Carbon monoxide (CO) is an extremely useful molecule with applications in industrial manufacturing, synthetic procedures as a C1 building block, and as a potential pharmaceutical to produce anti-inflammatory effects and vasodilation. However, the toxicity associated with CO has prevented its full utilization. In order to safely handle CO, compounds and molecules have been developed that act as storage materials for the gas. Ideal storage platforms only release CO upon stimulation via a trigger. Light activation is the most desirable trigger as it can be regulated in terms of the intensity and the wavelength of light used. The majority of light-induced CO-storage platforms that have been reported to date consist of metal carbonyl compounds where CO is bound directly to a metal center. However, disadvantages inherent to this motif, such as potential toxicity associated with the metal and lack of characterization of CO release remnant(s), has pushed the research community to search for alternative CO storage structures. The research presented in this dissertation outlines our approach toward the development of safe-to-handle, light-induced CO release platforms. We use a flavonol structure similar to those found in fruits and vegetables, such as quercetin, as a light-induced CO release unit. Through changes in the structure of the flavonol and its surrounding environment in chemical compounds, we have found ways to strategically control the light-induced CO release reactivity of the flavonol. Chemical compounds developed in this project are of interest for studying the effects of CO in biological systems and applications in synthetic processes.

The basal ganglia (BG) is known for its function not only in motor modulation but also in action selection and reward learning. There are two major anatomical pathways through the BG, the direct and the indirect pathways. The direct pathway starts from the striatum and then directly projects to the globus pallidus, pars interna (GPi) and the substantia nigra, pars reticulata (SNr) respectively, while the indirect pathway starts from the striatum but then indirectly projects to GPi and SNr through the globus pallidus, pars externa and then to the subthalamic nucleus. In addition, the output from GPi not only projects to the thalamus where it has been proposed to function in motor control, but also to the lateral habenula (LHb) where it has been proposed to function in outcome evaluation. Previous studies have found that there are three major genetically distinct neuron groups in the entopeduncular nucleus (EP) (rodent homologue of the primate GPi): 1) purely glutamatergic neurons projecting to LHb neurons expressing parvalbumin (PVLHb); 2) purely GABAergic neurons projecting to motor thalamic neurons expressing parvalbumin (PVThal); 3) GABA/glutamate co-releasing neurons projecting to LHb neurons expressing somatostatin (SstLHb). In this study, we knocked out the vesicular glutamate transporter 2 in SstLHb neurons through an adeno-associated virus in mice to test for the impact on goal-directed behavior using a probabilistic switching, two-armed bandit task (2ABT). Results obtained from the freely moving, water-restricted somatostatin-cre mice with the vesicular glutamate transporter 2 ablated in SstLHb neurons showed that: 1) there was neither improvement nor decline in their performance on the task; 2) they might be more distracted between trials while more concentrated within a trial; 3) they had an increase in the probability of switching between ports on consecutive trials when uncertainty in the location of the highly rewarded port was maximum compared to the control animals with intact glutamate release from SstLHb neurons to LHb. The success of the viral expression was then confirmed through whole-cell voltage-clamp recordings of postsynaptic neurons of the LHb, receiving projections from SstLHb neurons. In conclusion, our study has suggested that the glutamate release from the GABA/glutamate co-releasing neurons of EP projecting to LHb may play a role in reinforcement learning and motivation to obtain rewards, and the loss of glutamate in the GABA/glutamate co-releasing vesicles results in increasing uptake of GABA into these vesicles, leading to possible rebound burst firing of SstLHb neurons that eventually increases the sensitivity towards low rate of reward-delivery dramatically. / 2026-03-13T00:00:00Z

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.

The Basal Ganglia consist of a number of different nuclei that form a diverse circuitry of GABAergic, dopaminergic and glutamatergic neurons. This complex network is further organized in subcircuits that govern limbic and motor functions in humans and other vertebrates. Due to the interconnection of the individual structures, dysfunction in one area or cell population can affect the entire network, leading to synaptic and molecular alterations in the circuitry as a whole. The studies in this doctoral thesis aimed at characterizing restricted subpopulations of neurons in the Basal Ganglia circuitry and their importance in the wider function of the network. To this end, we identified subpopulations of neurons in the subthalamic nucleus (STN), substantia nigra (SN) and ventral tegmental area (VTA), characterized their molecular profile and investigated their physiological role in the circuitry. Within the mouse STN, reduction of glutamatergic neurotransmission in a subpopulation expressing Paired-like homeodomain transcription factor 2 (Pitx2) led to structural alterations in the nucleus as well as biochemical alterations of the dopaminergic system in the Nucleus accumbens (NAc) and changes in reward-related behavior. In the ventral midbrain, we identified and characterized novel marker genes selective to the VTA or SN. Of these, transient receptor potential cation channel subfamily V member 1 (TrpV1) marks a population of mainly glutamatergic neurons in the VTA which project to the NAc, while gastrin releasing peptide (Grp) is expressed in a population of dopaminergic neurons neuroprotected in Parkinson's disease. Furthermore, we discovered that disruption of glutamatergic co-release of dopaminergic neurons expressing dopamine transporter (DAT), diminishes fast EPSCs and glutamate release but does not affect the acquisition of reward-related behavioral tasks. To selectively quantify glutamate release from specific subpopulations, we devised a technique combining glutamate-amperometry and optogenetics. This was used to measure glutamate released from Pitx2-expressing synaptic terminals in the Globus pallidus as well as DAT- or TrpV1-expressing terminals in the NAc. In summary, this doctoral thesis has furthered understanding of the function and importance of specific subpopulations within the Basal Ganglia circuitry and provides a novel means to investigate glutamate in the intact rodent brain within clearly defined, restricted cell populations.

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