Dopamine and serotonin are two neurotransmitters known to both play a very important role in the human brain. For example, the death of dopamine producing neurons in a region of the brain known as the substantia nigra are known to cause the motor symptoms of Parkinson's disease. Also, many antidepressants are believed to work by increasing the extracellular level of serotonin in the brain. For the first time, it is now possible to measure the release of these two chemicals at sub-second time resolution in a human brain using a technique known as fast-scan cyclic voltammetry, for example from patients undergoing deep brain stimulation (DBS) electrode implantation surgery. In this work, we aimed to assess the feasibility of obtaining veridical dual measurements of serotonin and dopamine from substrates with mixtures of both chemicals. In the wet lab, data was collected on known concentrations of dopamine and serotonin and then used to make models capable of estimating the concentration of both chemicals from the voltammograms recorded in the patients. A method of linear regression known as the elastic net was used to make models from the wet lab data. The wetlab data was used to compare the performance of univariate and multivariate type models over various concentration ranges from 0-8000nM of dopamine and serotonin. Cross validation revealed that the multivariate model outperformed the univariate model both in terms of the linear correlation between predictions and actual values, and pH induced noise. The pH induced noise for the univariate model was 3.4 times greater for dopamine and 4.1 times greater for serotonin than the multivariate model. Raman spectroscopy was also investigated as a possible alternative to fast-scan cyclic voltammetry. Raman spectroscopy could have several benefits over fast-scan cyclic voltammetry, including the ability to chronically implant the measurement probe into a patient's brain and make observations over a long period of time. Raman spectroscopy data was collected on known concentrations of dopamine to investigate its potential in making in vivo measurements, however this data collection failed. Therefore, simulations were made which revealed the potential of the elastic net algorithm to determine the Raman spectra of several neurotransmitters simultaneously, even when they are in mixtures and the spectra are obstructed by the noisy background. The multivariate type model outperformed the univariate type model on Raman spectroscopy data and was able to predict dopamine with an error of 805nM RMS and serotonin with an error of 475nM RMS after being trained on concentrations smaller than 5uM of both dopamine and serotonin. In addition, the original Raman spectra of both neurotransmitters was extracted from the noise and reproduced very accurately by this method. / Master of Science

Dopamine (DA) is a key striatal neuromodulator central to normal functioning of the basal ganglia. Identifying and characterizing circuits governing striatal DA transmission is necessary for understanding DA involvement in adaptive behaviour and pathology. Properties of evoked striatal DA release can be examined using fast-scan cyclic voltammetry at carbon fibre microelectrodes, a technique enabling live monitoring of transmitter release events with sub-millisecond resolution. Experimental work presented in this thesis employed this approach to study regulation of striatal DA by acetylcholine (ACh) in health and disease in acute brain slices. Synchronous activity in a small population of striatal cholinergic interneurons (ChIs) was previously shown to directly drive striatal DA release. Here using optogenetic approach I explore physiological relevance of ChI-evoked drive of striatal DA by examining whether corticostriatal and thalamostriatal afferents to ChIs can trigger ACh-evoked DA events. Following floxed vector injections in motor cortex or caudal intralaminar thalamus of CaMK2a-Cre mice I examine the properties of evoked DA upon light activation of channelrhodopsin-2-transduced inputs to striatal ChIs. These experiments revealed that both cortical and thalamic afferents are capable of driving ACh-evoked DA release, but operate using a different complement of post-synaptic ionotropic glutamate receptors and display distinct release recovery profiles. I further explore if rebound excitation in a population of striatal ChIs could drive DA events by examining whether ACh-evoked DA release follows optical inhibition of striatal ChIs selectively expressing hyperpolarizing halorhodopsin 3.0 or archaerhodopsin 3.0 in ChAT-Cre mice. This work showed that hyperpolarizing ion pumps were not successful in triggering ChI-evoked DA release. I also investigate whether cholinergic brainstem innervation of striatum could contribute to or drive ACh-evoked striatal DA events in ChAT-Cre rat, concurrently showing that ChI-evoked DA release is not a species artefact, and is present in mouse and rat alike. Current results also suggest that cholinergic brainstem afferents do not drive or contribute to striatal ACh-evoked DA events. Close interaction between DA and ACh systems further indicates that ACh could impact dopaminergic dysfunction. To explore this I examined the state of ACh transmission in a mouse model of Parkinson’s disease overexpressing human wild type alpha–synuclein protein. These animals present with impaired striatal DA release from young age, but DA deficits could be mediated by changes in ACh tone. Here I show that impaired striatal DA release is the results of primary DA axon dysfunction, although in ventral striatum DA release deficits could be partially compensated by increased ACh tone at nicotinic receptors. I further show that the functional state of muscarinic ACh receptors in not altered following decreased DA transmission, although the data from aged animals suggest that alpha–synuclein-dependent changes in vesicle handling could contribute to impaired DA releasability. Finally, I show that vesicle handling may indeed be altered in this mouse model as impaired DA release is evident with short stimulation protocols, while with prolonged depolarization of DA axon terminals alpha–synuclein-overexpressor mice are better able to sustain evoked DA release. Overall, the main body of work presented in this thesis examined the processes regulating striatal DA transmission via ACh system. In particular, I show that ChI-evoked drive of striatal DA release can be recruited physiologically and further establish that changes in ACh transmission are not the primary drivers of impaired DA releasability in a mouse model of Parkinson’s disease overexpressing human alpha–synuclein protein.

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Synucleins are highly conserved presynaptic proteins with unknown function. α-synuclein plays a key role regulating dopamine homeostasis and is intimately involved in Parkinson’s disease (PD) pathogenesis. However, the normal/pathological role of α-synuclein remains unidentified. Studies exploring its function are limited as current transgenic mouse models do not fully recapitulate PD pathology. This thesis reports the functional characterisation of two novel synuclein-based mouse models. I report the molecular and functional characterisation of transgenic mouse lines with wild-type or A30P-mutant human α-synuclein genomic locus carried within a bacterial artificial chromosome. SNCA-A30P^+Snca-/- mice exhibited a highly physiologically relevant expression pattern of the transgene, including expression in the substantia nigra pars compacta (SNpc) and a specific, age-related loss of TH^+ cells in the SNpc, the key region of preferential cell loss in PD, compared with non-transgenic Snca -/- littermate controls. Analysis of dopamine signalling using fast-scan cyclic voltammetry (FCV) showed young adult SNCA-A30P^+Snca-/- mice had an approximately 20% lower evoked extracellular dopamine concentration ([DA]o) compared with non-transgenic Snca -/- littermate controls, a decrease specific to the dorsal striatum. This difference diminished with age and could not be attributed to changes in dopamine reuptake/content. I detail the behavioural and neurochemical phenotype in mice lacking all three synucleins (α/β/γ). Functional compensation between synucleins emphasises the importance of studying their effects by removing all three proteins simultaneously. Triple-null mice exhibited hyperactivity in a novel environment reminiscent of a hyperdopaminergic-like phenotype, but showed no phenotype in anxiety or motor related tests. FCV revealed synuclein triple-null mice had a two-fold increase in [DA]o, specific to the dorsal striatum and not attributable to changes in dopamine reuptake/content, changes in striatal nicotinic receptor activity nor calcium-dependent changes in dopamine exocytosis. Together, the analysis from these two novel mouse models reveal synucleins play an important role in altering synaptic function in the dorsal striatum (the region selectively affected in PD) and contributes to growing evidence suggesting synucleins are negative regulators of synaptic dopamine release.

Introduction: Over recent decades, there has been an increase in the availability and use of Novel Psychoactive Substances (NPS) all over the world. They include several classes of chemicals that mimic the effects of illicit drugs and have been purposefully introduced into the market to circumvent or undermine the purpose of legal regulation. Currently, there is information lacking on the pharmacology of these substances; however, the increasing number of cases and outbreaks of intoxications/deaths is becoming a cause for deepening concern. Multi-disciplinary research in the fields of biology, chemistry, clinical medicine and web analysis is needed to develop responses against this tidal wave. Aim: The overall aim of this project is to gain insights into pharmacological, neurochemical and molecular properties of selected NPS to provide a reliable background needed for detection, assessment, and management of NPS-related harms. A range of approaches and methodologies was employed and a spectrum of different fields of knowledge has been engaged to gain some understanding into the complex multi-faceted phenomenon of NPS. Methods: Different substances have been selected as targets for the present project according to the clinical pattern of toxicity raised by their worldwide use and the lack of scientific knowledge available about them. The methods employed were: in vitro quantitative autoradiography (to evaluate the binding properties of the novel SCs BB-22, 5F-PB-22, 5F-AKB-48 and STS-135 at the cannabinoid receptor type 1 and N-methyl-D-aspartate receptor; and the binding properties of the synthetic stimulants 5-IT and 2-DPMP at the dopamine transporter in rat brain slices); in vitro Fast Scan Cyclic Voltammetry (to assess the effects of BB-22 on evoked dopamine efflux and dopamine re-uptake half-life in nucleus accumbens brain slices); in vivo microdialysis (to monitor dopamine release in terminal areas of the reward system after acute administration of the synthetic cannabinoids BB-22, 5F-PB-22, 5F-AKB-48 and STS-135; the dieting aid compound 2,4-DNP; the synthetic stimulants 2-DPMP and D2PM in freely moving animals); in silico molecular docking (to investigate the intermolecular interactions of the SCs BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, and other referent compounds, with a homology model of the rodent cannabinoid receptor type 1 (CB1R) and the crystal structure of the human CB1R); and a web-based analysis approach (to analyse the information provided by a range of fora communities on 4,4'-DMAR use, additionally critical reviewing the available evidence-based literature on this topic). Results: Our in vitro quantitative autoradiography studies, confirmed that the index compounds BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, behave as highly potent CB1R ligands able to compete with the radioligand [3H]CP-55,940 in cortical and striatal brain slices. On the other hand, all synthetic cannabinoids tested were unable to compete with the radioligand [3H]MK-801 in the same cerebral areas, rejecting the hypothesis of their potential binding to the N-methyl-D-aspartate receptor (NMDAR) at all concentrations investigated. Consistent with previous in vitro studies, 5-IT and 2-DPMP behaved as highly potent dopamine transporter (DAT) ligands able to compete with the radioligand [125 I]RTI-121 in a concentration-dependent way in the Caudate Putamen (CPu) and Nucleus Accumbens (NAc) brain slices. Notably, 2-DPMP was able to displace the radioligand in both cerebral regions, starting from lower concentrations compared to 5-IT. In vitro Fast Scan Cyclic Voltammetry findings demonstrated that local application of the synthetic cannabinoid BB-22 in brain slices, was unable to change evoked dopamine efflux and dopamine reuptake time-constant in the NAc shell at any doses tested. The results obtained would suggest the relative contributions of complex neuronal circuits, either within or outside the NAc, whose modulation would interfere with the interactions between BB-22 and dopaminergic neurons and represent critical pathways accounting for some of the rewarding properties of BB-22 exposure. In vivo microdialysis outcomes suggested that all SCs tested could increase dopamine release in the NAc shell at specific doses, while no changes in dopamine output were observed in other areas of the reward system, namely NAc core and medial prefrontal cortex (mPFCx) after BB-22 administration. These outcomes provided a circumstantial pre-clinical evidence for a greater putative abuse liability of SCs compared to the natural compound found in cannabis (Δ9‐THC). Furthermore, the acute treatment with 2,4-DNP did not cause any change in dopamine release in the NAc shell and CPu rejecting the hypothesis of psychoactivity of this substance at the dose tested. On the other hand, the synthetic stimulant 2-DPMP elicited a comparable increase of dopamine (DA) release in the NAc shell and CPu at the higher doses tested, while D2PM caused a selective increase of DA release in the NAc shell, providing a circumstantial preclinical evidence for a putative abuse liability of this compound at the highest dose assessed. The in silico molecular docking studies demonstrated that the SCs BB-22, 5F-PB-22, 5F-AKB-48 and STS-135 interact with CB1 receptor residues that, according to previous mutation and computational studies, are considered crucial for synthetic cannabinoid binding recognition. Additionally, they share some interacting residues with other aminoalkylindole derivatives (e.g. WIN-55,212-2). The web-based analysis focused on 4,4'-DMAR, suggested that fora members co-operate in exchanging an extensive body of knowledge about this drug, and the recurring topics of discussion include: routes of administration and dosages; desired and undesired effects; comparison and association with other drugs and medications; overall impression; provision of harm reduction advice. This approach has been useful to better understand some of the clinical and psychopharmacological issues pertaining to 4,4'-DMAR. Conclusions: Overall, these studies provided new pharmacological, neurochemical and molecular knowledge on a range of Novel Psychoactive Substances essential for identifying potential therapeutical approaches against their use/abuse. The novelty of this project lies in the adoption of a multi-disciplinary approach involving a range of methodologies from different areas of expertise (neurobiology, pharmacology, chemistry, netnography) all integrated to clarify some aspects of the index NPS, which were not yet available in the current literature. Additional studies are needed to better explain short and long-term effects of the index NPS, their abuse potential, and their interactions with other drugs of abuse.

The work presented in the thesis is about the evaluation of Organic Electrochemical Transistors (OECTs) for fast scan cyclic voltammetry (FSCV). FSCV is a method which has been used for real time dopamine sensing both in vivo and in vitro. The method is sensitive to noise and could therefore benefit from signal preamplification at the point of sensing, which could be achieved by incorporation of OECTs. In this study the OECTs are based on the conductive polymer poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS). The gate consists of gold microelectrodes of different sizes to be used one at a time. When dopamine is reacted at the gate electrode, the redox state of the PEDOT:PSS OECT channel is modulated and the resulting change in drain current can be measured. The gate current, which contains the sensing information, is after filtering obtained by differentiating the channel potential with respect to time. The derived gate current is plotted in cyclic voltammogram for different dopamine concentrations and the amplitude of the oxidation/reduction peaks can be used to determine the dopamine concentration. In this thesis for the first time it is demonstrated that OECTs can be used for FSCV detection of dopamine. The results are discussed and an outlook on future work is given.

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