Development of PET and SPECT Probes for Glutamate Receptors

L-Glutamate and its receptors (GluRs) play a key role in excitatory neurotransmission within the mammalian central nervous system (CNS). Impaired regulation of GluRs has also been implicated in various neurological disorders. GluRs are classified into two major groups: ionotropic GluRs (iGluRs), which are ligand-gated ion channels, and metabotropic GluRs (mGluRs), which are coupled to heterotrimeric guanosine nucleotide binding proteins (G-proteins). Positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging of GluRs could provide a novel view of CNS function and of a range of brain disorders, potentially leading to the development of new drug therapies. Although no satisfactory imaging agents have yet been developed for iGluRs, several PET ligands for mGluRs have been successfully employed in clinical studies. This paper reviews current progress towards the development of PET and SPECT probes for GluRs

Targeting the type 5 metabotropic glutamate receptor in a mouse model of terminal neurodegeneration

The type 5 metabotropic glutamate receptor (mGlu5) plays an important role in learning and memory processes and has been identified as a potential drug target for the treatment of neurodegenerative diseases (NDD). Given the limited treatment options available to patients living with NDD, there is a pressing need for novel therapeutic interventions that treat both the symptomatic and progressive components of neurodegeneration. Promisingly, the pharmacological and genetic blockade of mGlu5 has been shown to reduce disease pathology and improve cognition in several preclinical mouse models of neurodegeneration (Budgett et al., 2022). Expressed in both neurons and glia, mGlu5 has been shown to play a role in neuroinflammation and, therefore, represents a potential target for regulating neuroinflammation in disease (Byrnes et al., 2009). Therefore, this thesis aimed to evaluate the role of mGlu5 in the modulation of neuroinflammation and the progression of NDD in a model of terminal neurodegeneration, murine prion disease. To provide insights into the signalling mechanisms of mGlu5 and the characteristics of novel compounds, in vitro functional assays were conducted on cell lines expressing mGlu5. Flp-in cells expressing mouse mGlu5 and mouse primary cortical astrocytes were found to be suitable systems for investigating the signalling properties of mGlu5 ligands. In these systems, VU0424238 was confirmed to be a mGlu5 negative allosteric modulator (NAM) that binds to the main allosteric binding site on mGlu5 (Felts et al., 2017). Furthermore, target engagement and efficacy in vivo were established. Recent studies have shown close correlates between murine prion disease and human NDDs, including hippocampal-based cognitive deficits, neuroinflammation, and terminal neurodegeneration (Bourgognon et al., 2018; Bradley et al., 2016). Here, the model of murine prion disease was investigated using histological and biochemical studies to characterise the neuroinflammatory response throughout disease progression. The astrocytic and microglial markers GFAP, vimentin, Iba-1, and CD68 were confirmed to be upregulated in murine prion disease, in addition to several pro-inflammatory cytokines. This is similar to human NDDs characterised by chronic neuroinflammation. Subsequently, this thesis aimed to define the impact of mGlu5 blockade, both pharmacologically and genetically, on the progression of murine prion disease. Firstly, mGlu5 was inhibited pharmacologically using the NAM VU0424238. Although chronic VU0424238 treatment significantly reduced the expression of GFAP in the hippocampus of female prion-diseased mice, mGlu5 antagonism did not alter the overall progression of murine prion disease. There were no alterations to the accumulation of misfolded prion protein, symptom onset, survival, or behaviour after chronic VU0424238 treatment. Next, mGlu5-deficient mice were inoculated with prion disease. Although mGlu5 deficiency resulted in the reduced expression of the inflammatory markers Iba-1 and GFAP in early-stage disease, it did not affect overall disease progression. Overall, these findings suggest that mGlu5 may represent a potential approach by which neuroinflammatory processes in NDDs might be modulated. However, the results also suggest that mGlu5 may not play a significant role in the progression of prion disease and may function differently in prion disease compared to other NDDs

Mécanismes cérébraux de la régulation de la douleur : perception de la douleur et hypoalgésie induite psychologiquement

Objectif : Cette thèse a pour but de préciser les mécanismes neuropsychologiques de la douleur, de la régulation endogène de la douleur et de l'hypoalgésie induite psychologiquement (HIP) par la synthèse de près de trente ans de recherche imagerie cérébrale fonctionnelle. Méthodologie : Étant donné l'abondance des études sur le sujet et le manque d'intégration de leurs résultats, la technique de métaanalyse quantitative basée sur les coordonnées d'activation cérébrale fut privilégiée dans cette thèse, telle qu’implémentée dans l'algorithme ALE (Activation Likelyhood Estimate). Une force supplémentaire de cette thèse repose sur la rigueur du processus de sélection des articles. En effet, les études incluses dans les métaanalyses devaient satisfaire des critères stricts d'inclusion, ceci dans le but de favoriser la précision et la validité des conclusions subséquentes. Étude 1 : Le premier article visait à identifier les aires cérébrales impliquées dans la réduction de la douleur par des méthodes psychologiques d'interventions. Les articles retenus portent sur une variété de méthodes d'intervention, telles que le placebo, l'hypnose, la méditation, la perception de contrôle sur la stimulation douloureuse et l'induction d'émotions. Les résultats indiquent que l'HIP implique un vaste réseau d'activation qui comprend le cortex cingulaire antérieur, l'insula antérieure, les zones orbitofrontale et préfrontale latérale, ainsi que les régions pariétale, temporale et souscorticales. Ces activations reflèteraient l'implication des mécanismes neuropsychologiques cognitifs et émotionnels sous-tendent les interventions psychologiques ciblées par ces études, incluant la conscience de soi et la motivation. De plus, les divergences de patron d'activation entre les approches ont été explorées, notamment pour le placebo et la distraction. Étude 2 : Le deuxième article a identifié des patrons d'activations préférentiellement associés à la perception de la douleur, à l'HIP, ainsi que des activations communément associées à la douleur et l'HIP. Les résultats indiquent que 1) la perception de la douleur est associée à l'activation d'aires somatosensorielles et motrices, ce qui pourrait être le reflet de la préparation d'une action adaptative, 2) l'HIP est liée à l'engagement de régions préfrontales antéromédianes et orbitales, possiblement en lien avec des processus motivationnels et émotionnels, et 3) la douleur et l'HIP sont associés à l'activation d'aires préfrontales dorsolatérales, de l'insula antérieure et du cortex cingulaire moyen, ce qui pourrait refléter l'engagement spontané pendant la douleur de mécanismes endogènes de régulation descendante. Conclusion : Par ces études, cette thèse fait le point sur les mécanismes cérébraux impliqués différentiellement dans la perception de la douleur, dans sa régulation endogène et dans l'hypoalgésie induite psychologiquement.Objective: This thesis aims to clarify the neuropsychological mechanisms of pain, of the endogenous regulation of pain and of psychologically induced hypoalgesia (PIH), through the synthesis of almost thirty years of functional brain imaging research. Methodology: Given the abundance of studies in this domain and the lack of integration of their results, we used the quantitative meta-analysis technique based on brain activation using the ALE (Activation likelihood Estimate) statistic. The strength of this thesis lies in the globalized perspective of the litterature, and in the rigor of the article selection process from which results were extracted. Indeed, the studies included in the meta-analyses needed to meet strict inclusion criteria in order to strengthen the accuracy and the validity of subsequent conclusions. Study 1: The first article is aimed at identifying brain areas involved in pain reduction through psychological methods of intervention. Chosen articles that covered a variety of approaches, such as placebo, hypnosis, meditation, perception of control over the stimulation, and induction of emotions. Analysis across these various studies indicated that PIH involves a broad network of activation that includes the anterior cingulate cortex, anterior insulae, orbital and lateral prefrontal and frontal areas, as well as parietal, temporal and subcortical regions. This activation network may reflect the involvement of diverse neuropsychological mechanisms in the various affective, self-awareness, cognitive and motivational processes underlying the psychological interventions targeted by these studies. In addition, we explored some specific patterns of brain activity related to placebo and distraction, in comparison to other approaches. We propose several hypotheses regarding the distinctive neuropsychological processes underlying these approaches. Study 2: The second article aimed at investigating patterns of brain activity preferentially associated with pain perception or with PIH. First we assessed patterns of increased and decreased activity during experimental pain in healthy volunteers. Second we determined the brain regions preferentially activated during pain perception or during PIH with subtraction analyses. Using a conjunction analysis, we also determined a set of brain regions possibly involved in regulatory processes activated spontaneously during acute of pain. Our results indicate that 1) somatosensory and motor areas are preferentially related to pain perception, which may reflect the preparation of a motor response, 2) dorsolateral prefrontal areas, anterior insula and the anterior midcingulate cortex were associated with both pain and PIH and may reflect the spontaneous activation of top-down regulation mechanisms during pain, and 3) antero-medial and orbital prefrontal regions were preferentially associated with PIH, which may indicate motivational and emotional processes associated with the engagement of an externally driven hypoalgesic procedure

Development of a Microarray Biosensor for Real-Time and Continuous Measurement of Neurochemicals

Continuous simultaneous measurement of glutamate (GLU), an excitatory neurochemical, and γ-aminobutyric acid (GABA), an inhibitory neurochemical, constitutes one of the major challenges in neuroscientific research. Maintaining appropriate levels of GLU and GABA is important for normal brain functions. Abnormal levels of GLU and GABA are responsible for various brain dysfunctions, like epilepsy and traumatic brain injury. GLU and GABA being non-electroactive are challenging to detect in real-time. To date, GABA is detected mainly via microdialysis with a high performance liquid chromatography (HPLC) system that employs electrochemical (EC) and spectroscopic methodologies. However, these systems are bulky and unsuitable for real-time continuous monitoring. As opposed to microdialysis, biosensors are easy to miniaturize and are highly suitable for in-vivo studies. Unfortunately, this method requires a rather cumbersome process that relies on externally applied pre-reactors and reagents. Here, we report the design and implementation of a GABA microarray probe that operates on a newly conceived principle. It consists of two microbiosensors, one for GLU and one for GABA detection, modified with glutamate oxidase and GABASE enzymes, respectively. The detection of GABA by this probe is based upon the in-situ generation of α-ketoglutarate from the GLU oxidation that takes place at both microbiosensor sites. By simultaneously measuring and subtracting the H2O2 oxidation currents of GLU microbiosensor from GABA microbiosensor, GABA and GLU can be detected continuously in real-time in vitro and ex vivo. This mechanism happens without the addition of any externally applied reagents. We optimized our novel approach in commercially available ceramic-based probes. The GABA probe was successfully tested in an adult rat brain slice preparation. However, those electrodes are geometrically limited (we cannot have a sentinel site at the same spatial level as GLU and GABA sites). Keeping theseissues in mind, we have developed a microwire array sensor that is not only capable of simultaneous measurement of GLU and GABA, but is also able to track signal resulting from interferents (e.g. Ascorbic Acid, AA). The unique geometry enables these microwire probes to measure GLU, GABA and interferents in the same spatial level. A Simple fabrication procedure and easy integration with the existing amperometric systems allow us to use them in cell culture, brain tissue, and in vivo recordings as an inexpensive alternative to our planar electrodes. We demonstrated the effectiveness of the probes in rat brain tissue. We were able to get. Additionally, we determined the excitation/inhibition (E/I) ratios for different stimulations which have clinical relevance. Our results about this E/I balance can help refine electrical stimulation parameter for different clinical purposes (e.g. deep brain stimulation). Finally, we successfully tested our probe in awake-free behaving rats. In summary, our results suggest that microwire probes have the potential to become a powerful tool for measuring GLU and GABA in various ex-vivo and in-vivo disease models, such as epilepsy