Sandrine Parrot

A2A adenosine receptor deletion is protective in a mouse model of Tauopathy

Consumption of caffeine, a non-selective adenosine A2A receptor (A2AR) antagonist, reduces the risk of developing Alzheimer’s disease (AD) in humans and mitigates both amyloid and Tau burden in transgenic mouse models. However, the impact of selective A2AR blockade on the progressive development of AD-related lesions and associated memory impairments has not been investigated. In the present study, we removed the gene encoding A2AR from THY-Tau22 mice and analysed the subsequent effects on both pathological (Tau phosphorylation and aggregation, neuro-inflammation) and functional impairments (spatial learning and memory, hippocampal plasticity, neurotransmitter profile). We found that deleting A2ARs protect from Tau pathology-induced deficits in terms of spatial memory and hippocampal long-term depression. These effects were concomitant with a normalization of the hippocampal glutamate/gamma-amino butyric acid ratio, together with a global reduction in neuro-inflammatory markers and a decrease in Tau hyperphosphorylation. Additionally, oral therapy using a specific A2AR antagonist (MSX-3) significantly improved memory and reduced Tau hyperphosphorylation in THY-Tau22 mice. By showing that A2AR genetic or pharmacological blockade improves the pathological phenotype in a Tau transgenic mouse model, the present data highlight A2A receptors as important molecular targets to consider against AD and Tauopathies.

A rapid and sensitive method for the analysis of brain monoamine neurotransmitters using ultra-fast liquid chromatography coupled to electrochemical detection.

Electrochemical detection is often used to detect catecholamines and indolamines in brain samples that have been separated by conventional reverse-phase high performance liquid chromatography (HPLC). This paper presents the transfer of an existing chromatographic method for the determination of monoamines in brain tissues using 5 μm granulometry HPLC columns to columns with a particle diameter less than 3 μm. Several parameters (repeatability, linearity, accuracy, limit of detection, and stability of samples) for this new ultrafast high performance liquid chromatography (UHPLC) method were examined after optimization of the analytical conditions. The separation of seven compounds, noradrenaline, dopamine and three of its metabolites, dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine, and serotonin and its metabolite, 5-hydroxyindole-3-acetic acid was analyzed using this UHPLC-electrochemical detection method. The final method, which was applied to brain tissue extracts from mice, rats, and cats, decreased analysis time by a factor of 4 compared to HPLC, while guaranteeing good analytical performance.

Coupling on-line brain microdialysis, precolumn derivatization and capillary electrophoresis for routine minute sampling of O-phosphoethanolamine and excitatory amino acids

In previous papers, we described the analysis of excitatory amino acids (EAAs) and catecholamines in microdialysis samples using capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD). In the present paper, we report that an automated analysis of such samples can be easily achieved by on-line coupling of the microdialysis probe with a continuous flow derivatization system and a commercially available CE-LIFD apparatus. Because of the short analysis time (less than 2 min) and high separation efficiency (100-200,000 theoretical plates), high temporal resolution of microdialysis (minute range) is preserved as compared to off-line systems, while both EAAs and O-phosphoethanolamine (PEA) can be simultaneously detected. This new method has been applied to the measurement of these compounds in microdialysis samples from hippocampal slice cultures and striatum of anesthetized rats. Extracellular concentrations of EAAs, but not PEA, increased during perfusion of a solution containing high K+ or a glutamate uptake inhibitor. However, after in vitro ischemia on hippocampal slices, both EAAs and PEA concentrations increased, but with different temporal patterns.

Analysis of microdialysate monoamines, including noradrenaline, dopamine and serotonin, using capillary ultra-high performance liquid chromatography and electrochemical detection

Electrochemical methods are very often used to detect catecholamine and indolamine neurotransmitters separated by conventional reverse-phase high performance liquid chromatography (HPLC). The present paper presents the development of a chromatographic method to detect monoamines present in low-volume brain dialysis samples using a capillary column filled with sub-2μm particles. Several parameters (repeatability, linearity, accuracy, limit of detection) for this new ultrahigh performance liquid chromatography (UHPLC) method with electrochemical detection were examined after optimization of the analytical conditions. Noradrenaline, adrenaline, serotonin, dopamine and its metabolite 3-methoxytyramine were separated in 1μL of injected sample volume; they were detected above concentrations of 0.5-1nmol/L, with 2.1-9.5% accuracy and intra-assay repeatability equal to or less than 6%. The final method was applied to very low volume dialysates from rat brain containing monoamine traces. The study demonstrates that capillary UHPLC with electrochemical detection is suitable for monitoring dialysate monoamines collected at high sampling rate.

Ultra high performance liquid chromatography as a tool for the discovery and the analysis of biomarkers of diseases: a review.

The development and use of UHPLC-based methods for the identification, validation and analysis of biomarkers for diseases is reviewed. The currents trends in types of stationary phases and modes of detection are discussed. Afterwards, examples are provided on the use of UHPLC-MS for finding novel biomarkers in samples from in vitro or in vivo animal models of human diseases, as well as in biofluid samples (mainly urine and plasma) obtained from patients. Molecular profiling and targeted analysis are considered, providing an overview of recent experimental or clinical works carried out using UHPLC analysis of compounds from various chemical classes, such as low molecular weight metabolites, hormones, lipids, peptides and proteins.

In-capillary derivatization and capillary electrophoresis separation of amino acid neurotransmitters from brain microdialysis samples

A new in-capillary derivatization method with naphtalene-2,3-dicarboxyaldehyde (NDA)/CN(-) has been developed for capillary electrophoresis with laser-induced fluorescence detection of brain microdialysate amino acids. Samples are sandwiched between two plugs of reagent mixture at the capillary inlet and subsequently separated. Highest derivatization yields are obtained by using a reagent to sample plug length ratio equal to 4, performing a first electrophoretic mixing followed by a zero potential amplification step before applying the separation voltage and using a NaCN to NDA concentration ratio equal to 1. This new single-step methodology allows the analysis of amino acid neurotransmitters in rat brain microdialysis samples.

Glutamatergic alterations in the cortex of genetic absence epilepsy rats

BackgroundIn absence epilepsy, the neuronal hyper-excitation and hyper-synchronization, which induce spike and wave discharges in a cortico-thalamic loop are suspected to be due to an imbalance between GABA and glutamate (GLU) neurotransmission. In order to elucidate the role played by GLU in disease outcome, we measured cortical and thalamic extracellular levels of GLU and GABA. We used an in vivo quantitative microdialysis approach (no-net-flux method) in an animal model of absence epilepsy (GAERS). In addition, by infusing labelled glutamate through the microdialysis probe, we studied in vivo glutamate uptake in the cortex and thalamus in GAERS and non-epileptic control (NEC) rats. Expression of the vesicular glutamate transporters VGLUT1 and VGLUT2 and a synaptic component, synaptophysin, was also measured.ResultsAlthough extracellular concentrations of GABA and GLU in the cortex and thalamus were not significantly different between GAERS and NEC rats, cortical GLU uptake was significantly decreased in unrestrained awake GAERS. Expression of VGLUT2 and synaptophysin was increased in the cortex of GAERS compared to NEC rats, but no changes were observed in the thalamus.ConclusionThe specific decrease in GLU uptake in the cortex of GAERS linked to synaptic changes suggests impairment of the glutamatergic terminal network. These data support the idea that a change in glutamatergic neurotransmission in the cortex could contribute to hyperexcitability in absence epilepsy.

In vivo temporal sequence of rat striatal glutamate, aspartate and dopamine efflux during apomorphine, nomifensine, NMDA and PDC in situ administration.

In vivo microdialysis was used to investigate the interactions between dopamine (DA), glutamate (Glu) and aspartate (Asp) in anaesthetised-rat striatum. The combination of brain microdialysis and capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) allows the simultaneous monitoring of the efflux of these neurotransmitters up to every 10 s. DA and Glu reuptake inhibitors, nomifensine and L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and, dopaminergic and glutamatergic receptor agonists, apomorphine and NMDA respectively, were administered by reverse dialysis. Reverse dialysis of 20 micro M nomifensine induced a rapid and marked increase (+3200% at 5 min) in extracellular DA, while a decrease in Glu and Asp (-11 and -25%, respectively) was observed simultaneously. Reverse dialysis of 10 micro M apomorphine led to progressive changes: -63% decrease in DA and +25% Glu increase at 36 min. Reverse dialysis of 1 mM NMDA induced a simultaneous increase in DA, Glu and Asp which peaked at +2 min (+840%, +40% and +150%, respectively). Surprisingly, a second increase in Glu was observed 5 min after the end of NMDA perfusion. Reverse dialysis of PDC (1 mM and 10 mM) induced a rapid increase in Glu and Asp levels, while DA increased with a 26-s delay. These findings indicate that, in the striatum, endogenous DA and Glu may act in opposition to regulate each others efflux. These results have been obtained due to unique features offered by microdialysis coupled with CE-LIFD.

Application of histamine or serotonin to the hypoglossal nucleus increases genioglossus muscle activity across the wake-sleep cycle

The decrease in genioglossus (GG) muscle activity during sleep, especially rapid eye movement (REM) or paradoxical sleep, can lead to airway occlusion and obstructive sleep apnoea (OSA). The hypoglossal nucleus innervating the GG muscle is under the control of serotonergic, noradrenergic and histaminergic neurons that cease firing during paradoxical sleep. The objectives of this study were to determine the effect on GG muscle activity during different wake–sleep states of the microdialysis application of serotonin, histamine (HA) or noradrenaline (NE) to the hypoglossal nucleus in freely moving cats. Six adult cats were implanted with electroencephalogram, electro‐oculogram and neck electromyogram electrodes to record wake–sleep states and with GG muscle and diaphragm electrodes to record respiratory muscle activity. Microdialysis probes were inserted into the hypoglossal nucleus for monoamine application. Changes in GG muscle activity were assessed by power spectrum analysis. In the baseline conditions, tonic GG muscle activity decreased progressively and significantly from wakefulness to slow‐wave sleep and even further during slow‐wave sleep with ponto‐geniculo‐occipital waves and paradoxical sleep. Application of serotonin or HA significantly increased GG muscle activity during the wake–sleep states when compared with controls. By contrast, NE had no excitatory effect. Our results indicate that both serotonin and HA have a potent excitatory action on GG muscle activity, suggesting multiple aminergic control of upper airway muscle activity during the wake–sleep cycle. These data might help in the development of pharmacological approaches for the treatment of OSA.

Differential dynamics of amino acid release in the amygdala and olfactory cortex during odor fear acquisition as revealed with simultaneous high temporal resolution microdialysis

Although the amygdala seems to be essential to the formation and storage of fear memories, it might store only some aspects of the aversive event and facilitate the storage of more specific sensory aspects in cortical areas. We addressed the time course of amygdala and cortical activation in the context of odor fear conditioning in rats. Using high temporal resolution (1-min sampling) intracerebral microdialysis, we investigated the dynamics of glutamate and GABA fluctuations simultaneously in basolateral amygdala (BLA) and posterior piriform cortex (pPCx) during the course of the acquisition session, which consisted of six odor (conditioned stimulus)-footshock (unconditioned stimulus) pairings. In BLA, we observed a transient increase in amino acid concentrations following the first odor-shock pairing, after which concentrations returned to baseline levels or slightly below. In pPCx, transient increases were seen after each pairing and were also observed after the last odor-shock pairing, corresponding to the predicted times of anticipated trials. Furthermore, we observed that for the first pairing, the increase in BLA occurred earlier than the increase in pPCx. These data suggest that the amygdala is engaged early during acquisition and precedes the activation of the olfactory cortex, which is maintained until the end of the session. In addition, our data raise the challenging idea that the olfactory cortex might store certain aspects of fear conditioning related to the timing of the associations.