Neurochemical Measurements in Animal Models of Neurodegeneration and Neurotoxicity

Abstract

This dissertation is a compilation of work in which selected analytical methods, including fast-scan cyclic voltammetry at carbon-fiber microelectrodes (FSCV), were used to determine neurotransmitter release and uptake properties in animals that model neurodegenerative disease and neurotoxicity. Alterations in the release and uptake of dopamine (DA), a central nervous system neurotransmitter that plays an important role in motor function and cognition, could contribute to, as well as be a consequence of, abnormal syndromes associated with neurodegeneration and neurotoxicity. First, we describe the application of FSCV to measure DA release and uptake in animals that model post chemotherapy cognitive impairment (PCCI). PCCI is a complication of chemotherapy treatment that is characterized by a general decline in cognition affecting visual and verbal memory, attention, complex problem solving skills, and motor function. It is estimated that one third of patients who undergo chemotherapy treatment will experience cognitive impairment. To investigate how chemotherapy treatment affects these systems, FSCV at carbon-fiber microelectrodes was used to measure dopamine release and uptake in coronal brain slices of the striatum. Here, we report on two PCCI studies with rats treated with carboplatin or a cocktail containing cyclophosphamide, methotrexate, and 5-fluorouracil (CMF), both of which are composed of clinically relevant chemotherapeutic compounds. Measurements were taken from rats treated weekly with selected doses of chemotherapeutic agent and from control rats treated with saline. It was found that DA release in the striatum is attenuated in chemotherapy-treated rats. Nevertheless, overall dopamine content, measured in striatal brain lysates by high performance liquid chromatography, and reserve pool DA, measured by FSCV after pharmacological manipulation, did not significantly change, suggesting that chemotherapy treatment impairs the dopamine release and uptake processes. Second, we report on regional differences in DA dysregulation in transgenic Huntington’s disease model mice. Huntington’s disease (HD) is a fatal, neurodegenerative movement disorder that is characterized by degeneration of the striatum. It has been determined previously that electrically-evoked dopamine (DA) release is severely attenuated in the dorsolateral striatum of R6/2 HD model mice. Here, we have used fast-scan cyclic voltammetry to uncover regional differences of DA release in the striatum of R6/2 mice. We found a dorsal-to-ventral progressive gradient in single pulse DA release in 6 to 14 week-old R6/2 mice. Moreover, when applying a 120 stimulation pulse-train, we found that DA release was only significantly attenuated in the dorsal striatum. In order to see if regional differences of release were caused by the density of viable dopamine terminals, autoradiographic labeling of the dopamine transporter (DAT) with [3H]WIN 35,428 was performed. It was found that the density distribution of DAT is significantly less in R6/2 mice in comparison to their WT controls; however, there were no significant regional differences. These data collectively suggest that the genetic mutation involved in HD leads to the increased vulnerability of the dorsal striatum in comparison to the ventral striatum, therefore providing insight to the disease mechanism The final project presented here involves the development of a method to combine FSCV measurements with caged compound photo-activation. Caged compounds have been used extensively to investigate neuronal function in a variety of preparations, including cell culture, ex vivo tissue samples, and in vivo. We describe electrochemical measurements used to determine the extent of caged compound photo-activation while simultaneously measuring DA in vitro.