials and Methods...67 Results...69 Locomotion...71 Materials and Methods...71 Results...72 Tail Suspension Test...72 Materials and Methods...72 Results...74 Conclusions...77 Figures Figure 12: Arial view of marble burying arena...81 Figure 13: Marble burying schematic...82

Figure 14: Pilot test of marble burying with escitalopram oxalate...83

Figure 15: Pilot test of marble burying with fluoxetine HCl...84

Figure 16: Marble burying with escitalopram oxalate in knockin mice...85

Figure 17: Marble burying with fluoxetine HCl in knockin mice...86 Figure 18: Locomotion during pilot escitalopram oxalate MB...87 Figure 19: Locomotion during pilot fluoxetine HCl MB...88 Figure 20: Tail suspension test view...89 Figure 21: Tail suspension test schematic...90

Figure 22: Tail suspension test escitalopram oxalate pilot in C57BL/6J mice...91

Figure 23: Tail suspension test fluoxetine HCl pilot in C57BL/6J mice...92

Figure 24: Tail suspension test fluvoxamine maleate pilot in C57BL/6J mice...93

Figure 25: Tail suspension test with escitalopram oxalate in knockin mice, duration immobile...94

Figure 26: Tail suspension test with fluvoxamine maleate in knockin mice, duration immobile...95

Figure 27: Tail suspension test with escitalopram oxalate in knockin mice, latency to first immobility event...96

Figure 28: Tail suspension test with fluvoxamine maleate in knockin mice, latency to first immobility event...97

Chapter 6: Discussion and Conclusions

Abstract...98 General Discussion...99 Conclusions...102 Future Directions...103 Final Remarks...107 Appendix A: Knockin Gene Insertion...108 Figures

Figure 29: Cartoon of homologous recombination of the hSERT construct into the mouse genome...111

Figure 30: Chimeric pups born summer 2007...112 Appendix B: Genotyping Protocol...113 Figure Figure 31: Representative agarose gel of PCR products...116 References...117

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