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This Ph.D. thesis contributes towards the neural computational modelling and analysis, and functional connectivity analysis of the serotonergic system. The thesis starts with a concise review of the known neurobiological functions of the serotonergic system. Different experimental measurements of the system are described, with focus on electrophysiological, optogenetic and voltammetry recordings. Further, the neuronal signalling of serotonergic systems under reward and punishment tasks are described. This is followed by a brief review on computational modelling of the serotonergic system, with focus on mechanistic and biologically based models. Then, signal processing and data analytical approaches of electrophysiological data are discussed. While reviewing these, research questions are formulated regarding the serotonergic system. It is amply clear from the reviews that, it is not known whether neural circuits encompassing serotonergic neurons can be degenerate (i.e. different structures performing the same functions), and how population of serotonin neurons in the dorsal raphe nucleus interact with themselves and with the cortex. Following the literature review are three original contributing chapters. In the first contributing chapter, biologically based mean-field network models of serotonergic and dopaminergic neural interactions under reward and punishment tasks are developed and simulated to evaluate the possibility of network structure degeneracy. Non-serotonergic and non-dopaminergic neural populations are considered to evaluate multiple possible indirect serotonin-dopamine connections. The modelling results reveal the possibility that serotonin-dopamine neural circuits can be degenerate, at least under reward/punishment tasks. In the next contributing chapter, the stability of these degenerate neural circuits under tonic and phasic activity modes are evaluated using dynamical systems theory. The analyses show that all the considered degenerate neural circuits are stable in both activity modes. In the third contributing chapter, signal processing and analysis are performed on electrophysiological data (neuronal spike trains and electrocorticography, ECoG) from experimental collaborators. In particular, analyses were conducted on ECoG activities in the frontal cortices and the visual cortex, and neuronal firing activities from the dorsal raphe nucleus (DRN), a main source of serotonergic neurons. Then coherence-based method is used to identify the functional connectivity among simultaneously recorded neurons in the DRN, and between the DRN neuronal activity and the ECoG activity. The coherence analyses show that interactions of the DRN neurons are generally weak and sparse, and that the slow-firing DRN neurons (putative serotonergic neurons) exhibit relatively stronger interactions with each other. Further, unlike the strong corticocortical ECoG interactions, the DRN neuronal to ECoG interactions are generally weak, and that slow, regular firing DRN (putative serotonergic) neurons have relatively stronger interactions with the right frontal ECoG activity. Finally, this thesis concludes with a discussion on all the chapters and proposed future work.

During flexible behaviour, dopamine is thought to carry reward prediction errors (RPEs), which update values and hence modify future behaviour. However, in real-world situations where the statistical relationships in the environment can be learned, continuously adapting values is not always the most efficient way of adapting to change. Moreover, the environment is not always fully observable, and observations may provide only partial information about the current state of the world. In such partial observable structured environments, as is found in many real-world situations, it is not well understood what kind of information dopamine conveys or its causal role in shaping adaptive behaviour. To probe dopamine's involvement in goal-directed behavioural flexibility in such environments, we measured and manipulated dopamine while mice solved a partially observable structured sequential decision task. In chapter 3 we show that mice solve such a task using state inference. In chapter 4, we recorded calcium activity from dopaminergic cell bodies in the ventral tegmental area and dopamine axonal projections in the ventral and dorsomedial striatum, as well as dopaminergic concentrations in the same striatal regions. Dopamine multiplexed a wide range of information. At different timescales dopamine signalling was consistent with carrying information about choice-specific RPEs, choice-independent reward history and lateralised movement signals. RPE computations were shaped by task structure and the inferred state of the task. Nonetheless, in chapter 5, we show that although dopamine responded strongly to rewards, optogenetically activating or inhibiting dopamine at the time of trial outcome had no effect on subsequent choice. However, in a different task context, we could show that the same stimulation had a substantial effect on animals' choices. Therefore, we conclude that when inference guides choice, rewards have a dopamine-independent influence on policy through the information they carry about the world's state.

A recent pathological hypothesis of Parkinson's disease suggests that at the time of disease onset, midbrain dopaminergic cells lose their synaptic connectivity before dying. The preposition that dopaminergic axons degenerate initially and predominantly in PD offers new critical opportunities for the development of neuroprotective and restorative therapies. However, we know little about how dopaminergic neurons innervate their target areas. Because of their functional importance in PD, understanding the mechanisms underlying the development and function of midbrain dopaminergic circuits is of considerable interest. Therefore, the initial focus of my research was on exploring novel pathways governing the development of dopaminergic circuitry. Using a mouse model, my work defined a novel role for Nolz1, a developmentally expressed transcription factor, in regulating aspects of dopaminergic axon growth and target innervation. I show that Nolz1 regulates critical aspects of striatal development in the mouse embryo and that aberrant striatal patterning alters the outgrowth of nigrostriatal projection neurons (Chapter 2). The premature degeneration of axonal projections in PD has been linked to changes in mitochondrial function. Impairment of mitochondrial dynamics, transport, and loss of plasticity of axon terminals precedes the onset of neuronal degeneration in this neurodegenerative disease. PINK1 is a mitochondrial kinase that ensures mitochondrial health and mutations in PINK1 cause PD. My work explored how mutations in PINK1 alter cellular metabolism, using a Drosophila model of PD. Multi-omics analysis of pink1 mutant flies uncovered metabolic and transcriptional modifications in cysteine metabolism which coincided with defects in mitochondrial respiration. These findings confirmed PINK1's canonical role in mitochondrial function, while highlighting the relevance of cysteine metabolism to compensate for early and selective defects in mitochondrial respiration and ATP production (Chapter 3). To answer if we can recapitulate these changes in a human model, I then used human induced pluripotent stem cells (iPSCs). I differentiated human induced pluripotent stem cells (iPSCs) carrying a recently discovered I368N mutation in PINK1 into neural precursor cells (NPCs) and examined their metabolic profile. I found that the PINK1 I368N mutation causes global metabolic changes that broadly validate the comprehensive number of hits recovered in pink1 mutant flies, suggesting a significant overlap between my integrated networks. This convergence of disease phenotypes points to a common disease mechanism that may offer a unifying perspective on early-stage PD pathology (Chapter 4). Together, these results shed light on novel genes and pathways that could be exploited for therapeutic intervention.

Altered states of consciousness (ASCs) provide models through which the necessary and sufficient neural underpinnings for consciousness can be characterised. Indeed, the study of ASCs through human functional neuroimaging has identified large-scale cortical networks (such as the default mode network; DMN) whose disintegration is common across both pharmacological transient and pathological chronic ASCs. However, these cortico-centric network accounts have largely been unable to fully characterise fundamental neurobiological processes which may underpin disruptions of consciousness and/or network changes. Instead, such work has mainly been performed in preclinical animal experiments and in lesion studies, which have identified that brainstem neurotransmitter nuclei - among them in particular the monoaminergic source nuclei and transmitter systems - are consistently implicated in both pharmacological and pathological perturbations of wakefulness/consciousness. Thus, the dysfunction of these nuclei could underpin human ASCs and network changes, and their assessment might correspondingly hold translational, diagnostic and therapy-informing utility. To address this central question, in this thesis I used resting state fMRI recordings to analyse how the dopaminergic ventral tegmental area (VTA) and the serotonergic raphe nuclei are affected in pharmacological (propofol sedation, lysergic acid diethylamide & psilocybin administration) and pathological (disorders of consciousness, DOC) contexts, and how these findings relate to network-level impairments, as well as previous animal findings concerning the same nuclei. In Chapter II, I assessed how functional connectivity of the VTA is altered across both propofol sedation and DOC patients. I found that in both sedation and DOC, VTA connectivity is disrupted from the main node of the DMN in the precuneus/posterior cingulate cortex, revealing a connectivity impairment universal to both ASCs. The extent of this VTA connectivity impairment was associated with DMN disconnectivity, as well as behaviour and/or outcome measures. Furthermore, I found that this connectivity could be upregulated by a dopaminergic agonist. Chapter III further developed the findings of the previous chapter by aiming to integrate these with a pre-existing DOC disease framework called the anterior forebrain mesocircuit, which regards thalamic impairment as central to DOC pathoyphysiology. I found that VTA-thalamus connectivity is also impaired in DOC patients, but that this was undetectable in seed-to-voxel contrasts due to a sub-group of patients showing control-like connectivity levels. Patients who showed such preserved VTA-thalamus connectivity also were found to be responsive in an fMRI volitional task (Tennis task), and the greater this VTA-thalamus connectivity strength was, the less impairment of whole brain complexity of the fMRI signal was observed, which supports the concept that thalamic and mesocircuit impairment in DOC might be at least partially mediated through dopaminergic dysfunction. Chapter IV focussed on psychedelic ASCs, which are elicited by serotonergic psychedelic drugs, such as lysergic acid diethylamide (LSD). I found that serotonergic raphe nuclei connectivity is powerfully disrupted in acute LSD administrations - strikingly once more to the posterior DMN node in the precuneus/posterior cingulate. The extent of this disruption was again associated with DMN disintegration, and showed causal mediation effects on hallmarks of the psychedelic experience. I also demonstrated that the cortical ratio of 5HT2A receptors over 5HT1A receptors might be driving this raphe uncoupling effect. Based on these results and pre-existing literature, I hypothesised that this observed connectivity disruption in acute psychedelic administration may have adaptive sub-/post-acute effects on raphe functionality that might underpin psychedelic therapeutic effects, e.g. in depression. Chapter V tested said hypothesis of psychedelic-induced sub-/post-acute raphe changes in two depression cohorts who received psilocybin. In data-driven analyses, I found that only post-acute connectivity of the raphe to the precuneus was associated with strength of depression symptom reduction across both trials. Psychedelic treatment induced significant sub- and post-acute upregulations of raphe-precuneus coupling, whereas SSRI treatment did not. Additionally, raphe-precuneus connectivity enhancements also positively covaried with DMN integrity and were accompanied by increases in spontaneous raphe BOLD activity. This provides preliminary evidence that a post-acute upregulation of raphe functionality might underpin psychedelic anti-depressant effects. Altogether, these studies demonstrate that monoaminergic nuclei dysfunction might be a hallmark common to various different ASCs - and that functionality of these brainstem sources of non-classical transmitters might plausibly be associated with behavioural, clinical and network-level phenomena associated with perturbed consciousness. This provides various necessary translational bridges between preclinical and clinical work, and hints at the possibility of non-invasive neurotransmitter systems-level assessments with diagnostic and therapeutic utility.

The dopaminergic system plays a crucial role in learning, reward-processing, motivation, motor control and attention. The population of midbrain dopamine neurons projects extensively to many areas of the brain. This system is maintained in a homeostatic way, and it can self-regulate to maintain behavioural function in extreme cases such as Parkinson's disease. However, Huttunen (2016) discovered a significant correlation between midbrain dopamine neuron number in male rats and their behavioural performances in a gambling task. It was unexpected that such non-clinical variation in dopamine neuron number can lead to differences in observable behaviours. Our project aims to investigate the link between dopamine structure and function in risk decision-making, and to explore possible factors that can manipulate the brain and behaviour. Chapter 1 reviewed the dopaminergic system and its adaptive role in conditions such as environmental enrichment and stress. Chapter 2 described the experiment using a modified risk task which failed to replicate the original results from Huttunen (2016). Chapter 3 examined the possible sex difference in the brain of behaviour of rats. Chapter 4 and 5 explored factors that can influence this dopamine-loss-stay relationship, including environmental enrichment and neurogenesis. We demonstrated that dopamine-expressing neurons in the rat midbrain may underlie loss-stay behaviours, which may reflect the animals' risk-attitude. Moreover, the correlation was significant only when the age of the animal was considered. Therefore, this correlation may be a result of aging, or the compensatory mechanisms that often accompany ageing. Future research may shed light on possible treatment and interventions for neural decline in ageing and other clinical conditions.

Impulsivity is a multidimensional trait in humans and other mammalian species. It is widely regarded as the tendency to act rapidly, without appropriate foresight and underlies many psychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD), mood disorders such as depression and mania, and substance use disorder (SUD). Research has shown that impulsivity is a non-unitary construct, characterised by distinct behavioural manifestations and, accordingly, distinct neural substrates. This thesis focuses on elucidating the psychological and neural mechanisms of waiting impulsivity in experimental rats trained on the 5-choice serial reaction time task (5CSRTT). The psychological processes involved in this behaviour are yet to be fully understood. However, aspects of sustained attention, urgency, motivation, and delay-aversion are all likely to play a role in shaping this behaviour. Although the neural substrates and neurotransmitter systems underlying waiting impulsivity have been extensively investigated, there are still many unanswered questions. Some of the questions that this work aims to address are: 1) can we refine our understanding of the psychological mechanism that influence premature responses, specifically the extent to which these are driven by motivated behaviour or, instead, negative urgency? 2) Does waiting impulsivity confer advantages in specific experimental contexts and what are the neurotransmitter mechanisms regulating this? 3) Can we refine our understanding of the precise neural circuits involved in the execution of a premature response? 4) How does impulsivity and attention on the 5CSRTT compare with performance on other tasks of sustained attention? Are there any shared neural processes? To investigate these questions, I used a range of experimental approaches spanning behavioural testing to systemic and local (intra-cerebral) pharmacology, brain lesions and chemogenetics. In chapter 2, I show that premature responses are influenced by reinforcement rate and motivation, and that negative urgency does not seem to play an important role in the genesis of these responses. In chapter 3, I found that high levels of trait impulsivity confer an advantage in contexts that require rapid focusing and action. I also showed that this advantage might be conferred putatively by elevated striatal dopamine (DA) release in the striatum. In chapter 4, I 6 show that inhibition of the mesolimbic DA system reduces premature responses but was unable to pinpoint the midbrain-striatal loop responsible for this effect. Finally, in chapter 5, two tasks were compared that assess sustained visual attention. PFC lesions effected using the excitotoxin quinolinic acid profoundly affected performance on the 5CSRTT but had negligible effects on a signal detection attentional task. Taken together, these findings suggest that impulsive (premature) responding in the 5CSRTT is linked with the motivation to perform the task but does appear to depend on negative urgency. Trait impulsivity confers an advantage in specific contexts and depends on striatal DA function. Finally, sustained attention on the 5CSRTT is distinct from other forms of sustained attention both at the psychological and neural levels. Some of these findings are consistent with studies on impulsive individuals, thus highlighting the potential for translational research between rodents and humans. Ultimately, this work expands our understanding of the psychological and neural circuit mechanisms underlying waiting impulsivity.

The experimental work presented in this thesis explores the suitability of boron-doped diamond (BDD) electrodes for the electrochemical sensing of compounds important to health. Various forms of BDD electrode have found increased application to electroanalysis in the past two decades, but there is still room to explore more complex molecules with synthetic BDD. The aim of this thesis is to examine whether the known chemical and material advantages-wide potential window, low background capacitance, chemical inertness, and physical robustness-can be realised. Although BDD electrodes generally have higher resistance to fouling by analyte media than other carbon and metallic electrode materials, electrode poisoning can still be very problematic. The first study investigates ways to restore or improve the electrode response reliably and quickly after it has been fouled. The fouling analyte was dopamine (DA), known to form a wide variety of polymers and is one of the most fouling chemicals that exists. After 3 cyclic scans, a thick adsorbed layer was observed by scanning electron microscopy (SEM), which negatively impacted the accuracy of further measurements. In a solution of 1 mM DA, the heavily fouled BDD can be efficiently returned to high activity by using 20 pulses of alternating ± 10 mA/cm^2 current density with 100 ms duration. If only anodic pulses are used, then this is not observed. This treatment in situ increased the quality of the signal to beyond the initial value for an acid-cleaned electrode. This finding provides confidence for exploring other heavily fouling analytes. The second study examined the voltammetric behaviour of the genotoxic pollutant 2-amino-9-fluorenone (2-AFN) and its interaction with double-stranded DNA (dsDNA). Cyclic voltammetry revealed complex adsorptive behaviour of 2-AFN onto the BDD. Applying 60 pulses of alternating ± 10 mA/cm^2 current density with 100 ms duration in situ gave consistent and reproducible voltammetry. With the aid of SEM and X-ray photoelectron spectroscopy to study the nature of the adsorption, it was shown that 2-AFN diffuses to the electrode and undergoes proton-dependent (below pH 10.0) irreversible oxidation in the anodic region to form dimers or longer polymer chains. These and other monomers can then also take part in the reversible reduction of the carbonyl group. By optimising the parameters of differential pulse voltammetry, the limit of detection of 2-AFN was found to be 10 nM-an order of magnitude lower than previously reported. After exposing dsDNA to 2-AFN, preferential damage to the guanosine moiety was observed, suggesting the intercalation of 2-AFN into the planar aromatic structure of dsDNA. Extensive square wave voltammetry showed that 2-AFN causes a disproportionate amount of damage to dsDNA, observed as significant deviations from the expected peak currents and potentials. In the third study, bare BDD electrodes were used in the pulsed voltammetric and amperometric quantification of five pesticides in buffered solutions and in river water: namely, the four carbamates carbaryl (CB), carbofuran (CF), fenobucarb (FC), and aldicarb (AC), along with malathion (MT). After exposure to 1 mM MT, a reduction feature was observed on subsequent scans that is highly sensitive to pH. This is attributed to a complex carbon-oxygen adsorbate that is robust to aggressive cleaning methods and only removable by hydrogen-plasma etching. For the carbamates, detection limits of 1 µM for FC, CB, CF (better than other undecorated planar electrodes), and 2.5 µM for AC were obtained. The same limits and calibration ranges were achieved via amperometric batch-injection in spiked river water for CB, and CF. The detection limit in river water for FC was 5 µM. The oxidised BDD electrode demonstrated linear sensitivity up to 100 µM. Alkaline hydrolysis applied prior to analyte mixing allowed for the simultaneous detection of CF, CB, and FC. The fourth study turns to a group of compounds that has recently come back into public interest: antibiotics. Amoxicillin (AMX), cefadroxil (CFX), ofloxacin (OFX), and tetracycline (TC) have all been used for growth promotion in industrial farming. These molecules are rarely investigated via electrochemistry, and here BDD electrodes were applied to the analysis of OFX for the first time, achieving a competitive detection limit of 1 µM. Because of peak-definition and sensitivity problems of AMX, this work focused on the other three analytes. Square-wave voltammetry was used to achieve a detection limit of 500 nM for CFX and 1 nM for TC, which is an order of magnitude lower than previously reported. All three exhibited a wide dynamic range, up to 200 µM. A major advantage of using a BDD electrode was realised for this project. Because of the inertness of the diamond surface, adsorption which dominates the electrochemistry of these compounds on non-diamond electrodes did not take place. The thesis concludes with a summary of key results and suggestions for future research topics.

Surface-enhanced Raman scattering (SERS) spectroscopy is a powerful analytical technique for ultrasensitive detection of chemicals and biomolecules. As the high sensitivity of SERS requires analytes to be in close contact with a plasmonic substrate, the detection of analyte molecules with low chemical affinity towards the substrate is thus limited, for example dopamine molecules as well as other neurotransmitters (NTs), which are the focus within this thesis. Two binding methods of NTs to SERS substrates will be covered: by Cucurbit[n]uril (CB[n]) and by Fe(III) ions. The fundamental resonance modes of NTs molecules and ultratrace NTs SERS sensing are discussed in detail. Further exploration of SERS substrates in the format of oil/water interfacial film will also be reported. The SERS application integrated with microfluidic techniques will be discussed in the final part of this thesis.

Cognitive flexibility refers to how individuals adapt their behaviour to changes in the environment. Although important for survival and wellbeing, cognitive flexibility is impaired in a wide range of neurological and neuropsychiatric disorders, including Parkinson’s Disease (PD) and Obsessive-Compulsive Disorder (OCD). Optimal flexibility is known to depend on dopamine (DA) neurotransmission in the central nervous system, but the precise mechanism and brain loci underlying the effects of DA on flexible decision-making remain unclear. In this thesis, cognitive flexibility was inferred in experimental rats by evaluating their performance on a reversal-learning task involving a simple discrimination between rewarded and non-rewarded stimuli. During reversal, subjects must adapt and respond to the formerly non-rewarded stimulus whilst ignoring the initially rewarded stimulus. Learning on this task thus requires constants shifts in behaviour in response to positive (rewarded) and negative (non-rewarded) feedback. The overarching hypothesis of my thesis is that DA modulates reversal learning performance by signaling positive and negative reward prediction errors (RPE) within the direct (rewarded) and indirect (non-rewarded) pathways, respectively. To investigate this hypothesis, I used a range of experimental approaches to interrogate the neuromodulation of the direct and indirect pathways by DA. In chapter 4, I found dissociable effects of D1 and D2 receptor antagonists during different stages of serial visual reversal learning when administered into the nucleus accumbens shell. In chapter 3, I used a recently developed valence-probe visual discrimination task to dissociate different components of reversal learning and tested the extent to which these were dependent on D2 receptors. We found that the D2 agonist quinpirole impaired reversal learning when given systemically, an effect that depended on decreased sensitivity to negative feedback, but improved performance when given directly into the nucleus accumbens. In chapter 5, the synaptic location of D2 receptors involved in the modulation of reversal learning was evaluated. Using a post-synaptic probe compound (an adenosine 2A receptor antagonist) evidence is presented for a predominately post-synaptic locus underlying the effects of D2 agents on reversal learning. Finally, in chapter 6, an in-vivo optogenetics intervention was used to simulate activity in the mesoaccumbal and nigrostriatal circuits during reversal learning. Here, activation of the mesoaccumbal, not nigrostriatal, circuit modulated reversal learning on trials when the expected reward was omitted. Taken together, these original results provide support for a dissociable role of DA receptors and striatal sub-regions in learning from positive and negative feedback in reversal learning. These findings expand our understanding of the neural circuit mechanisms underlying cognitive inflexibility and highlight potential therapeutic targets to improve flexible decision making in PD, OCD and a range of other brain disorders.

Impairments in emotional reactivity and behavioural flexibility are pervasive across disparate psychiatric conditions as traditionally defined. Here, I provide new evidence on how these processes are altered by neuromodulators in humans, with a primary focus on serotonin (5- HT; 5-hydroxytryptamine). Emotional reactions prepare the body for action. Some emotion is primitive, implicit, and critical for surviving threats, yet can inappropriately persist in times of safety. Other emotions are more complex, self-conscious and important in maintaining harmonious interpersonal relationships. At the same time, learned behaviours that are adaptive in the first instance, may become irrelevant or even disadvantageous as circumstances change. In Chapters 3 through 6, I report on experiments in healthy human volunteers that employed the dietary technique acute tryptophan depletion (ATD). ATD temporarily lowers serotonin synthesis and release by depleting its biosynthetic precursor tryptophan. Chapter 3 is a study of self-reported social emotion. ATD enhanced emotion in response to social injustice non-specifically; however, consideration of personality traits revealed that highly empathic participants reported more guilt under ATD, whereas individuals high in trait psychopathy demonstrated more annoyance. Chapter 4, in contrast, considers evolutionarily ancient automatic emotional reactions to threats. This was assayed instead by an objective measure, the skin conductance response (SCR). Here, ATD conversely attenuated the retention of Pavlovian conditioned emotional memory to threat. Traits again influenced this response: individuals more intolerant of uncertainty displayed the greatest attenuation of emotional reactions. Chapter 5 both extends the studies on emotion and bridges to the remaining empirical work by investigating reversal learning, an index of cognitive flexibility, in two experiments. Individuals again underwent Pavlovian (stimulus-outcome) threat conditioning, whereby one stimulus predicted threat, and another was safe. These contingencies then swapped (reversed). In a separate experiment, participants underwent instrumental (stimulus-response-outcome) conditioning on a deterministic schedule (the correct option was always correct), followed by reversal of the contingencies. ATD impaired both Pavlovian and instrumental reversal learning. Chapters 6 through 8 instead examine instrumental reversal learning that was probabilistic (the correct option was correct most but not all of the time), rather than deterministic. Chapter 6 expands on previous ATD studies of probabilistic reversal learning (PRL) in the literature, which had not found effects on choice behaviour. Despite nearly tripling the sample size, behaviour here assessed by conventional methods was unaffected, replicating previously published null results. Applying reinforcement learning (RL) models, however, revealed ATD elevated a basic perseverative tendency, referred to as “stimulus stickiness”; behaviour was more stimulus-bound and insensitive to the outcome of actions, consistent with the deterministic instrumental reversal impairment following ATD. Chapters 7 and 8 apply RL models as well, to existing datasets on PRL for comparison. Chapter 7 shows that healthy volunteers under lysergic acid diethylamide (LSD), which acts both at serotonin but also dopamine receptors, showed enhanced learning from positive feedback in particular, which was related to perseveration. Chapter 8 applies computational methods to PRL in clinical populations. RL modelling revealed a computational signature that dissociated PRL in stimulant use disorder (SUD) and obsessive-compulsive disorder (OCD): Individuals with SUD showed heightened stimulus stickiness, as occurred following ATD in healthy volunteers, whereas the OCD group (under serotonergic medication) demonstrated lower stimulus stickiness than healthy controls. Dopaminergic agents remediated a reward learning deficit in SUD, among other measures. The general discussion considers these various findings in terms of theories of central serotonin function, in relation to the animal literature, and its relevance to mental disorder. These results, collectively, advance knowledge of neurochemical and computational mechanisms underlying psychiatric conditions trans-diagnostically, with implications for revised psychiatric classifications in line with the Research Domain Criteria (RDoC).

Dopamine is a highly conserved neurotransmitter, and it is known to be involved reward, locomotion, cognition and motivation. Moreover, dysfunction or loss of dopamine neurons has been implicated in diseases such as addiction and Parkinson's disease, respectively. Dopamine neurons develop early in life, and zebrafish possess a complete compliment of dopaminergic neurons by 5 days post fertilisation. Previous anatomical and genetic studies have suggested the dopaminergic interneurons of the zebrafish subpallium are equivalent to mammalian midbrain DAergic neurons. In this thesis the physiology and functional role of the cluster of dopamine neurons found in the zebrafish subpallium have been examined. In the first results chapter, the anatomical, morphological and physiological development of subpallial dopaminergic neurons has been examined. This revealed that by 5 dpf, subpallial dopaminergic neurons innervate the telencephalon, thalamus and hypothalamus whilst receiving input from across the brain. Additionally, subpallial dopaminergic neurons receive excitatory and inhibitory synaptic input, are intrinsically excitable and exhibit endogenous firing. Together, these findings suggest the subpallial dopaminergic neurons are functionally integrated into the brain during early development. In the second and third results chapters, the functional role of subpallial dopaminergic neurons was investigated. To delineate their function, these neurons were targeted for laser ablation. These investigation demonstrated loss of subpallial dopaminergic neurons was sufficient to perturb foraging and startle behaviours in free-swimming fish without affecting locomotion. Physiological recordings revealed these neurons are active when zebrafish are exposed to attractive and aversive stimuli. In sum, these investigations provide insights into the role of dopamine signalling in modulating decision-making, approach, and avoidance behaviours.

Operant conditioning is the process by which animals learn to associate their own behaviour with positive or negative outcomes, biasing future action selection in order to maximise reward and avoid punishment. It is an important strategy to ensure survival in an ever-changing environment. Although operant conditioning has been observed across vertebrate and invertebrate species, the underlying neural mechanisms are still not fully understood. The Drosophila larva is an excellent model system to study neural circuits, since it is genetically tractable, with a variety of tools available. Although it is quite small, it is capable of a diverse range of behaviours and can achieve complex learning tasks. However, while the mechanisms underlying classical conditioning, where animals learn about the appetitive or aversive qualities of an external sensory cue, have been extensively studied in larvae, it has remained an open question whether they are capable of operant conditioning. This is in part due to the challenges which arise during the training process: in order to train an animal to associate its own actions with their outcomes, the experimenter needs to be able to deliver rewarding or punishing stimuli directly in response to behaviour. In this thesis, I introduce a novel high-throughput tracker suitable for training up to 16 larvae simultaneously. I have developed a customised software for real-time detection of various actions that larvae perform: left and right bend, forward crawl, roll and back-up. Light and heat stimuli can be administered at individual animals with minimal delay, enabling optogenetic or thermogenetic activation of circuits encoding reward or punishment in response to behaviour. Using this system, I show that Drosophila larvae are capable of operant conditioning. Pairing bends to one direction, e.g. the left, with optogenetic activation of a large group of reward-encoding dopaminergic and serotonergic neurons is sufficient to induce a learned preference for bending towards this side after training. I explore whether there are other types of actions which larvae can learn to associate with valence, and introduce a second operant conditioning paradigm, in which larvae modify their behaviour following pairing of the stimulus with forward crawls. To identify new candidate neurons signalling valence in a learning context, I also conduct a classical conditioning screen, in which I pair an odour with optogenetic activation of distinct neuron types covered by different driver lines. While activation of many types of gustatory sensory neurons paired with the odour was insufficient for memory formation, I find that the serotonergic neurons of the brain and the subesophageal zone (SEZ) can induce strong appetitive learning. Finally, I show that activity of serotonergic rather than dopaminergic neurons is sufficient for memory formation in the operant bend direction paradigm, and that operant conditioning is impaired when restricting activation to the serotonergic neurons of the brain and the SEZ. My results suggest a novel role of serotonergic neurons for learning in insects as well as the existence of learning circuits outside of the mushroom body. Different subsets of serotonergic neurons mediate classical and operant conditioning. This works lays a foundation for future studies of the function of serotonin and the mechanisms underlying operant conditioning at both circuit level and cellular level.

Reward follows work, but with finite energy, all life must decide what reward is worth the work. Economic theory provides a framework of how rational decision makers optimise this choice: agents should maximise the possible utility (subjective benefit) discounted by the disutility (subjective cost) of the required effort. The dopamine reward prediction error signal is a potential neuronal substrate of this utility calculation as it is known to capture subjective value of rewards, risk and time. Other work has demonstrated pathologies of midbrain dopamine neurons and drugs that target dopaminergic signalling affect the willingness to expend effort. However, it is not clear from previous studies how effort disutility is represented in the reward prediction error signal. To resolve this question, this thesis explores the subjective value of effort through behavioural experiments in rhesus macaques and relates this to neurophysiological experiments recording from midbrain dopamine neurons in the same animals. In the first experiments, a straight-forward behavioural task is developed and validated to measure the subjective value of effort in monkeys. Random utility models are used to independently estimate the juice utility and effort disutility functions, which are then used to provide experimental evidence for the subtractive model of effort cost discounting. Extending these experiments, the second set of experiments examines whether monkeys form an internal reference point of effort expenditure and how this affects their choice. A model in which an effort reference point is learnt from previous choices is developed and used to demonstrate, that like humans, the expectation of effort has a significant effect on the subjective value of effort in a predictable manner. In a different task, effort is presented not as an absolute quantity in the options but a per-unit (or ‘wage’) quantity. By presenting options with two rewards and manipulating the wage associated with one of the rewards, this thesis demonstrates monkeys choose consistently with the principles of revealed preference theory. In recordings of midbrain dopamine neurons in the awake animal, the results of the first two sets of experiments are related to the activity of dopamine neurons during effort valuation. At the presentation of effort-predicting stimuli, a reference-dependent effort cost is encoded. Introducing risky effort cues, I elicited effort prediction errors and by comparison to reward prediction errors demonstrated a common scale of encoding between effort and reward. Taken together these findings suggest subjective effort valuation is reference-based and encoded in midbrain dopamine neurons, supporting the hypothesis that dopamine neurons encode a net utility signal.

This thesis extends the understanding of the neural and neurochemical contributions to two forms of behavioural adaptation, reversal learning and contingency degradation, in which stimulus/action-reward contingencies are altered. The results are interpreted within the psychological framework of the compulsivity construct, and their implications for the pathological behaviour of obsessive-compulsive-disorder (OCD) are considered. The orbitofrontal cortex (OFC) and striatum are key brain structures involved in reversal learning, as are the neurotransmitters serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA) within those respective regions. However, there has been little empirical evidence of how these two structures and neurochemical systems interact, especially in the functional context of reversal learning. In Chapter Three, the impact of experimentally-induced reductions of 5-HT in the anterior OFC on monoamine levels in subcortical structures such as the striatum and amygdala was determined, DA being found to be significantly up-regulated in the amygdala. Functionally, 5-HT depletion of the OFC has previously been shown to induce deficits in reversal learning. To determine the possible causal significance of amygdala dopamine up-regulation for said reversal learning deficit, the effects of blocking the upregulation with the infusion of intra amygdala DA receptor antagonists following bilateral OFC 5-HT depletion were investigated in a reversal learning paradigm. In Chapter Four, the differential roles of regions of striatum were examined in visual reversal learning. Two recent investigations in non-human primates highlighted the role of the striatum in reversal learning,but pinpointed the critical region to be either the ventromedial caudate or the putamen. Marmosets were trained on a serial reversal task that allowed multiple acute neural manipulations, and the ventromedial caudate and putamen were then reversibly inactivated using the GABAA agonist muscimol. Results indicated dose-related impairments specifically in reversal learning within the putamen, with sparing of discrimination retention. By contrast, similar reversible inactivation of the caudate nucleus produced marked deficits in visual discrimination performance (retention). In Chapter Five, the neural basis of action-outcome contingency knowledge was investigated by inactivating distinct regions of the PFC, the perigenual ACC (pgACC; area 32) and the anterior OFC, and determining response sensitivity to the degradation of action-outcome contingencies. In previous work, excitotoxic lesions of either the pgACC or the OFC had been found to induce insensitivity to contingency degradation in marmosets. However, the design of that experiment did not allow specification of whether stimulus- or action-outcome associations were disrupted, and a precise neural locus could not be determined for the behavioural effects as the OFC lesions included parts of the lateral and medial OFC. I therefore developed a novel contingency degradation paradigm that distinguished between stimulus- and action-outcome associations to enable the study of acute pharmacological manipulations in both brain regions. The pgACC and OFC were reversibly inactivated using GABAA-GABAB agonists (muscimol-baclofen). Whereas the pgACC inactivation produced selective deficits in sensitivity to action-outcome contingency degradation, OFC inactivation reduced the suppressive effect of noncontingent reward on responding more generally but left intact sensitivity to degradation of the contingencies. These results are discussed in terms of different theories of the functions of the pgACC and OFC. In the final discussion the findings on the neural substrates of reversal learning and contingency degradation are drawn together in terms of their significance for theories of PFC involvement in cognitive control, and for the understanding of OCD and other neuropsychiatric disorders.

Chronic pain in Parkinson's disease (PD) is significantly higher than in the general population, and is often overlooked to be a side effect of the impaired movement and stiffness associated with the disease. There is scientific rational to link the degeneration which occurs in the PD brain to the increased prevalence of chronic pain. Although acute pain thresholds have been shown to be lower in PD, there is a lack of neuroimaging research which investigates the role of central processing in PD. Therefore, this thesis documents one of the first neuroimaging studies to investigate whether central pain processing is abnormal in people with PD. The perception of pain can be modulated by subject driven processing such as attention, salience assignment and cognition, which is regarded as top-down modulation and is often abnormal in chronic pain conditions. A technique to investigate top-down processing is to study the neural activity during the anticipation of acute pain stimulus. Hence, in this thesis, we used EEG to record the anticipatory response to acute pain stimuli delivered by a CO2 laser. Our first experiment compared people with PD whilst off their medication to a healthy age-matched cohort and concluded that the PD patients showed an amplified anticipatory response and a higher sensitivity to the laser. In contrast, following dopamine increasing medication, no difference was shown between the PD and healthy controls. To investigate the role of dopamine in altering pain perception in PD, the second study investigated the effect of manipulating the dopamine D2 receptor in young healthy volunteers. The study reported that the agonist and antagonist induced reductions in neural activity in the parietal and temporal lobes during pain perception, and the antagonist induced a reduction in the activity of the insula during pain perception, and intensified the effect of certainty during anticipation on the rating of pain. The patient and the D2R manipulation studies demonstrated that dopamine may play a role in the top-down processing of pain and hence explain why chronic pain in PD is prevalent. Importantly the pain rating and sensitivity were not changed by Levodopa administration or D2R manipulation and supports the notion that dopamine is central to modulating top-down processing, rather than directly coding the intensity of pain. Therefore, together with further neuroimaging research conducted during the PhD, there is strong evidence of a disruption in the central processing of pain in PD and promotes the use of alternative therapies in the treatment of chronic pain in PD.

Psychotic symptoms are prevalent in a wide variety of psychiatric and neurological disorders. Yet, despite decades of research, the neurobiological mechanisms via which these symptoms come to manifest themselves remain to be elucidated. I argue in this thesis that using a mechanistic approach towards understanding psychosis that borrows heavily from the predictive coding framework, can help us understand the relationship between neurobiology and symptomology. In the first results chapter I present new data on a biomarker that has often been cited in relation to psychotic disorders, which is glutamate levels in the anterior cingulate cortex (ACC), as measured with magnetic resonance spectroscopy. In this chapter I aimed to replicate previous results that show differences in glutamate levels in psychosis and health. However, no statistically significant group differences and correlations with symptomology were found. In order to elucidate the potential mechanism underlying glutamate changes in the anterior cingulate cortex in psychosis, I tested whether a pharmacological challenge of Bromocriptine or Sulpiride altered glutamate levels in the anterior cingulate cortex. However, no significant group differences were found, between medication groups. In the second results chapter I aimed to address a long-standing question in the field of computational psychiatry, which is whether prior expectations have a stronger or weaker influence on inference in psychosis. I go on to show that this depends on the origin of the prior expectation and disease stage. That is, cognitive priors are stronger in first episode psychosis but not in people at risk for psychosis, whereas perceptual priors seem to be weakened in individuals at risk for psychosis compared to healthy individuals and individuals with first episode psychosis. Furthermore, there is some evidence that these alterations are correlated with glutamate levels. In the third results chapter I aimed to elucidate the nature of reward prediction error aberrancies in chronic schizophrenia. There has been some evidence suggesting that schizophrenia is associated with aberrant coding of reward prediction errors during reinforcement learning. However it is unclear whether these aberrancies are related to disease years and medication use. Here I provide evidence for a small but significant alteration in the coding of reward prediction errors that is correlated with medication use. In the fourth results chapter I aimed to study the influence of uncertainty on the coding of unsigned prediction errors during learning. It has been hypothesized by predictive coding theorists that dopamine plays a role in the precision-weighting of unsigned prediction error. This theory is of particular relevance to psychosis research, as this might provide a mechanism via which dopamine aberrancies, might lead to psychotic symptoms. I found that blocking dopamine using Sulpiride abolishes precision-weighting of unsigned prediction error, providing evidence for a dopamine mediated precision-weighting mechanism. In the fifth results chapter I aimed to extend this research into early psychosis, to elucidate whether psychosis is indeed associated with a failure to precision-weight prediction error. I found that first episode psychosis is indeed associated with a failure to precision-weight prediction errors, an effect that is explained by the experience of positive symptoms. In the sixth results chapter I explore whether the degree of precision-weighting of unsigned prediction errors is correlated with glutamate levels in the anterior cingulate cortex. Such a correlation might be plausible given that psychosis has been associated with both. However, I did not find such a relationship, even in a sample of 137 individuals. Thus I concluded that anterior cingulate glutamate levels might be more related to non-positive symptoms associated with psychotic disorders. In summary, a mechanistic approach towards understanding psychosis can give us valuable insights into the disease mechanisms at play. I have shown here that the influence of expectations on perception is different across disease stage in psychosis. Furthermore, aberrancies in prediction error mechanisms might explain positive symptoms in psychosis, a process likely mediated by dopaminergic mechanisms, whereas evidence for glutamatergic mediation remains absent.

Depression is a highly prevalent clinical condition which has been estimated to affect a growing part of the population in western countries. Alongside expenditure on diagnostics and treatment, there is a high economic impact due to lost productivity. Although a range of treatments are available, diagnoses are currently costly and require subjective assessment by a specialist. Moreover, treatment selection can be lengthy and can involve trial and error. To develop better diagnostics, stratification, and treatments for depression, we need a better understanding of the condition across different levels - from neural mechanisms to cognition and behaviour. Computational modelling is an emergent theory-driven approach which can aid linking data across different levels of analysis - from neural mechanisms and computations in the brain, to cognitive algorithms and observable behaviour. Some models integrate diverse findings and make predictions, while others enable inference of clinical measures which are not obvious in raw data. Modelling can lead to better understanding of depression, and in turn to better stratification and treatments. On the other hand, machine learning and classification methods can help detect clinically-relevant patterns in experimental data in a purely data-driven manner. This can lead to development of better screening and diagnostic methods. In the current work, we first review some of the most prominent neurocognitive theories of depression, as well as existing studies which used computational modelling methods. Based on our review, we argue that modelling can provide a rich set of tools for a better understanding of the condition. We then develop two novel computational modelling accounts of depression. In the first account, we propose an explicit mechanistic link between a robust behavioural negative bias effect and some of the widely reported or theorised neural aspects of depression - hyperactive amygdala and inhibited dopamine release. In the second account, we attempt to better explain depressive cognitive deficits and show how they can arise from depression-relevant etiological factors - altered valuation and controllability estimates. Finally, in the third part of this work we attempt to develop a novel system for detecting depressive symptoms based on a combination of face-tracking, eye-tracking and cognitive performance measures. We evaluate the system in a pilot experiment and show that a combination of measures can achieve better results than measures from each domain separately.

Apathy or lack of motivation is increasingly recognized to be a major factor affecting quality of life and prognosis in many neurological conditions. It is particularly prevalent in Parkinson's disease, impacting on every disease stage, including de novo cases, and has been reported to affect up to 70% of cases. Despite the pervasiveness of apathy, challenges remain in its detection, clinical assessment and treatment. Several lines of evidence have implicated fronto-striatal reward related neural pathways in the genesis of apathy but the precise processes remain to be fully explained. This thesis examines the potential mechanisms of apathy using Parkinson's disease as a model to study the condition. Novel oculomotor tasks that used eye movement and pupillary responses were developed to help assess if insensitivity to incentives could be an underlying component of apathy. This was examined in healthy young and elderly participants as well as in patients with Parkinson's disease. Patients were tested both ON and OFF their normal dopaminergic medication so that the effect of dopamine could be assessed and the association with apathy determined. This was also performed in a pharmacological study in young participants with the use of Haloperidol, a dopaminergic D2-selective antagonist. Insensitivity to rewards modulated by dopamine was regarded to be a contributory mechanism of apathy in Parkinson's disease and also applicable to general mechanisms of motivation in healthy populations.

Apathy is a common, debilitating but poorly understood disorder of motivation that manifests in reduced goal directed behaviours. The integration of cost and reward information about potential actions is central to normal motivated behaviour. This thesis will argue that disruption of this normal process - often termed effort-based decision making - underlies the clinical syndrome of apathy. Two broad sets of questions are investigated to address this hypothesis, in clinically apathetic patients with either Parkinson's disease (PD) or a genetic form of cerebral small vessel disease (SVD). Firstly, are apathetic patients hypersensitive to effort costs, or insensitive to rewards when making decisions about potential actions? Do these changes associated with apathy map onto physiological alterations in reward processing and disruption of anatomical regions that underlie normal effort-based decision making? And do these behavioural changes occur along the same axis as those induced by dopamine depletion? Secondly, using an ecological framework of decision making in which participants must compare current (foreground) reward information to the background reward context to guide adaptive behaviour, this research explores whether apathy is associated with aberrant use of background reward information, and how this interacts with the dopaminergic system. The studies demonstrate effort-based decision making is disrupted in patients with apathy, driven by reduced incentivisation by the rewarding outcomes of actions rather than hypersensitivity to effort costs themselves. Reward insensitivity manifests in both behavioural choices and physiological responses to incentives, is associated with reduced white matter integrity in tracts connecting regions crucial for effort-based decision making, but occurs along a separate mechanistic axis to changes induced by dopamine depletion. Furthermore, whilst healthy human decision makers are remarkably sensitive to changes in background reward environment, apathy changed the influence of this information - dependent on dopaminergic tone. Together, the work in this thesis presents evidence that disruption of a specific cognitive process underlies the apathetic syndrome. This advances understanding of normal motivated behaviour and identifies potential therapeutic avenues to treat this important clinical syndrome.

Primary aldosteronism is the most common secondary cause of hypertension with a prevalence of about 10%. About half of PA cases are caused by aldosterone-producing adenomas (APA). Two APA subtypes, ZG-like and ZF-like APAs, have been described, according to the histological resemblance to normal zona glomerulosa (ZG) and zona fasciculata (ZF), underlying somatic mutations (KCNJ5 commonly found in ZF-like, CACN1AD, ATP1A1, ATP2B3, CTNNB1 in ZG-like APAs), and transcriptome profile. It is unknown if the process of tumorigenesis differs between ZG- and ZF-like APAs. In order to define ZG specific genes, we have compared the transcriptome of APAs and their adjacent adrenal glands by microarray assay. RNA was isolated by laser capture microdissection (LCM) from adjacent ZG, ZF and APAs from 14 patients with Conn’s and 7 patients with phaeocromocytoma. Two top hit genes from the comparison of ZG vs ZF were functionally studied, ANO4 and NEFM. NEFM, encoding neurofilament medium, was the fourth most up-regulated gene in ZG vs ZF, showing 14.8-fold-fold higher expression levels (p=9.16-12) in ZG than ZF. NEFM was also one of the most down-regulated genes in ZF-like vs ZG-like APAs. Immunohistochemistry (IHC) confirmed selective high expression of NEFM in ZG and ZG-like APAs. Silencing NEFM in H295R cells increased aldosterone secretion and cell proliferation. In addition, it increased stimulation and inhibition, respectively, of aldosterone secretion from H295R cells by the dopamine receptor D1R agonist fenoldopam and antagonist SCH23390. IHC showed predominantly intracellular staining for D1R in NEFM-rich ZG-like APAs, but membranous staining in NEFM-poor ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZG-like APAs was lower than in cells from ZF-like APAs. NEFM expression levels directly correlate with KCNJ5 phenotype: KCNJ5 mutations down-regulate NEFM mRNA and protein levels in H295R cells and in primary cells from ZG-like APAs. ANO4,encoding a Ca2+-activated chloride channel family member, was the third most upregulated gene, showing 19.9-fold higher expression levels (p=6.6x10-24) in ZG than ZF. IHC confirmed ZG selectivity of ANO4 protein in the adrenal cortex. The staining was mainly cytoplasmic. Unlike NEFM, there was no difference in expression of ANO4 between ZG- and ZF-like APAs, the levels being mid-way between those of ZF and ZG. Overexpression of ANO4 in H295R cells caused an increase in CYP11B2 and NR4A2 gene expression levels but basal aldosterone secretion was unchanged. In the presence of calcium agonists, ANO4 reduced aldosterone secretion. ANO4 subcellular localisation was confirmed as cytoplasmic by immunofluorescence microscopy of transfected cells. When exposed to calcium ionophores, ANO4 generated small chloride currents as detected by YFP assay. In summary, the comparison of transcriptome of ZG with paired ZF found unexpected up-regulated genes. Most of the highly up regulated genes in human ZG, including NEFM and ANO4, inhibit either basal or stimulated aldosterone secretion, and this may reflect an adaptive response to high salt intake. No clear-cut correspondence was found between transcriptome of APAs and their resembling zone of adrenal cortex. The down-regulation of NEFM following transfection of mutant KCNJ5 suggests that ZF-like properties may be a consequence of mutation, rather than tissue of origin.

Connectomics – the study of neural connectivity – is primarily concerned with the mapping and characterisation of wired synaptic links; however, it is well established that long-distance chemical signalling via extrasynaptic volume transmission is also critical to brain function. As these interactions are not visible in the physical structure of the nervous system, current approaches to connectomics are unable to capture them. This work addresses the problem of missing extrasynaptic interactions by demonstrating for the first time that whole-animal volume transmission networks can be mapped from gene expression and ligand-receptor interaction data, and analysed as part of the connectome. Complete networks are presented for the monoamine systems of Caenorhabditis elegans, along with a representative sample of selected neuropeptide systems. A network analysis of the synaptic (wired) and extrasynaptic (wireless) connectomes is presented which reveals complex topological properties, including extrasynaptic rich-club organisation with interconnected hubs distinct from those in the synaptic and gap junction networks, and highly significant multilink motifs pinpointing locations in the network where aminergic and neuropeptide signalling is likely to modulate synaptic activity. Thus, the neuronal connectome can be modelled as a multiplex network with synaptic, gap junction, and neuromodulatory layers representing inter-neuronal interactions with different dynamics and polarity. This represents a prototype for understanding how extrasynaptic signalling can be integrated into connectomics research, and provides a novel dataset for the development of multilayer network algorithms.

The midbrain dopamine system is involved in many aspects of purposeful behaviour and, when compromised, can have devastating effects on movement and cognition as seen in diseases like Parkinson's. In the healthy brain, dopamine neurons are thought to play particularly important roles in learning by signalling errors in reward prediction. The objective of this thesis was to investigate the diversity in the functional properties of the midbrain dopamine system, and how this is altered through genetic variation of relevance to Parkinson's and development of cell phenotype. This objective was addressed with a combination of behavioural experiments, in vivo single-cell recording and labelling (both in anaesthetised as well as awake rodents), immunofluorescence labelling, retrograde tracing and stereology. In a first set of experiments, it was demonstrated that chronic as well as acute genetic challenges can alter the firing patterns of midbrain dopamine neurons. Using a novel bacterial artificial chromosome-transgenic rat model, it was shown that the R1441C mutation in human leucine-rich repeat kinase 2, which is linked to Parkinson's, leads to motor deficits and an age-dependent reduction in the in vivo firing variability and burst firing of substantia nigra pars compacta (SNc) dopamine neurons. These findings help reveal processes of early, pre-degenerative dysfunction in dopamine neurons in Parkinson's. Similar effects on firing variability and burst firing of SNc dopamine neurons were found in a mouse model with conditional knock- out of the transcription factors Forkhead box A1 and A2 (FoxA1/2) in midbrain dopamine neurons. These findings indicate that FoxA1/2 are not only crucial for the early development of dopamine neurons, but also their function in the mature brain. In a second set of experiments in wildtype mice, it was demonstrated that midbrain dopamine neurons (located in SNc and ventral tegmental area) show diverse expression of the molecular markers Calbindin, Calretinin, Aldh1a1, Sox6, Girk2, SatB1 and Otx2. It was found that selective expression of these markers is of use for discriminating between midbrain dopamine neurons that project to dorsal striatum or nucleus accumbens. To elucidate whether the diverse molecular marker expression would map onto dopamine neurons whose firing correlates with distinct behavioural events, midbrain dopamine neurons were recorded and labelled in head-fixed awake mice either exposed to neutral sensory stimuli or performing a classical conditioning paradigm. The population activity of midbrain dopamine neurons was not modulated by neutral sensory stimuli. Interestingly, fewer than 50% of identified dopamine neurons showed phasic firing increases following reward- predicting cue and/or reward delivery, despite the common assumption that most (if not all) midbrain dopamine neurons signal reward prediction errors. Instead, firing was modulated by other explanatory factors, such as licking, or showed no modulation during the task. Response types of midbrain dopamine neurons were not correlated with their anatomical location nor the selective or combinatorial expression of the markers Aldh1a1, Calbindin and Sox6. In conclusion, the first set of experiments identified how different genetic burdens can alter the in vivo firing of midbrain dopamine neurons, and provide new insights into how circuits can change in pathological or compensatory ways at early disease stages in Parkinson's. The second set of experiments revealed striking heterogeneity of midbrain dopamine neurons in the intact system, and established further a functional diversity in the response types of identified midbrain dopamine neurons that is only partially consistent with canonical reward prediction error signalling.

The frontostriatal dopamine system modulates brain function and is affected by both genetic and environmental factors. Dysfunction of this system is associated with many pathological states, including schizophrenia. The enzyme catechol-O- methyltransferase (COMT) metabolises dopamine and its gene contains a polymorphism (Val158Met) that affects enzyme activity. Delta-9- tetrahydrocannabinol (THC), the main psychoactive component of cannabis, has been suggested to interact with this polymorphism to increase the risk for psychosis and cognitive impairments. Dopaminergic mechanisms are a plausible candidate for mediating this interaction. I used microdialysis coupled with high performance liquid chromatography (HPLC) to examine the effects of THC on extracellular dopamine and its metabolites in the nucleus accumbens, dorsal striatum and medial prefrontal cortex (mPFC) in freely moving mice. Following acute COMT inhibition with tolcapone, THC increased extracellular dopamine levels in the nucleus accumbens in tolcapone-, but not in vehicle-, treated mice. The introduction of the low activity Met allele into the COMT gene produced a highly specific, novel mouse model of the Val158Met polymorphism. In contrast to the effects of acute COMT inhibition, the Met allele protected against THC-induced changes in accumbal dopamine. No interactive neurochemical effects were observed in the dorsal striatum (pharmacological and genetic study) or in a preliminary study of the mPFC (genetic study only). On a progressive ratio task measuring motivational salience, the direction of the interactive effect between COMT genotype and THC differed between 2 independent cohorts and provided tentative leads that stress/arousal-dependent effects on COMT may have a confounding effect. My data provide evidence that COMT activity modulates the effect of THC on accumbal dopamine function, and suggest the mechanism through which this interaction is mediated differs between acute and lifelong reduction in COMT activity. Through the interactive effect on the dopaminergic system, the data provide a potential mechanism for the reported interaction between COMT and cannabis/THC in determining psychosis risk and cognitive impairments.

Dopamine has an important role in normal cognition and reward processing, both of which are impaired in disorders involving dopamine dysfunction such as addiction, schizophrenia, and Parkinson's disease. However, our understanding of the interplay between different aspects of the dopamine system and reward-guided behavior in humans is limited. Food is an important type of reward that is critical for survival and impacts the decisions we make every day. Here, we characterize the relationship between two food-reward related phenotypes and dopamine synthesis capacity (related to tonic dopamine) as well as dopamine D1 and D2 receptor availability in healthy humans. First, we examined the link between dopamine synthesis and receptor availability and body mass regulation in 117 individuals with body mass index (BMI) values ranging from normal to obese. We found that current BMI was related to a pattern of increased dopamine synthesis in the hypothalamus, a region important for homeostatic control of appetite, but decreased dopamine D2 receptor availability in the midbrain, where D2 autoreceptors regulate dopamine release throughout the brain. This suggests that increased BMI is related to a dopamine imbalance between homeostatic drivers of appetite and reward system regulatory control mechanisms that could result in an overactive, unregulated intake of food. Building on this finding, we studied the link between dopamine synthesis capacity and receptor availability and an important food-reward related behavior, foraging. Fifty-seven healthy volunteers completed a computer-based foraging task where we measured their threshold for leaving one group of rewards to search for another in four different reward environments varying from a low to high rate of reward receipt. We found that two particular patterns of dopamine synthesis and receptor availability in the anterior cingulate cortex and basal ganglia were linked to the amount that individuals changed their threshold based on the reward rate of the environment. Finally, since the prefrontal cortex is known to be important for reward-guided behavior, we implemented two methodological advancements aimed to address limitations that make it difficult to measure cortical dopamine in humans with PET imaging. The first method involves partial volume correction and surface-based smoothing in order to increase the signal to noise in the cortex. The second method is a data-driven PET data parcellation and automated reference region selection algorithm to optimize the voxels included in the reference region. In conclusion, we have characterized the dopaminergic contribution of two different foodreward guided phenotypes and have developed two techniques that will aid future research on the role of cortical dopamine. Understanding the neural mechanisms underlying these rewardguided behaviors helps us to not only understand normal behavior, but also serves as a reference for comparison when studying related pathological states.

Networks of interneurons within the spinal cord coordinate the rhythmic activation of muscles during locomotion. These networks are subject to extensive neuromodulation, ensuring appropriate behavioural output. Astrocytes are proposed to detect neuronal activity via Gαq-linked G-protein coupled receptors and to secrete neuromodulators in response. However, there is currently a paucity of evidence that astrocytic information processing of this kind is important in behaviour. Here, it is shown that protease-activated receptor-1 (PAR1), a Gαq-linked receptor, is preferentially expressed by glia in the spinal cords of postnatal mice. During ongoing locomotor-related network activity in isolated spinal cords, PAR1 activation stimulates release of adenosine triphosphate (ATP), which is hydrolysed to adenosine extracellularly. Adenosine then activates A1 receptors to reduce the frequency of locomotor-related bursting recorded from ventral roots. This entails inhibition of D1 dopamine receptors, activation of which enhances burst frequency. The effect of A1 blockade scales with network activity, consistent with activity-dependent production of adenosine by glia. Astrocytes also regulate activity by controlling the availability of D-serine or glycine, both of which act as co-agonists of glutamate at N-methyl-D-aspartate receptors (NMDARs). The importance of NMDAR regulation for locomotor-related activity is demonstrated by blockade of NMDARs, which reduces burst frequency and amplitude. Bath-applied D-serine increases the frequency of locomotor-related bursting but not intense synchronous bursting produced by blockade of inhibitory transmission, implying activity-dependent regulation of co-agonist availability. Depletion of endogenous D-serine increases the frequency of locomotor-related but not synchronous bursting, indicating that D-serine is required at a subset of NMDARs expressed by inhibitory interneurons. Blockade of the astrocytic glycine transporter GlyT1 increases the frequency of locomotor-related activity, but application of glycine has no effect, indicating that GlyT1 regulates glycine at excitatory synapses. These results indicate that glia play an important role in regulating the output of spinal locomotor networks.

The ‘synaptic tagging-and-capture’ (STC) theory of cellular memory consolidation holds that memory persistence can be altered by prior or subsequent patterns of neural activity (Redondo & Morris 2011). The aim of this thesis was to develop a realistic model of everyday memory for mice and use the optogenetic toolbox to investigate the neuromodulatory circuitry that modulates persistence of everyday spatial memories. The task involved learning a win-stay rule with the daily goal of finding the location of food in the event arena. Using the developed task, it was confirmed that unrelated novel experiences can facilitate the persistence of spatial memory in a manner sensitive to pharmacological blockade of hippocampal dopamine D1/D5 receptors. Further analysis focused on identifying the specific neuromodulatory systems that mediate this effect. An influential model called the ‘hippocampus- VTA loop’ (Lisman & Grace 2005) points to the critical role of dopaminergic neurons in the ventral tegmental area (VTA), but recent evidence also implicates locus coeruleus (LC) as a potential source of dopamine in the hippocampus (Smith & Greene 2012). In order to identify the dopaminergic structure(s) that may mediate the novelty effect on memory persistence, single unit activity of optogenetically identified catecholaminergic (CAergic) neurons in mouse VTA and LC was recorded in a novelty exploration paradigm. Using tyrosine hydroxylase-Cre knock-in mice and a Cre-dependent adeno-associated viral vectors, CAergic neurons in VTA and LC were selectively tagged with channelrhodopsin-2 (ChR2). Conditional ChR2 expression made it possible to reliably identify CAergic neurons during unit recording sessions in freely moving animals. The main conclusion of the study is that that CAergic neurons in both VTA and LC selectively increase their firing rates in novel environments, relative to both a familiar environment and a home cage baseline. When normalised to their average baseline firing rates, LC neurons are more strongly activated by novelty than VTA neurons. In the final experiment outlined in this thesis, another cohort of Th-Cre mice, in which ChR2 was expressed in CAergic neurons of both VTA and LC using a Cre-dependent adeno-associated virus, was trained on the everyday appetitive spatial memory task. ChR2-mediated photoactivation of CAergic neurons in LC but not in VTA 30 min after encoding, substituting for novelty, was successful in enhancing the persistence of memory. Paradoxically, the effect of optogenetic LC activation was blocked by hippocampal microinfusion of dopamine D1/D5 receptor antagonist but not β-adrenergic receptor antagonist. Results of experiments described in this thesis support the principle of STC theory and collectively indicate that dopamine released from hippocampal terminals of LC neurons mediates the novelty effect on memory persistence. Importantly, they also point to a more general of role of LC in gating of entry to long-term memory.

The zebrafish has an amazing capacity for regeneration which includes regeneration of neurons within the central nervous system (CNS) both during development and into adulthood. This attribute makes the zebrafish a valuable tool in the study of regeneration. In this thesis, the research focussed on the regeneration of a specific type of cell in the CNS, dopaminergic (DA) neurons. The DA system of the zebrafish is believed to be evolutionarily conserved with comparable DA populations found in the brain of mammals. Dissimilar to mammals, however, the zebrafish is capable of regenerating various types of neurons and their axons. Thus, the zebrafish DA system provides an excellent model to study replacement of this specific and important cell type in the adult CNS. We have developed a novel toxin ablation paradigm to specifically ablate select groups of DA neurons in the adult zebrafish diencephalon, leaving other DA populations unaffected. To do this a selective DA toxin, 6-hydroxydopamine, was used. One of the ablated DA diencephalic populations is the only source of dopaminergic spinal innervation in the zebrafish. Their ablation leads to a loss of DA spinal axons following our toxin ablation. The ascending projection of the diencephalic population ablated by the toxin has been suggested as the most likely candidate for a zebrafish equivalent of the mammalian nigro-striatal pathway. The loss of cells is very specific and reproducible, indicating that these cells are particularly vulnerable to the toxin. Quantification of affected populations at various time-points post ablation was carried out to determine the capacity for regeneration of DA neurons in the CNS of zebrafish. This revealed that in some populations neuron numbers returned to those seen in controls. However, in other populations neuron numbers only partially recovered even at late time points. We have shown that this recovery is due to neurogenesis; furthermore, by inducing inflammation after the toxin treatment the recovery of DA cell numbers was accelerated by 50%. Regenerated cells originated from Olig2 positive ependymo-radial glial cells found bordering the diencephalic ventricle. We aimed to investigate the function of this group of ablated neurons through a battery of behavioural tests. These tests revealed deficits in the toxin treated animals’ fine movement, such as is necessary for maintaining shoal cohesion and breeding behaviours, whereas general movement behaviours were not found to be impaired. Zebrafish embryos also present as a great resource in the screening of drugs. Their fast and well characterised early development makes them an ideal tool for investigating previously untested neuroprotectants. A reproducible ablation paradigm similar to that established in the adults was also established in the zebrafish embryo. This was then used as a tool to investigate potential novel neuroprotectants. This screen revealed two new flavonoid compounds which had the ability to induce full protection of the affected dopaminergic cells in the zebrafish embryonic brain. The embryonic ablation model therefore represents a vertebrate in vivo model system for future high throughput screening of neuroprotective compounds against toxin induced DA cell loss. Ultimately, understanding how zebrafish functionally regenerate dopaminergic neurons using this ablation model will likely provide a useful tool into the research of neurodegenerative diseases, such as PD.

The hallmark of an adult mammalian central nervous system is the inability to regenerate after an injury. Zebrafish, on the other hand, have an astounding regenerative capacity. After a spinal cord lesion, zebrafish can re-establish the damaged neuronal network and regain their swimming ability within weeks. This is partly due to the presence of the ependymal radial glia (ERGs), which line the wall of the central canal and act as the stem/progenitor cells of the spinal cord. Under homeostatic conditions the ERGs are largely quiescent, however, the lesion triggers them to proliferate and replace cells that have been lost due to the damage. Previous studies have shown that the regeneration of the motor neurons is affected by the signalling pathways similar to those governing the first development of these cells during embryogenesis, such as Sonic hedgehog, Notch and dopamine signalling. Serotonin (5-HT), similar to dopamine, is a monoaminergic neurotransmitter with a wide range of physiological and behavioural functions. It has also been shown to play a role during development of the nervous system. In this doctoral thesis I address the hypothesis that 5-HT has a positive effect on the development and adult regeneration of motor neurons. In addition, I expand on the previously discovered augmenting effect of dopamine on motor neuron development, by analysing the downstream pathways of its action. I show that during the development, incubating embryos in 5-HT increases the proliferation of the motor neuron progenitor (pMN) cells, which leads to augmented motor neuron production. RT-PCR on FAC sorted pMN cells highlights a number of serotonergic receptors that might be responsible for this effect. Although the downstream pathways are still unknown, 5-HT appears not to act on the sonic hedgehog canonical pathway, as shown by the unchanged expression of the hedgehog effector gene, patched2 after 5-HT treatment. I show that 5-HT does not affect the generation of vsx1+ or pax2a+ interneurons, suggesting that it has a predominant effect on motor neuron production. In the intact spinal cord of an adult zebrafish, the pMN-like ERGs express serotonergic receptors, indicating they are responsive to 5-HT stimulation. After a lesion, 5-HT administration enhances the proliferation of the pMN-like ERGs caudal to the lesion site resulting in an increase in the number of newborn motor neurons. Rostral to the lesion site, administration of exogenous 5-HT does not have an effect on the ERG proliferation, possibly due to the fact that the endogenous source of 5-HT, in the form of the descending axons, is still present and might already elicit a maximal response of the progenitor cells. 5-HT does not have an effect on the proliferation of the progenitor cells dorsal or ventral to the pMNlike domain, nor does it affect the regeneration of the serotonergic interneurons. These results suggest that 5-HT from the brain preferentially contributes to the regeneration of the motor neurons. Dopamine is another monoamine shown to enhance motor neuron production during the development and regeneration. To investigate the downstream pathways of dopamine signalling on motor neuron production during embryogenesis, RNA-sequencing was performed on FAC sorted pMN cells after a treatment with a dopamine agonist, pergolide. The results yielded 14 differentially expressed genes (FDR < 0.05) with diverse functions in the cell, indicating that dopamine might act on multiple targets to promote motor neuron production. Taken together, these results demonstrate the positive effect of monoaminergic stimulation on motor neuron development and regeneration. They provide an insight into the pathways that govern the proliferation of stem/progenitor cells in the embryonic and adult spinal cord, which might contribute to the research working on enhancing adult neurogenesis in mammals.

Fine temporal regulation of dopamine transmission is critical to its effects on behaviour. Dopamine can be cleared from the synapse either by recycling via the dopamine transporter (DAT) or by enzymatic degradation involving catechol-O-methyltransferase (COMT). DAT recycling predominates in striatum and contributes to dopaminergic regulation of reward-guided behaviour, while COMT degradation predominates in cortex and modulates executive functions. However, human functional imaging studies demonstrate interactive effects of DAT and COMT genotype, suggesting that the traditional division between DAT and COMT is not so clear-cut. Given the interdependence of mesolimbic and mesocortical circuitry and the presence of COMT in the striatum, it is possible that DAT and COMT interact to a greater extent than previously thought. We investigated the contributions of DAT and COMT to regulation of dopamine transmission and reward-guided behaviour by combining in vivo electrochemical recording, pharmacology, and behavioural testing in mice. Using fast scan cyclic voltammetry to record evoked dopamine release in anaesthetised animals, we found that systemic DAT blockade increased the size of dopamine transients in the nucleus accumbens (NAc) but not in the medial frontal cortex (MFC), demonstrating that DAT regulates phasic striatal dopamine release and confirming that DAT makes little contribution to regulation of cortical dopamine transmission. Unexpectedly, COMT inhibition did not affect evoked dopamine transients in either the NAc or the MFC. In agreement with these findings, systemic administration of a DAT blocker, but not of a COMT inhibitor, increased motivation to work for reward in a progressive ratio paradigm. COMT inhibition also had little effect on reinforcement learning (RL) strategies during reward-guided decision making. Intriguingly, however, we found that DAT blockade both decreased the influence of model-free RL and increased the influence of model-based RL on behaviour. Our study confirms that DAT regulates dopamine transmission in striatum but not in cortex and indicates that sub-second changes in dopamine transmission in both regions are largely insensitive to COMT. However, our behavioural data reveal the importance of striatal dopamine in multiple components of reward-guided behaviour, including both motivational aspects traditionally associated with striatum as well as cognitive aspects heretofore mainly associated with cortical function. Together, these findings emphasise that reward processing occurs across corticostriatal circuits and contribute to our understanding of how striatal dopamine transmission regulates reward-guided behaviours.

The ability to take effective decisions is fundamental for successful environmental adaptation and survival. In this thesis, I investigated situations in which decisions appear irrational, at least from certain standpoints. I conducted a behavioural decision-making experiment in two groups of macaques: controls and a group with ventromedial prefrontal cortex/medial orbitofrontal cortex (vmPFC/ mOFC) lesions. Some choices lead to compound outcomes composed of different constituent parts. Control macaques' decisions suggested their estimates of the value of the compound were biased away from the sum of the values of the constituents and towards their mean. Lesions of vmPFC/mOFC diminished the size of the effect so that macaques in some ways appeared to make more rational decisions. Based on the results of this experiment I devised a similar Functional Magnetic Resonance Imaging (fMRI) paradigm with the control animals. This demonstrated strong vmPFC/mOFC activity when similar decisions were made and suggested a value comparison process. In addition, I investigated the role of dopamine in learning using Fast-Scan Cyclic Voltammetry (FSCV), while rats performed a simple decision-making task. Theories about the role of dopamine in learning have focused on the possibility that it codes scalar reward value prediction errors. Less consideration has been given to the possibility that dopamine might reflect prediction errors about reward identities regardless of value. I measured dopamine in the nucleus accumbens when unexpected changes in reward value or identity occurred while rats executed a two-choice two-reward instrumental task. Dopamine levels in the nucleus accumbens reflected reward value prediction errors. In addition, however, they also reflected some information about reward identity under some circumstances. Further investigation suggested that this might be due to differences in the nutritional value of different reward types that did not have clear measurable impacts of behaviour in the tasks that I used.

Reward prediction errors are quantitative signed terms that express the difference between the value of an obtained outcome and the expected value that was placed on it prior to its receipt. Positive reward prediction errors constitute reward, negative reward prediction errors constitute punishment. Reward prediction errors have been shown to be powerful drivers of reinforcement learning in formal models and there is thus a strong reason to believe they are used in the brain. Isolating such neural signals stands to help elucidate how reinforcement learning is implemented in the brain, and may ultimately shed light on individual differences, psychopathologies of reward such as addiction and depression, and the apparently non-normative behaviour under risk described by behavioural economics. In the present thesis, I used the event related potential technique to isolate and study electrophysiological components whose behaviour resembled reward prediction errors. I demonstrated that a candidate component, “feedback related negativity”, occurring 250 to 350 ms after receipt of reward or punishment, showed such behaviour. A meta-analysis of the existing literature on this component, using a novel technique of “great grand averaging”, supported this view. The component showed marked asymmetries however, being more responsive to reward than punishment and more responsive to appetitive rather than aversive outcomes. I also used novel data-driven techniques to examine activity outside the temporal interval associated with the feedback related negativity. This revealed a later component responding solely to punishments incurred in a Pavlovian learning task. It also revealed numerous salience-encoding components which were sensitive to a prediction error’s size but not its sign.

Based on the belief that it is a locomotor control structure, the pedunculopontine tegmental nucleus (PPTg) has been considered a potential target for deep brain stimulation (DBS) for Parkinson's disease (PD) patients with symptoms refractory to medication and/or stimulation of established target sites. To date, a number of patients have been implanted with PPTg electrodes with mostly disappointing results. Exact target site in PPTg, possible mechanisms of PPTg-DBS and likely potential benefits need to be systematically explored before consideration of further clinical application. The research described here approaches these questions by (i) investigating the role of the PPTg in gait per se; (ii) developing a refined model of PD that mimics the underlying pathophysiology by including partial loss of the PPTg itself; (iii) adapting a wireless device to let rats move freely while receiving DBS; and (iv) investigating the effect of DBS at different sites in the PPTg on gait and posture in the traditional and refined model of PD. Underlining the concern that understanding the PPTg as a locomotor control structure is inadequate, the experiments showed that neither partial nor complete lesions of PPTg caused gait deficits. The refined model showed hardly any differences compared to the standard one, but the effect of DBS in each was very different, highlighting the need to take degeneration in the PPTg into consideration when investigating it as a DBS target. The differential results of anterior and posterior PPTg-DBS show the critical importance of intra-PPTg DBS location: Anterior PPTg electrodes caused severe freezing and worsened gait while some gait parameters improved with stimulation of posterior PPTg. The results suggest mechanisms of PPTg-DBS beyond the proposed activation of over-inhibited PPTg neurons, including aggravation of already dysfunctional inhibitory input by anterior PPTg-DBS and activation of ascending projections from posterior PPTg to the forebrain.

Dopamine is an important neurotransmitter that is involved in several human functions such as reward, cognition, emotions and movement. Abnormalities of the neurotransmitter itself, or the dopamine receptors through which it exerts its actions, contribute to a wide range of psychiatric and neurological disorders such as Parkinson’s disease and schizophrenia. Thus far, despite the great interest and extensive research, the exact role of dopamine and the causalities of dopamine related disorders are not fully understood. Here we have developed multimodal imaging methods, to investigate the release of dopamine and the distribution of the dopamine D2-like receptor family in-vivo in healthy humans. We use the [11C]PHNO PET ligand, which enables exploration of dopamine-related parameters in striatal regions, and for the first time in extrastriatal regions, that are known to be associated with distinctive functions and disorders. Our methods involve robust approaches for the manual and automated delineation of these brain regions, in terms of structural and functional organisation, using information from structural and diffusion MRI images. These data have been combined with [11C]PHNO PET data for quantitative dopamine imaging. Our investigation has revealed the distribution and the relative density of the D3R and D2R sites of the dopamine D2-like receptor family, in healthy humans. In addition, we have demonstrated that the release of dopamine has a functional rather than a structural specificity and that the relative densities of the D3R and D2R sites do not drive this specificity. We have also shown that the dopamine D3R receptor is primarily distributed in regions that have a central role in reward and addiction. A finding that supports theories that assigns a primarily limbic role to the D3R.