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Cardiovascular disease is the leading cause of mortality in the developed world with up to fifty percent of cases being due to sudden cardiac death. Changes in sympatho-vagal balance underpin many cardiovascular conditions including heart failure and myocardial infarction. Neuraxial modulation of the autonomic nervous system is an emerging therapy to prevent ventricular arrhythmias, the main cause of sudden cardiac death. <b>Chapter One</b> reviews our current understanding of how the cardiac autonomic nervous system influences ventricular arrhythmogenesis. A particular focus was on the controversial role of cholinergic receptors and nitric oxide (NO) in parasympathetic protection from ventricular arrhythmias. Tetrahydrobiopterin (BH₄), a critical cofactor for both tyrosine hydroxylase and NO synthases, and the co-transmitter neuropeptide-Y (NPY) may also influence sympathetic triggering of ventricular arrhythmias. This leads to the specific aims of the thesis which were to determine the mechanisms of the cholinergic antifibrillatory effect, investigate the role of cotransmission in arrhythmogenesis and, the mechanistic role of BH4 in autonomic cardiovascular control. <b>Chapter Two</b> detailed the experimental approach taken to investigate the hypotheses. A novel Langendorff heart preparation was developed with intact autonomic nerves to investigate how the stable analogue of acetylcholine, carbamylcholine (CCh) raises ventricular fibrillation threshold (VFT) and whether exogenous or endogenously released NPY lowers VFT. These actions are further investigated using optical mapping, dye free imaging of ventricular cell monolayers, immunohistochemistry, ELISA assays and measurements of NO metabolite production. To investigate the role of BH4 in the sympathetic control of the heart, an IRES-cre recombinase strategy was used to produce genomic deletion of GCH1 (the gene encoding BH4) in sympathetic neurons. Biopterins and plasma catecholamines were measured using HPLC, and blood pressure and heart rate via tail cuff plethysmography. <b>Chapter 3</b> showed that CCh increased VFT, prolonged action potential duration and flattened the electrical restitution curve. This effect required stimulation of both muscarinic and nicotinic receptors and the generation of nNOS derived NO utilising a cGMP dependent pathway. These observations are in keeping with established evidence demonstrating the obligatory role of the muscarinic receptor and indicate that the role of NO is likely to be via modulation of cholinergic neurotransmission. <b>Chapter 4</b> studied the role of the sympathetic co-transmitter NPY. NPY has been shown to increase ventricular myocyte calcium dynamics. Plasma levels are also increased post myocardial infarction and during heart failure, and correlate with outcomes. Perfusion of NPY decreased VFT via a Y1 receptor dependent mechanism and increased arrhythmic activity in myocyte monolayers. Direct sympathetic stimulation resulted in NPY release and remained pro-arrhythmic despite &beta;-blockade, an effect that could be abolished by combined &beta;-Y₁ receptor blockade. These observations indicated that NPY may be a novel, pro-arrhythmic trigger amenable to therapeutic pharmacological modulation. <b>Chapter 5</b> details the generation and phenotyping of two tissue specific Gch1 knockout mouse models. Whilst one model failed to produce significant lowering of BH₄ in sympatho-adrenal tissue, the other did result in a marked neuro-motor phenotype. A biochemical rescue or alternative genomic modification approach would be required to study the cardiovascular phenotype of sympathetic Gch1 deletion in more detail. <b>Chapter 6</b> is a concluding discussion summarising the main findings of the thesis, placing them in a clinical context and discussing avenues for further research.

Neuronal communication in the mammalian brain relies on the presynaptic release of neurotransmitters which bind to ligand-gated ion channels found on postsynaptic neurons to modulate neuronal excitability. One such neurotransmitter is acetylcholine (ACh), a small molecule that is the signalling messenger of the cholinergic system. The cholinergic system is involved in a variety of behavioural functions including motor activity, sensory function, and higher executive commands. Dopaminergic neurons in the substantia nigra pars compacta (SNc) and the basal ganglia in general have long been implicated in initiation and completion of voluntary movement. Studies have shown that cholinergic neurons from two brainstem nuclei, the laterodorsal tegmental nucleus and the pedunculopontine nucleus, project onto substantia nigra dopaminergic (DA) neurons in the midbrain and release ACh, GABA or both to modulate motor behaviours. However, with prior research primarily focused on demonstrating the phenomenon of co-transmission itself, the subcellular distribution and dynamics of ACh and GABA release onto SN DA neurons receiving co-transmitted inputs largely remains to be investigated. The present study investigates the spatial and physiological properties of ACh/GABA co- transmission from brainstem cholinergic axons synapsing onto medial SN DA neurons to understand its role in tuning the neuron’s excitatory-inhibitory balance. To that end, we developed a channelrhodopsin (ChR2)-based functional input mapping technique with high spatial resolution to probe the dendritic distribution of ACh and GABA synaptic inputs onto DA neurons in ChATcre::ChR2 mice. Using this technique, we discovered three different types of monosynaptic inputs from cholinergic axons onto DA cells: co-transmitted ACh/GABA, GABA only, and ACh only. Furthermore, we revealed a somatodendritic patterning of cholinergic input distribution onto DA cells with a predominant GABA conductance along the lateral dendrites and a soma-centered mix ACh/GABA transmission. Physiological findings were corroborated using immunolabeling against VGAT and VAChT, which showed many closely spatially clustered ACh and GABA- specific cholinergic terminals and few truly colocalized VAChT and VGAT terminals. This result revealed that true co-transmission represents a minority of the presynaptic mode of release from cholinergic axons onto medial SN DA neurons, and that the majority actually share closely spatially clustered ACh and GABA-specific cholinergic terminals. To investigate the dynamic properties of soma-centered ACh/GABA transmission, we restricted our stimulation field to the cell body to measure the contribution of nAChR and GABAR-mediated conductances without recruiting the lateralized population of primary GABA inputs. We then employed a deconvolution method to understand the relative plasticity of contributions of nAChRs and GABARs to ACh/GABA transmission onto DA cells. We confirmed an initial dominant GABAergic component of ACh/GABA transmission that was previously reported. However, we found that the GABAergic contribution had a greater decay compared to the ACh component with repeated stimulations. As such the predominant initial inhibition is followed by a subsequent equalization of excitatory and inhibitory conductances. Finally, we performed similar experiments to compare the short-term plasticity of the isolated GABA conductance during 15 Hz stimulation between the populations of mix ACh/GABA inputs proximally and the population of primary GABA inputs found on the lateral dendrites 160 μm from the cell body. Interestingly, the lateral GABA component was more sustained across repeated stimulations compared to the proximal GABA conductance, suggesting a differential contribution to excitation/inhibition balance by spatially distributed populations of ACh and GABA inputs from cholinergic axons onto the dendrites of medial SN DA neurons. To our knowledge, this is the first study to examine the distribution and dynamics of ACh/GABA transmission onto midbrain DA system using fine-scale ChR2-assisted subcellular input mapping and conductance deconvolution. / Graduate / 2022-04-12

Des travaux récents démontrent que certains neurones dopaminergiques du mésencéphale ont la capacité de libérer du glutamate en plus de la dopamine (DA). Ce phénomène de « co-transmission » requiert l’expression du transporteur vésiculaire du glutamate de type 2 (VGLUT2) dans les neurones dopaminergiques. Certaines observations montrent que l’expression de VGLUT2 dans les neurones dopaminergiques survient tôt durant leur développement et est essentiellement limitée aux neurones de l’aire tegmentaire ventrale (VTA). De plus, cette libération de glutamate se retrouve principalement au niveau des terminaisons de ces neurones dans le striatum ventral, mais pas dans le striatum dorsal. Ces données suggèrent d’une part un rôle développemental possible du glutamate dans les neurones dopaminergiques, et d’autre part, que les signaux dérivés des neurones cibles puissent réguler le double phénotype des neurones dopaminergiques menant ainsi à une plasticité phénotypique. Par ailleurs, il est toujours inconnu si cette libération de glutamate se fait à partir des terminaisons qui relâchent la DA ou à partir de terminaisons axonales distinctes. De plus, le rôle physiologique de ce surprenant phénomène de co-transmission reste également inconnu. Ainsi, dans cette étude, nous avons d’abord démontré in vitro et in vivo que l’expression de VGLUT2 est nécessaire pour la survie et la croissance d’une sous-population de neurones dopaminergiques. En utilisant une lignée de souris ayant une délétion génique spécifique de VGLUT2 dans les neurones dopaminergiques, nous avons observé une diminution du nombre de terminaisons dopaminergiques et glutamatergiques dans le striatum, une baisse de libération de DA dans le striatum ventral, une diminution de la coordination motrice ainsi qu’une diminution de l’activité locomotrice induite par les drogues d’abus. D’autre part, nous avons démontré in vitro et in vivo que les neurones dopaminergiques au double phénotype établissent des terminaisons distinctes afin de relâcher le glutamate et la DA. De plus, nous démontrons que ce phénomène de ségrégation des sites de libération semble être induit par une interaction avec les neurones du striatum ventral. Ces travaux démontrent le rôle physiologique déterminant de la co-transmission DA-glutamate pour l’homéostasie du système DAergique et dévoile une caractéristique fondamentale de l’établissement des terminaisons axonales de ces neurones. Ces travaux permettent ainsi de mieux comprendre les rôles physiologiques de la co-libération de glutamate par les neurones du système nerveux central et présentent une nouvelle perspective sur les dysfonctions potentielles de ces neurones dans les maladies du cerveau. / A subset of midbrain dopamine (DA) neurons has been shown to express the type 2 vesicular glutamate transporter (VGLUT2) supporting their capacity for glutamate co-release from some of their axon terminals. However, the physiological significance of this phenomenon is presently unknown. VGLUT2 expression by DA neurons occurs early during their development and is mainly found in DA neurons localized to the ventral tegmental area (VTA). Glutamate release by DA neurons can be detected at terminals contacting ventral but not dorsal striatal neurons. Together, these findings suggest the possibility glutamate co-release by DA neurons plays a developmental role and that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is undetermined. Moreover, the physiological role of glutamate release by DA neurons is essentially unknown. Using a conditional gene knock-out approach to selectively disrupt the Vglut2 gene in mouse DA neurons, we obtained in vitro and in vivo evidence demonstrating reduced growth and survival of mesencephalic DA neurons, associated with a decrease in the density of DA innervation in the nucleus accumbens, reduced activity-dependent DA release, decreased motor coordination and impaired locomotor activation induced by drugs of abuse. In this study we also provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals are mostly segregated and that striatal neurons regulate the co-phenotype of midbrain DA neurons and the segregation of release sites. These findings provide strong evidence for a functional role of the glutamatergic cophenotype in the development of mesencephalic DA neurons, unveils a fundamental feature of dual neurotransmission and plasticity of the DA system and open new perspectives into the pathophysiology of brain diseases implicatingthe DA system.

La microscopie optique stochastique de reconstruction (STORM) contourne la limite de diffraction en enregistrant des signaux monomoléculaires spatialement et temporellement séparés, atteignant une résolution de ~10-40 nm. Dans mon étude, j'ai développé une stratégie d'imagerie et d'analyse de données dSTORM bicolore afin d'étudier l'ultrastructure des synapses inhibitrices mixtes. Mes résultats ont montré que les GlyRs, les GABAARs, la géphyrine et RIM1/2 présentent une organisation intra-synaptique hétérogène et forment des domaines sous-synaptiques (SSDs). Les GlyR et les GABAAR ne sont pas complètement mélangés, mais peuvent occuper des espaces différents à la densité post-synaptique (PSD). De plus, les SSD de géphyrine postsynaptique sont alignées avec les SSD de RIM1/2 pré-synaptiques, formant des nanocolonnes trans-synaptiques. Au cours d'une activité neuronale élevée par traitement 4-AP, la corrélation spatiale entre les GlyRs, les GABAARs et la géphyrine a augmentée au PSD. De plus, la corrélation spatiale des GlyRs et RIM1/2 a également augmenté, tandis que celle des GABAARs et RIM1/2 n'a pas changé. Le nombre de SSD par synapse pour ces protéines synaptiques n'est pas modifié par 4-AP. Cette étude fourni un nouvel angle de compréhension des mécanismes sous-jacents à la co-transmission GABAergique/glycinergique. / Stochastic optical reconstruction microscopy (STORM) bypasses the diffraction limit by recording spatially and temporally separated single molecule signals, achieving a resolution of ~10-40 nm. In my study, I have developed a two-color dSTORM imaging and data analysis strategy, in order to investigate the ultrastructure of mixed inhibitory synapses. My results show that GlyRs, GABAARs, gephyrin and RIM1/2 exhibit a heterogeneous intra-synaptic organization and form sub-synaptic domains (SSDs). GlyRs and GABAARs were not fully intermingled, but sometimes occupied different spaces at the post-synaptic density (PSD). In addition, post-synaptic gephyrin SSDs were aligned with pre-synaptic RIM1/2 SSDs, forming trans-synaptic nanocolumns. During elevated neuronal activity by 4-AP treatment, the spatial correlation between GlyRs, GABAARs and gephyrin was increased at the PSD. Moreover, the spatial correlation of GlyRs and RIM1/2 was also increased, while that of GABAARs and RIM1/2 did not change. The number of SSDs per synapse for these synaptic proteins was not changed by 4-AP. My study thus provides a new angle for understanding the mechanisms underlying GABAergic/glycinergic co-transmission.

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To survive, animals must adapt to a complex and challenging world in a way that is flexible and responsive, while maintaining internal homeostasis. Neuromodulators provide a means to systemically alter behavioral or physiological state based on intrinsic or extrinsic cues, however dysregulated neuroendocrine signaling has negative consequences for fitness and survival. Here I examine neuroendocrine function and dysfunction using the escape response in Caenorhabditis elegans. The RFamide neuropeptide FLP-18 is a co-transmitter with the monoamine tyramine and functions both synergistically and antagonistically to tyramine in coordinating escape behavior. Using behavioral analysis and calcium imaging, I show that FLP-18 functions primarily through the G-protein coupled receptor (GPCR) NPR-5 to increase calcium levels in muscle, enhancing locomotion rate, bending and reversal behavior during the escape response. Furthermore, I examine the relationship between persistent acute stress and resilience using repeated activation of the escape response as a model of neuroendocrine dysregulation. Repeated activation of the escape response shortens lifespan and renders animals more susceptible to thermal, oxidative, and nutritional stress. Tyramine release is necessary and sufficient for this effect and activity of the tyraminergic RIM neurons is differentially regulated by acute versus long-term stressors. Impaired stress resistance requires both the GPCR TYRA-3 in the intestine and intestinal neuropeptide release. Activation of the insulin receptor DAF-2 is downstream of TYRA-3 and inhibits the transcription factors DAF-16/FOXO, SKN-1/Nrf2 and HSF-1, linking monoamine signaling in acute stress to the insulin signaling pathway and impaired resilience to long-term stressors.

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