Pallidin function in Drosophila surface glia regulates sleep and is dependent on amino acid availability.

The Pallidin protein is a central subunit of a multimeric complex called biogenesis of lysosome-related organelles complex 1 (BLOC1) that regulates specific endosomal functions and has been linked to schizophrenia. We show here that downregulation of Pallidin and other members of BLOC1 in the surface glia, the Drosophila equivalent of the blood-brain barrier, reduces and delays nighttime sleep in a circadian-clock-dependent manner. In agreement with BLOC1 involvement in amino acid transport, downregulation of the large neutral amino acid transporter 1 (LAT1)-like transporters JhI-21 and mnd, as well as of TOR (target of rapamycin) amino acid signaling, phenocopy Pallidin knockdown. Furthermore, supplementing food with leucine normalizes the sleep/wake phenotypes of Pallidin downregulation, and we identify a role for Pallidin in the subcellular trafficking of JhI-21. Finally, we provide evidence that Pallidin in surface glia is required for GABAergic neuronal activity. These data identify a BLOC1 function linking essential amino acid availability and GABAergic sleep/wake regulation.

Serotonin and the ventilatory effects of etonogestrel, a gonane progestin, in a murine model of congenital central hypoventilation syndrome.

Introduction: Congenital Central Hypoventilation Syndrome, a rare disease caused by PHOX2B mutation, is associated with absent or blunted CO2/H+ chemosensitivity due to the dysfunction of PHOX2B neurons of the retrotrapezoid nucleus. No pharmacological treatment is available. Clinical observations have reported non-systematic CO2/H+ chemosensitivity recovery under desogestrel. Methods: Here, we used a preclinical model of Congenital Central Hypoventilation Syndrome, the retrotrapezoid nucleus conditional Phox2b mutant mouse, to investigate whether etonogestrel, the active metabolite of desogestrel, led to a restoration of chemosensitivity by acting on serotonin neurons known to be sensitive to etonogestrel, or retrotrapezoid nucleus PHOX2B residual cells that persist despite the mutation. The influence of etonogestrel on respiratory variables under hypercapnia was investigated using whole-body plethysmographic recording. The effect of etonogestrel, alone or combined with serotonin drugs, on the respiratory rhythm of medullary-spinal cord preparations from Phox2b mutants and wildtype mice was analyzed under metabolic acidosis. c-FOS, serotonin and PHOX2B were immunodetected. Serotonin metabolic pathways were characterized in the medulla oblongata by ultra-high-performance liquid chromatography. Results: We observed etonogestrel restored chemosensitivity in Phox2b mutants in a non-systematic way. Histological differences between Phox2b mutants with restored chemosensitivity and Phox2b mutant without restored chemosensitivity indicated greater activation of serotonin neurons of the raphe obscurus nucleus but no effect on retrotrapezoid nucleus PHOX2B residual cells. Finally, the increase in serotonergic signaling by the fluoxetine application modulated the respiratory effect of etonogestrel differently between Phox2b mutant mice and their WT littermates or WT OF1 mice, a result which parallels with differences in the functional state of serotonergic metabolic pathways between these different mice. Discussion: Our work thus highlights that serotonin systems were critically important for the occurrence of an etonogestrel-restoration, an element to consider in potential therapeutic intervention in Congenital Central Hypoventilation Syndrome patients.

DM1 Transgenic Mice Exhibit Abnormal Neurotransmitter Homeostasis and Synaptic Plasticity in Association with RNA Foci and Mis-Splicing in the Hippocampus.

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disease mediated by a toxic gain of function of mutant RNAs. The neuropsychological manifestations affect multiple domains of cognition and behavior, but their etiology remains elusive. Transgenic DMSXL mice carry the DM1 mutation, show behavioral abnormalities, and express low levels of GLT1, a critical regulator of glutamate concentration in the synaptic cleft. However, the impact of glutamate homeostasis on neurotransmission in DM1 remains unknown. We confirmed reduced glutamate uptake in the DMSXL hippocampus. Patch clamp recordings in hippocampal slices revealed increased amplitude of tonic glutamate currents in DMSXL CA1 pyramidal neurons and DG granule cells, likely mediated by higher levels of ambient glutamate. Unexpectedly, extracellular GABA levels and tonic current were also elevated in DMSXL mice. Finally, we found evidence of synaptic dysfunction in DMSXL mice, suggestive of abnormal short-term plasticity, illustrated by an altered LTP time course in DG and in CA1. Synaptic dysfunction was accompanied by RNA foci accumulation in localized areas of the hippocampus and by the mis-splicing of candidate genes with relevant functions in neurotransmission. Molecular and functional changes triggered by toxic RNA may induce synaptic abnormalities in restricted brain areas that favor neuronal dysfunction.

Defects in Mouse Cortical Glutamate Uptake Can Be Unveiled In Vivo by a Two-in-One Quantitative Microdialysis.

Extracellular glutamate levels are maintained low by efficient transporters, whose dysfunction can cause neuronal hyperexcitability, excitotoxicity, and neurological disease. While many methods estimate glutamate uptake in vitro/ex vivo, a limited number of techniques address glutamate transport in vivo. Here, we used in vivo microdialysis in a two-in-one approach combining reverse dialysis of isotopic glutamate to measure uptake ability and zero-flow (ZF) methods to quantify extracellular glutamate levels. The complementarity of both techniques is discussed on methodological and anatomical basis. We used a transgenic mouse model of human disease, expressing low levels of the EAAT-2/GLT1 glutamate transporter, to validate our approach in a relevant animal model. As expected, isotopic analysis revealed an overall decrease in glutamate uptake, while the ZF method unveiled higher extracellular glutamate levels in these mice. We propose a sensitive and expedite two-in-one microdialysis approach that is sufficiently robust to reveal significant differences in neurotransmitter uptake and extracellular levels through the analysis of a relatively low number of animals.

Dorsal striatum and the temporal expectancy of an aversive event in Pavlovian odor fear learning.

Interval timing, the ability to encode and retrieve the memory of intervals from seconds to minutes, guides fundamental animal behaviors across the phylogenetic tree. In Pavlovian fear conditioning, an initially neutral stimulus (conditioned stimulus, CS) predicts the arrival of an aversive unconditioned stimulus (US, generally a mild foot-shock) at a fixed time interval. Although some studies showed that temporal relations between CS and US events are learned from the outset of conditioning, the question of the memory of time and its underlying neural network in fear conditioning is still poorly understood. The aim of the present study was to investigate the role of the dorsal striatum in timing intervals in odor fear conditioning in male rats. To assess the animal's interval timing ability in this paradigm, we used the respiratory frequency. This enabled us to detect the emergence of temporal patterns related to the odor-shock time interval from the early stage of learning, confirming that rats are able to encode the odor-shock time interval after few training trials. We carried out reversible inactivation of the dorsal striatum before the acquisition session and before a shift in the learned time interval, and measured the effects of this treatment on the temporal pattern of the respiratory rate. In addition, using intracerebral microdialysis, we monitored extracellular dopamine level in the dorsal striatum throughout odor-shock conditioning and in response to a shift of the odor-shock time interval. Contrary to our initial predictions based on the existing literature on interval timing, we found evidence suggesting that transient inactivation of the dorsal striatum may favor a more precocious buildup of the respiratory frequency's temporal pattern during the odor-shock interval in a manner that reflected the duration of the interval. Our data further suggest that the conditioning and the learning of a novel time interval were associated with a decrease in dopamine level in the dorsal striatum, but not in the nucleus accumbens. These findings prompt a reassessment of the role of the striatum and striatal dopamine in interval timing, at least when considering Pavlovian aversive conditioning.

In vivo γ-aminobutyric acid increase as a biomarker of the epileptogenic zone: An unbiased metabolomics approach.

OBJECTIVE: Following surgery, focal seizures relapse in 20% to 50% of cases due to the difficulty of delimiting the epileptogenic zone (EZ) by current imaging or electrophysiological techniques. Here, we evaluate an unbiased metabolomics approach based on ex vivo and in vivo nuclear magnetic resonance spectroscopy (MRS) methods to discriminate the EZ in a mouse model of mesiotemporal lobe epilepsy (MTLE). METHODS: Four weeks after unilateral injection of kainic acid (KA) into the dorsal hippocampus of mice (KA-MTLE model), we analyzed hippocampal and cortical samples with high-resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS). Using advanced multivariate statistics, we identified the metabolites that best discriminate the injected dorsal hippocampus (EZ) and developed an in vivo MEGAPRESS MRS method to focus on the detection of these metabolites in the same mouse model. RESULTS: Multivariate analysis of HRMAS data provided evidence that γ-aminobutyric acid (GABA) is largely increased in the EZ of KA-MTLE mice and is the metabolite that best discriminates the EZ when compared to sham and, more importantly, when compared to adjacent brain regions. These results were confirmed by capillary electrophoresis analysis and were not reversed by a chronic exposition to an antiepileptic drug (carbamazepine). Then, using in vivo noninvasive GABA-edited MRS, we confirmed that a high GABA increase is specific to the injected hippocampus of KA-MTLE mice. SIGNIFICANCE: Our strategy using ex vivo MRS-based untargeted metabolomics to select the most discriminant metabolite(s), followed by in vivo MRS-based targeted metabolomics, is an unbiased approach to accurately define the EZ in a mouse model of focal epilepsy. Results suggest that GABA is a specific biomarker of the EZ in MTLE.

Hyperexcitability and seizures in the THY-Tau22 mouse model of tauopathy.

Epileptic seizures constitute a significant comorbidity of Alzheimer's disease (AD), which are recapitulated in transgenic mouse models of amyloidogenesis. Here, we sought to evaluate the potential role of tau pathology regarding seizure occurrence. To this end, we performed intra-hippocampal electroencephalogram (EEG) recordings and PTZ (pentylenetetrazol) seizure threshold tests in THY-Tau22 transgenic mice of AD-like tau pathology. We demonstrate that despite a lack of spontaneous epileptiform activity in Tau22 mice, the animals display increased PTZ-induced seizure susceptibility and mortality. The increased propensity for induced seizures in THY-Tau22 mutants correlates with astrogliosis and increased expression of adenosine kinase, consistent with increased network excitability. These data support an impact of tau pathology toward AD-associated seizures and suggest that tau pathology may contribute to seizure generation in AD independent of Aß pathology.

Microdialysis Unveils the Role of the α2-Adrenergic System in the Basolateral Amygdala during Acquisition of Conditioned Odor Aversion in the Rat.

Previous work has shown that ß-adrenergic and GABAergic systems in the basolateral amygdala (BLA) are involved in the acquisition of conditioned odor aversion (COA) learning. The involvement of α2-adrenoreceptors, however, is poorly documented. In a first experiment, male Long-Evans rats received infusions of 0.1 µg of the selective α2-antagonist dexefaroxan (Dex) in the BLA before being exposed to COA learning. In a second experiment, levels of norepinephrine (NE) were analyzed following Dex retrodialysis into the BLA. While microdialysis data showed a significant enhancement of NE release in the BLA with Dex, behavioral results showed that pre-CS infusion of Dex impaired, rather than facilitated, the acquisition of COA. Our results show that the NE system in the BLA is involved in the acquisition of COA, including a strong α2-receptor modulation until now unsuspected. Supported by the recent literature, the present data suggest moreover that the processes underlying this learning are probably mediated by the balanced effects of NE excitatory/inhibitory signaling in the BLA, in which interneurons are highly involved.

LAT1-like transporters regulate dopaminergic transmission and sleep in Drosophila.

Amino acid transporters are involved in functions reportedly linked to the sleep/wake cycle: neurotransmitter synthesis and recycling, the regulation of synaptic strength, protein synthesis, and energy metabolism. In addition, the existence of bidirectional relationships among extracellular content, transport systems, and sleep/wake states is receiving emerging support. Nevertheless, the connection between amino acid transport and sleep/wake regulation remains elusive. To address this question, we used Drosophila melanogaster and investigated the role of LAT1 (large neutral amino acid transporter 1) transporters. We show that the two Drosophila LAT1-like transporters: Juvenile hormone Inducible-21 and minidiscs (Mnd) are required in dopaminergic neurons for sleep/wake regulation. Down-regulating either gene in dopaminergic neurons resulted in higher daily sleep and longer sleep bout duration during the night, suggesting a defect in dopaminergic transmission. Since LAT1 transporters can mediate in mammals the uptake of L-DOPA, a precursor of dopamine, we assessed amino acid transport efficiency by L-DOPA feeding. We find that downregulation of JhI-21, but not Mnd, reduced the sensitivity to L-DOPA as measured by sleep loss. JhI-21 downregulation also attenuated the sleep loss induced by continuous activation of dopaminergic neurons. Since LAT1 transporters are known to regulate target of rapamycin (TOR) signaling, we investigated the role of this amino acid sensing pathway in dopaminergic neurons. Consistently, we report that TOR activity in dopaminergic neurons modulates sleep/wake states. Altogether, this study provides evidence that LAT1-mediated amino acid transport in dopaminergic neurons is playing a significant role in sleep/wake regulation and is providing several entry points to elucidate the role of nutrients such as amino acids in sleep/wake regulation.

External Influences on Invertebrate Brain Histamine and Related Compounds via an Automated Derivatization Method for Capillary Electrophoresis.

Histamine has been shown to modulate visual system and photic behavior in arthropods. However, few methods are available for the direct quantification of histamine and its precursor and metabolites in arthropod brain. In this work, a method for the separation of histamine, its precursor histidine, and its metabolite N-methyl-histamine from brain extracts of a freshwater crustacean has been developed using capillary electrophoresis with laser-induced fluorescence detection. Molecules were tagged on their primary amine function with naphthalene-2,3-dicarboxaldehyde, but derivatized histamine and N-methyl-histamine exhibited poor stability in contrast to derivatized histidine. To overcome this limitation, an automated derivatization performed within the capillary electrophoresis instrument was optimized and quantitatively validated. The limits of detection were 50, 30, and 60 nmol/L for histidine, histamine, and N-methyl-histamine, respectively. This study reports, for the first time, the amounts of histamine and its related compounds in brain extracts from populations of the freshwater amphipod Gammarus fossarum, and shows that these amounts vary mainly according to population and season, but are not affected by an experimental electrical shock.

Novel routes to either racemic or enantiopure α-amino-(4-hydroxy-pyrrolidin-3-yl)acetic acid derivatives and biological evaluation of a new promising pharmacological scaffold.

Cycloaddition between (+) or (-)-menthone-derived nitrones and N-benzyl-3-pyrroline afforded enantiopure spiro-fused heterocycles. The reaction occurred enantio- and diastereo-selectively on the less hindered side of the nitrone, the 3-pyrroline N-benzyl group being oriented outwards, thus controlling the configurations of three simultaneously created chiral centers. From either (+) or (-)-menthone, both enantiomeric cycloadducts were synthesized in excellent yield. Removing the chiral auxiliary and the N-benzyl group delivered a series of enantiopure 4-hydroxy-3-glycinyl-pyrrolidine derivatives in 3-5 steps and 36 to 81 overall yields. Using two other achiral nitrones, shorter routes to racemic analogues were developed. Two of the synthesized compounds markedly lowered extracellular glutamate level and modestly interacted with cannabinoid type-1 receptors. As these two neuroactive compounds were devoid of in vitro toxicity and did not cross the blood brain interface, they might represent potential pharmacological agents to target peripheral organs.

Why Optogenetics Needs in Vivo Neurochemistry.

In neuroscience, the consequences of optogenetic manipulation are often studied using in vivo electrophysiology and by observing behavioral changes induced by light stimulation in genetically targeted rodents. In contrast, reports on the in vivo neurochemical effects of optogenetic stimulation are scarce despite the improving quality of analytical techniques available to monitor biochemical compounds involved in neurotransmission. This intriguing lack of neurochemical information suggests the existence of unknown or misunderstood factors hampering the expected rise of a novel specialty putatively be termed "neurochemical optogenetics".

Noradrenergic influences in the basolateral amygdala on inhibitory avoidance memory are mediated by an action on α2-adrenoceptors.

The role of norepinephrine (NE) in the consolidation of inhibitory avoidance learning (IA) in rats is known to involve α1- and ß-adrenoceptor systems in the basolateral nucleus of the amygdala (BLA). However, the amygdala also contains α2-adrenoceptor subtypes, and local microinfusions of the selective α2-adrenoceptor antagonist idazoxan and agonist UK 14,304 respectively into the BLA enhance and inhibit IA performances when administered before acquisition. The present study investigated whether the effects of idazoxan and UK 14,304 on IA were associated with changes in NE release within the BLA before and after one-trial inhibitory avoidance training. Male Sprague-Dawley rats were unilaterally implanted with a microdialysis probe in the BLA and were administered idazoxan (0.1mM) or UK 14,304 (10 µM) by retrodialysis infusion 15 min before the acquisition of IA. Dialysates were collected every 15 min for analysis of NE. Retrodialysis of idazoxan potentiated the release of NE induced by footshock application, whereas UK 14,304 decreased NE release to the extent that the footshock failed to induce any measurable effect on NE levels. Idazoxan infusion enhanced IA retention tested 24h later and this effect was directly related to the level of NE release in the BLA measured during IA acquisition. In contrast, the infusion of UK 14,304 did not modify IA performances in comparison to control animals, possibly due to compensatory activity of the contralateral BLA. These results are consistent with previous evidence that amygdala NE is involved in modulating memory consolidation, and provide evidence for an involvement of presynaptic α2-autoceptors in the BLA in this process.

Differential involvement of amygdala and cortical NMDA receptors activation upon encoding in odor fear memory.

Although the basolateral amygdala (BLA) plays a crucial role for the acquisition of fear memories, sensory cortices are involved in their long-term storage in rats. However, the time course of their respective involvement has received little investigation. Here we assessed the role of the glutamatergic N-methyl-d-aspartate (NMDA) receptors in the BLA and olfactory cortex at discrete moments of an odor fear conditioning session. We showed that NMDA receptors in BLA are critically involved in odor fear acquisition during the first association but not during the next ones. In the cortex, NMDA receptor activation at encoding is not necessary for recent odor fear memory while its role in remote memory storage needs further investigation.

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.

Detection and quantification of neurotransmitters in dialysates.

Sensitive analytical methods are needed for the separation and quantification of neurotransmitters obtained in microdialysate studies. This unit describes methods that permit quantification of nanomolar concentrations of monoamines and their metabolites (high-performance liquid chromatography [HPLC] electrochemical detection), acetylcholine (HPLC-coupled to an enzyme reactor), and amino acids (HPLC-fluorescence detection, capillary electrophoresis with laser-induced fluorescence detection).

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.

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.

A new animal model of obstructive sleep apnea responding to continuous positive airway pressure.

STUDY OBJECTIVES: An improved animal model of obstructive sleep apnea (OSA) is needed for the development of effective pharmacotherapies. In humans, flexion of the neck and a supine position, two main pathogenic factors during human sleep, are associated with substantially greater OSA severity. We postulated that these two factors might generate OSA in animals. DESIGN: We developed a restraining device for conditioning to investigate the effect of the combination of 2 body positions-prone (P) or supine (S)-and 2 head positions-with the neck flexed at right angles to the body (90°) or in extension in line with the body (180°)-during sleep in 6 cats. Polysomnography was performed twice on each cat in each of the 4 sleeping positions-P180, S180, P90, or S90. The effect of continuous positive airway pressure (CPAP) treatment was then investigated in 2 cats under the most pathogenic condition. SETTING: NA. PATIENTS OR PARTICIPANTS: NA. INTERVENTIONS: NA. MEASUREMENTS AND RESULTS: Positions P180 and, S90 resulted, respectively, in the lowest and highest apnea-hypopnea index (AHI) (3 ± 1 vs 25 ± 2, P < 0.001), while P90 (18 ± 3, P<0.001) and S180 (13 ± 5, P<0.01) gave intermediate values. In position S90, an increase in slow wave sleep stage 1 (28% ± 3% vs 22% ± 3%, P<0.05) and a decrease in REM sleep (10% ± 2% vs 18% ± 2%, P<0.001) were also observed. CPAP resulted in a reduction in the AHI (8 ± 1 vs 27 ± 3, P<0.01), with the added benefit of sleep consolidation. CONCLUSION: By mimicking human pathogenic sleep conditions, we have developed a new reversible animal model of OSA.

Fluorescence enhancement of a Meisenheimer complex of adenosine by gamma-cyclodextrin: a thermodynamic and kinetic investigation.

The fluorescent properties of a trinitrophenylated Meisenheimer complex of adenosine (TNP-Ado) in water were examined in the presence of alpha-, beta-, and gamma-cyclodextrins (CDs). The TNP-Ado complex exhibits minimal fluorescence in water, whereas addition of 10 mM alpha-CD, beta-CD, and gamma-CD enhances fluorescence by factors of 2, 7, and 110, respectively. The large enhancement by gamma-CD is attributed to its larger hydrophobic cavity, which is able to accommodate the TNP moiety of TNP-Ado. (1)H NMR spectra demonstrate 1:1 stoichiometry of the complex, which undergoes slow exchange on the NMR time scale. (1)H NMR and 2D ROESY spectra reveal substantial interaction of the TNP hydrogens with gamma-CD. Equilibrium constants were determined by fluorimetry from 10 to 20 degrees C by nonlinear curve fitting. Fluorescence is temperature dependent, with maximum fluorescence increasing with decreasing temperature. Complexation is exothermic with large negative entropy, consistent with formation of a tight complex between TNP-Ado and gamma-CD. Rate constants and activation parameters for both complexation and dissociation were determined by a combination of fluorimetry and 2D NMR exchange spectroscopy (EXSY).