Using chronoamperometry to investigate serotonin and dopamine transporter function

The global hypothesis of this thesis is that modest but biologically important alterations in neurotransmission underlie phenotypic alterations in mice (and humans) with altered gene expression. Chronoamperometry with carbon fiber microelectrodes was utilized to investigate changes in serotonin and dopamine uptake in SERT knockout mice and in mutant human A53T alpha-synuclein transgenic mice. Four primary findings are described in detail. The first is that partial reductions in SERT expression lead to similar magnitude decreases in SERT function in mice, similar to what has been shown to occur in humans with differential SERT expression. Characterization of uptake by chronoamperometry in neurosynaptosomes demonstrated that transport of serotonin was enhanced 7-fold in the presence of oxygen and abolished when synaptosomes were stirred. The results indicated that 50% reductions in SERT expression in SERT +/- mice resulted in 60% decreases in serotonin uptake rates measured in brain stem, frontal cortex and striatum compared to SERT +/+ mice. Uptake of serotonin was not detected in SERT-/- mice in any of these brain regions. These findings contradict those from previous radiochemical assay experiments in which differential uptake was not detected between SERT+/+ and SERT+/- mice.; The second main finding addresses the lack of sensitivity of radiochemical methods for detecting differential changes in transporter function in mice with partial reductions in SERT expression. The data indicate that absence of oxygen in the incubation medium and most importantly, the disruption of synaptosomal plasma membranes during the filtration step used in radiochemical assays together contribute to low uptake rates and the poor resolution of this method. Specifically, uptake rates measured by radiochemical methods are 200-fold lower than those determined by chronoamperometry even in the presence of oxygen. In addition, differential uptake rates between SERT +/+ and SERT+/- mice were only observed in frontal cortex and striatum but not in brain stem synaptosomes after oxygenation of the incubation medium in radiochemical uptake experiments. Most striking, the results indicate that the filtration of synaptosomal suspensions results in a 75% loss of serotonin taken up contributing to the lower uptake rates calculated by this method and to its poor ability to detect differential uptake rates between SERT+/+ and SERT+/- mice.; The third major finding is that reductions in SERT expression and function do not lead to neuroadaptive changes in dopamine transporter expression and function. Furthermore, we did not find evidence for promiscuous uptake of dopamine by the SERT in wildtype mice. Thus, behavioral changes in SERT knockout mice appear to be a direct consequence of alterations in serotonergic neurotransmission in SERT knockout mice.; Finally, the results of experiments in alpha-synuclein transgenic mice demonstrate that expression of A53T mutant human alpha-synuclein leads to reduced dopamine uptake rates in striatum. These alterations in dopamine neurotransmission are associated with adult onset locomotor hyperactivity in A53T alpha-synuclein transgenic mice.