Calcium imaging of odor-evoked responses in the Drosophila antennal lobe.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions. Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP and Cameleon, the bioluminescent calcium indicator GFP-aequorin, or a reporter of synaptic transmission, synapto-pHluorin has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS, LexA/LexAop, Q system) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed. Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(2+) and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca(2+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.

Differential transgene expression profiles from rAAV2/1 vectors using the tetON and CMV promoters in the rat brain.

The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors using the tetON expression cassette in comparison with the CMV promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although GFP was mainly expressed into neurons with both vectors, the relative proportions of DARPP-32+ projection neurons and parvalbumin+ interneurons were respectively 13:1 and 2:1 for the CMV and tetON vectors. DARP32+ neurons projecting to the globus pallidus were strongly GFP+ with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV but poorly by the tetON vector. Numerous GFP+ cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP+ neurons were observed with the CMV but not the tetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-tetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase+ neurons by the tetON vector whereas with the CMV vector, GFP+ cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-tetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.

Metabolic Flux and Compartmentation Analysis in the Brain In vivo.

Through significant developments and progresses in the last two decades, in vivo localized nuclear magnetic resonance spectroscopy (MRS) became a method of choice to probe brain metabolic pathways in a non-invasive way. Beside the measurement of the total concentration of more than 20 metabolites, (1)H MRS can be used to quantify the dynamics of substrate transport across the blood-brain barrier by varying the plasma substrate level. On the other hand, (13)C MRS with the infusion of (13)C-enriched substrates enables the characterization of brain oxidative metabolism and neurotransmission by incorporation of (13)C in the different carbon positions of amino acid neurotransmitters. The quantitative determination of the biochemical reactions involved in these processes requires the use of appropriate metabolic models, whose level of details is strongly related to the amount of data accessible with in vivo MRS. In the present work, we present the different steps involved in the elaboration of a mathematical model of a given brain metabolic process and its application to the experimental data in order to extract quantitative brain metabolic rates. We review the recent advances in the localized measurement of brain glucose transport and compartmentalized brain energy metabolism, and how these reveal mechanistic details on glial support to glutamatergic and GABAergic neurons.

Redox dysregulation in the pathophysiology of schizophrenia and bipolar disorder: insights from animal models.

Abstract Significance: Schizophrenia (SZ) and bipolar disorder (BD) are classified as two distinct diseases. However, accumulating evidence shows that both disorders share genetic, pathological, and epidemiological characteristics. Based on genetic and functional findings, redox dysregulation due to an imbalance between pro-oxidants and antioxidant defense mechanisms has been proposed as a risk factor contributing to their pathophysiology. Recent Advances: Altered antioxidant systems and signs of increased oxidative stress are observed in peripheral tissues and brains of SZ and BD patients, including abnormal prefrontal levels of glutathione (GSH), the major cellular redox regulator and antioxidant. Here we review experimental data from rodent models demonstrating that permanent as well as transient GSH deficit results in behavioral, morphological, electrophysiological, and neurochemical alterations analogous to pathologies observed in patients. Mice with GSH deficit display increased stress reactivity, altered social behavior, impaired prepulse inhibition, and exaggerated locomotor responses to psychostimulant injection. These behavioral changes are accompanied by N-methyl-D-aspartate receptor hypofunction, elevated glutamate levels, impairment of parvalbumin GABA interneurons, abnormal neuronal synchronization, altered dopamine neurotransmission, and deficient myelination. Critical Issues: Treatment with the GSH precursor and antioxidant N-acetylcysteine normalizes some of those deficits in mice, but also improves SZ and BD symptoms when given as adjunct to antipsychotic medication. Future Directions: These data demonstrate the usefulness of GSH-deficient rodent models to identify the mechanisms by which a redox imbalance could contribute to the development of SZ and BD pathophysiologies, and to develop novel therapeutic approaches based on antioxidant and redox regulator compounds. Antioxid. Redox Signal. 18, 1428-1443.

Redox dysregulation, neurodevelopment, and schizophrenia.

In schizophrenia, a developmental redox dysregulation constitutes one 'hub' on which converge genetic impairments of glutathione synthesis and environmental vulnerability factors generating oxidative stress. Their timing at critical periods of neurodevelopment could play a decisive role in inducing impairment of neural connectivity and synchronization as observed in schizophrenia. In experimental models, such redox dysregulation induces anomalies strikingly similar to those observed in patients. This is mediated by hypoactive NMDA receptors, impairment of fast-spiking parvalbumin GABA interneurons and deficit in myelination. A treatment restoring the redox balance without side effects yields improvements of negative symptoms in chronic patients. Novel interventions based on these mechanisms if applied in early phases of the disease hold great therapeutic promise.

Selective neurodegeneration induced in rotation-mediated aggregate cell cultures by a transient switch to stationary culture conditions: a potential model to study ischemia-related pathogenic mechanisms.

Aggregating brain cell cultures at an advanced maturational stage (20-21 days in vitro) were subjected for 1-3 h to anaerobic (hypoxic) and/or stationary (ischemic) conditions. After restoration of the normal culture conditions, cell loss was estimated by measuring the release of lactate dehydrogenase as well as the irreversible decrease of cell type-specific enzyme activities, total protein and DNA content. Ischemia for 2 h induced significant neuronal cell death. Hypoxia combined with ischemia affected both neuronal and glial cells to different degrees (GABAergic neurons>cholinergic neurons>astrocytes). Hypoxic and ischemic conditions greatly stimulated the uptake of 2-deoxy-D-glucose, indicating increased glucose consumption. Furthermore, glucose restriction (5.5 mM instead of 25 mM) dramatically increased the susceptibility of neuronal and glial cells to hypoxic and ischemic conditions. Glucose media concentrations below 2 mM caused selective neuronal cell death in otherwise normal culture conditions. GABAergic neurons showed a particularly high sensitivity to glucose restriction, hypoxia, and ischemia. The pattern of ischemia-induced changes in vitro showed many similarities to in vivo findings, suggesting that aggregating brain cell cultures provide a useful in vitro model to study pathogenic mechanisms related to brain ischemia.

An olfactory receptor for food-derived odours promotes male courtship in Drosophila.

Many animals attract mating partners through the release of volatile sex pheromones, which can convey information on the species, gender and receptivity of the sender to induce innate courtship and mating behaviours by the receiver. Male Drosophila melanogaster fruitflies display stereotyped reproductive behaviours towards females, and these behaviours are controlled by the neural circuitry expressing male-specific isoforms of the transcription factor Fruitless (FRU(M)). However, the volatile pheromone ligands, receptors and olfactory sensory neurons (OSNs) that promote male courtship have not been identified in this important model organism. Here we describe a novel courtship function of Ionotropic receptor 84a (IR84a), which is a member of the chemosensory ionotropic glutamate receptor family, in a previously uncharacterized population of FRU(M)-positive OSNs. IR84a-expressing neurons are activated not by fly-derived chemicals but by the aromatic odours phenylacetic acid and phenylacetaldehyde, which are widely found in fruit and other plant tissues that serve as food sources and oviposition sites for drosophilid flies. Mutation of Ir84a abolishes both odour-evoked and spontaneous electrophysiological activity in these neurons and markedly reduces male courtship behaviour. Conversely, male courtship is increased--in an IR84a-dependent manner--in the presence of phenylacetic acid but not in the presence of another fruit odour that does not activate IR84a. Interneurons downstream of IR84a-expressing OSNs innervate a pheromone-processing centre in the brain. Whereas IR84a orthologues and phenylacetic-acid-responsive neurons are present in diverse drosophilid species, IR84a is absent from insects that rely on long-range sex pheromones. Our results suggest a model in which IR84a couples food presence to the activation of the fru(M) courtship circuitry in fruitflies. These findings reveal an unusual but effective evolutionary solution to coordinate feeding and oviposition site selection with reproductive behaviours through a specific sensory pathway.

The role of Bmi1 in CNS development and in retinal degeneration

SUMMARY : The present work addresses several aspects of cell cycle regulation, cell fate specification and cell death in the central nervous system (CNS), specifically the cortex and the retina. More precisely, we investigated the role of Bmi1, a polycomb family gene required for stem cell proliferation and self-renewal, in the development of the cerebral cortex, as well as in the genesis of the retina. These data, together with studies published during the last two decades concerning cell cycle re-activation in apoptotic neurons in the CNS, raised the question of a possible link between regulation of the cell cycle during development and during retinal degeneration. 1. The effects of Bmi1 loss in the cerebral cortex : Consistently with our and others' observations on failure of Bmi9-/- stem cells to proliferate and self-renew in vitro, the Bmi9-/- cerebral cortex presented slight defects in proliferation in stem/progenitor cells compartments in vivo. This was in accordance with the pattern of Bmi1 expression in the developing forebrain. The modest proliferation defects, compared to the drastic consequences of Bmi9 loss in vitro, suggest that cell-extrinsic mechanisms may partially compensate for Bmi1 deletion in vivo during cortical histogenesis. Nevertheless, we observed a decreased proliferating activity in neurogenic regions of the adult telencephalon, more precisely in the subventricular zone, showing that Bmi1 controls neural stem/progenitor proliferation during adulthood in vivo. Our data also highlight an increased production of astrocytes at birth, and a generalized gliosis in the adult Bmi9-/- brain. Importantly, glial progenitors and astrocytes retained the ability to proliferate in the absence of Bmi1. 2. The effects of Bmi1 loss in the retina : The pattern of expression of Bmi1 during development and in the adult retina suggests a role for Bmi1 in cell fate specification and differentiation rather than in proliferation. While the layering and the global structure of the retina appear normal in Bmi1 /adult mice, immunohistochemìcal analysis revealed defects in the three major classes of retinal interneurons, namely: horizontal, bipolar and amacrine cells. Electroretinogram recordings in Bmi9-/- mice are coherent with the defects observed at the histological level, with a reduced b-wave and low-profile oscillatory potentials. These results show that Bmi1 controls not only proliferation, but also cell type generation, as previously observed in the cerebellum. 3. Cell cycle events and related neuroprotective strategies in retinal degeneration : In several neurodegenerative disorders, neurons re-express cell cycle proteins such as cyclin dependent kinases (Cdks) prior to apoptosis. Here, we show for the first time that this is also the case during retinal degeneration. Rd1 mice carry a recessive defect (Pdeóbrd/rd) that causes retinal degeneration and serves as a model of retinitis pigmentosa. We found that photoreceptors express Cdk4 and Cdk2, and undergo DNA synthesis prior to cell death. To interfere with the reactivation of Cdk-related pathways, we deleted E2fs or Brni1, which normally allow cell cycle progression. While deleting E2f1 (downstream of Cdk4/6) in Rd1 mice provides only temporary protection, knocking out Bmi1 (upstream of Cdks) leads to an extensive neuroprotective effect, independent of p16ink4a or p19arf, two tumor suppressors regulated by Bmi1. Analysis of Cdks and the DNA repair-related protein Ligase IV showed that Bmi1 acts downstream of DNA repair events and upstream of Cdks in this neurodegenerative mechanism. Expression of Cdks during an acute model of retinal degeneration, light damage-induced photoreceptor death, points to a role for Bmi1 and cell cycle proteins in retinal degeneration. Considering the similarity with the cell cycle-related apoptotic pathway observed in other neurodegenerative diseases, Bmi1 is a possible general target to prevent or delay neuronal death. RESUME : Ce travail aborde plusieurs aspects de la régulation du cycle cellulaire, de la spécification du devenir des cellules et de la mort cellulaire dans le système nerveux centrale (SNC), plus particulièrement dans le cortex cérébral et dans la rétine. Nous nous sommes intéressés au gène Bmi1, appartenant à la famille polycomb et nécessaire à la prolifération et au renouvellement des cellules souches. Nous avons visé à disséquer son rôle dans le développement du cortex et de la rétine. Ces données, ainsi qu'une série de travaux publiés au cours des deux dernières décennies concernant la réactivation du cycle cellulaire dans les neurones en voie d'apoptose dans le SNC, nous ont ensuite poussé à chercher un lien entre la régulation du cycle cellulaire pendant le développement et au cours de la dégénérescence rétinienne. 1. Les effets de l'inactivation de Bmi1 dans le cortex cérébral : En accord avec l'incapacité des cellules souches neurales in vitro à proliférer et à se renouveler en absence de Bmi1, le cortex cérébral des souris Bmi1-/- présente de légers défauts de prolifération dans les compartiments contenant les cellules souches neurales. Ceci est en accord avec le profil d'expression de Bmi1 dans le télencéphale. Les conséquences de la délétion de Bmi1 sont toutefois nettement moins prononcées in vivo qu'in vitro ; cette différence suggère l'existence de mécanismes pouvant partiellement compenser l'absence de Bmi1 pendant la corticogenèse. Néanmoins, l'observation d'une réduction de la prolifération dans la zone sous-ventriculaire, la zone majeure de neurogenèse dans le télencéphale adulte, montre que Bmi1 contrôle la prolifération des cellules souche/progénitrices neurales chez la souris adulte. Nos résultats démontrent par ailleurs une augmentation de la production d'astrocytes à la naissance ainsi qu'une gliose généralisée à l'état adulte chez les souris Bmi1-/-. Les progéniteurs gliaux et les astrocytes conservent donc leur capacité à proliférer en absence de Bmi1. 2. Les effets de l'inactivation de Bmi1 dans la rétine : Le profil d'expression de Bmi1 pendant fe développement ainsi que dans la rétine adulte suggère un rôle de Bmi1 dans la spécification de certains types cellulaires et dans la différentiation plutôt que dans la prolifération. Alors que la structure et la lamination de la rétine semblent normales chez les souris Bmi1-/-, l'analyse par immunohistochimie amis en évidence des défauts au niveau des trois classes d'interneurones rétiniens (les cellules horizontales, bipolaires et amacrines). Les électrorétinogrammes des souris Bmi1-/- sont cohérents avec les défauts observés au niveau histologique et montrent une réduction de l'onde « b » et des potentiels oscillatoires. Ces résultats montrent que Bmi1 contrôle la génération de certaines sous-populations de neurones, comme démontré auparavant au niveau de cervelet. 3. Réactivation du cycle cellulaire et stratégies théraoeutiaues dans les dégénérescences rétiniennes : Dans plusieurs maladies neurodégénératives, les neurones ré-expriment des protéines du cycle cellulaire telles que les kinases cycline-dépendantes (Cdk) avant d'entrer en apoptose. Nous avons démontré que c'est aussi le cas dans les dégénérescences rétiniennes. Les souris Rd1 portent une mutation récessive (Pde6brd/rd) qui induit une dégénérescence de la rétine et sont utilisées comme modèle animal de rétinite pigmentaire. Nous avons observé que les photorécepteurs expriment Cdk4 et Cdk2, et entament une synthèse d'ADN avant de mourir par apoptose. Pour interférer avec la réactivation les mécanismes Cdk-dépendants, nous avons inactivé les gènes E2f et Bmi1, qui permettent normalement la progression du cycle cellulaire. Nous avons mis en évidence que la délétion de E2f1 (en aval de Cdk4/6) dans les souris Rd1 permet une protection transitoire des photorécepteurs. Toutefois, l'inactivation de Bmi1 (en amont des Cdk) est corrélée à une neuroprotection bien plus durable et ceci indépendamment de p16ink4a et p19arf, deux suppresseurs de tumeurs normalement régulés par Bmi1. L'analyse des Cdk et de la ligase IV (une protéine impliquée dans les mécanismes de réparation de l'ADN) a montré que Bmi1 agit en aval des événements de réparation de l'ADN et en amont des Cdk dans la cascade apoptotique dans les photorécepteurs des souris Rd1. Nous avons également observé la présence de Cdk dans un modèle aigu de dégénérescence rétinienne induit par une exposition des animaux à des niveaux toxiques de lumière. Nos résultats suggèrent donc un rôle général de Bmi1 et des protéines du cycle cellulaire dans les dégénérescences de la rétine. Si l'on considère la similarité avec les événements de réactivation du cycle cellulaire observés dans d'autres maladies neurodégénératives, Bmi1 pourrait être une cible thérapeutique générale pour prévenir la mort neuronale.

Distribution of neuronal and metabolic markers in the human auditory cortex

SUMMARY The human auditory cortex, located on the supratemporal plane of the temporal lobe, is divided in a primary auditory area and several non-primary areas surrounding it. These different areas show anatomical and functional differences. Many studies have focussed on auditory areas in non-human primates, using investigation techniques such as electrophysiological recordings, tracing of neural connections, or immunohistochemical and histochemical staining. Some of these studies have suggested parallel and hierarchical organization of the cortical auditory areas as well as subcortical auditory relays. In humans, only few studies have investigated these regions immunohistochemically, but activation and lesion studies speak in favour of parallel and hierarchical organization, very similar to that of non-human primates. Calcium-binding proteins and metabolic markers were used to investigate possible correlates of hierarchical and parallel organization in man. Calcium-binding proteins, parvalbumin, calretinin and calbindin, modulate the concentration of intracellular free calcium ions and were found in distinct subpopulations of GABAergic neurons in non-human primates species. In our study, their distribution showed several differences between auditory areas: the primary auditory area was darkly stained for both parvalbumin and calbindin, and their expression rapidly decreased while moving away from the primary area. This staining pattern suggests a hierarchical organization of the areas, in which the more darkly stained areas could correspond to an earlier integration level and the areas showing light staining may correspond to higher level integration areas. Parallel organization of primary and non-primary auditory areas was suggested by the complementarity, within a given area, between parvalbumin and calbindin expression across layers. To investigate the possible differences in the energetic metabolism of the cortical auditory areas, several metabolic markers were used: cytochrome oxidase and LDH1 were used as oxidative metabolism markers and LDH5 was used as glycolytic metabolism marker. The results obtained show a difference in the expression of enzymes involved in oxidative metabolism between areas. In the primary auditory area the oxidative metabolism markers were maximally expressed in layer IV. In contrast, higher order areas showed maximal staining in supragranular layers. The expression of LDH5 varied in patches, but did not differ between the different hierarchical auditory areas. The distribution of the two LDH enzymes isoforms also provides information about cellular aspects of metabolic organization, since neurons expressed the LDH1 isoform whereas astrocytes express primarily LDH5, but some astrocytes also contained the LDH1 isoform. This cellular distribution pattern supports the hypothesis of the existence of an astrocyte-neuron lactate shuttle, previously suggested in rodent studies, and in particular of lactate transfer from astrocytes, which produce lactate from the glucose obtained from the circulation, to neurons that use lactate as energy substrate. In conclusion, the hypothesis of parallel and hierarchical organization of the auditory areas can be supported by CaBPs, cytochrome oxidase and LDH1 distribution. Moreover, the two LDHs cellular distribution pattern support the hypothesis of an astrocyte-neuron lactate shuttle in human cortex.

Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to study the maturation-dependent sensitivity of brain cells to two organophosphorus pesticides (OPs), chlorpyrifos and parathion, and to their oxon derivatives. Immature (DIV 5-15) or differentiated (DIV 25-35) brain cells were treated continuously for 10 days. Acetylcholinesterase (AChE) inhibitory potency for the OPs was compared to that of eserine (physostigmine), a reversible AChE inhibitor. Oxon derivatives were more potent AChE inhibitors than the parent compounds, and parathion was more potent than chlorpyrifos. No maturation-dependent differences for AChE inhibition were found for chlorpyrifos and eserine, whereas for parathion and paraoxon there was a tendency to be more effective in immature cultures, while the opposite was true for chlorpyrifos-oxon. Toxic effects, assessed by measuring protein content as an index of general cytotoxicity, and various enzyme activities as cell-type-specific neuronal and glial markers (ChAT and GAD, for cholinergic and GABAergic neurons, respectively, and GS and CNP, for astrocytes and oligodendrocytes, respectively) were only found at more than 70% of AChE inhibition. Immature compared to differentiated cholinergic neurons appeared to be more sensitive to OP treatments. The oxon derivates were found to be more toxic on neurons than the parent compounds, and chlorpyrifos was more toxic than parathion. Eserine was not neurotoxic. These results indicate that inhibition of AChE remains the most sensitive macromolecular target of OP exposure, since toxic effects were found at concentrations in which AChE was inhibited. Furthermore, the compound-specific reactions, the differential pattern of toxicity of OPs compared to eserine, and the higher sensitivity of immature brain cells suggest that the toxic effects and inhibition of AChE are unrelated.

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Evidence of altered antioxidant systems and signs of elevated oxidative stress are reported in peripheral tissue and brain of schizophrenic patients, including low levels of glutathione (GSH), a major thiol antioxidant and redox buffer. Functional and genetic data indicate that an impaired regulation of GSH synthesis is a vulnerability factor for the disease. Impaired GSH synthesis from a genetic origin combined with environmental risk factors generating oxidative stress (e.g., malnutrition, exposure to toxins, maternai infection and diabetes, obstetrical complications, and psychological stress) could lead to redox dysregulation. This could subsequently perturb normal brain development and maturation with delayed functional consequences emerging in early adulthood. Depending on the nature and the time of occurrence of the environmental insults, the structural and functional delayed consequences could vary, giving rise to various endophenotypes. The use of animal models of GSH deficit represents a valuable approach to investigate how interactions between genetic and environmental factors lead to the emergence of pathologies found in the disease. Moreover, these models of GSH can be useful to investigate links between schizophrenia and comorbid somatic disorders, as dysregulation of the GSH system and elevated oxidative stress are also found in cardiovascular diseases and diabetes. This chapter reviews pharmacological and genetic rodent models of GSH synthesis dysregulation used to address some of the aforementioned issues. Up to date, these models revealed that GSH deficits lead to morphological, physiological, and behavioral alterations that are quite analogous to pathologies observed in patients. This includes hypofunction of NMDA receptors, alteration of dopamine neurotransmission, anomalies in parvalbumin-immunoreactive fast-spiking interneurons, and reduced myelination. In addition, a GSH deficit affects the brain in a region-specific manner, the anterior cingulate cortex and the ventral hippocampus being the most vulnerable regions investigated. Interestingly, a GSH deficit during a limited period of postnatal development is sufficient to have long-lasting consequences on the integrity of PV-IR interneurons in the anterior cingulate cortex and impairs cognitive functions in adulthood. Finally, these animal models of GSH deficit display behavioral impairments that could be related to schizophrenia. Altogether, current data strongly support a contributing role of a redox dysregulation on the development of pathologies associated with the illness and demonstrate the usefulness of these models to better understand the biological mechanisms leading to schizophrenia.

Differential transgene expression profiles in rat brain, using rAAV2/1 vectors with tetracycline-inducible and cytomegalovirus promoters.

The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for the safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors (recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1) using the tetracycline-inducible (TetON) expression cassette in comparison with the cytomegalovirus (CMV) promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although green fluorescent protein (GFP) was expressed mainly in neurons with both vectors, the relative proportions of DARPP-32-positive projection neurons and parvalbumin-positive interneurons were, respectively, 13:1 and 2:1 for the CMV and TetON vectors. DARP32-positive neurons projecting to the globus pallidus were strongly GFP positive with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV vector but poorly by the TetON vector. Numerous GFP-positive cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP-positive neurons were observed with the CMV vector but not the TetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-TetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase-positive neurons by the TetON vector whereas with the CMV vector, GFP-positive cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-TetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.

Immunodensity and mRNA expression of A2A adenosine, D2 dopamine, and CB1 cannabinoid receptors in postmortem frontal cortex of subjects with schizophrenia: effect of antipsychotic treatment.

RATIONALE: Dopamine D2 receptors are the main target of antipsychotic drugs. In the brain, D2 receptors coexpress with adenosine A2A and CB1 cannabinoid receptors, leading to functional interactions. OBJECTIVES: The protein and messenger RNA (mRNA) contents of A2A, D2, and CB1 receptors were quantified in postmortem prefrontal cortex of subjects with schizophrenia. MATERIALS AND METHODS: The study was performed in subjects suffering schizophrenia (n=31) who mainly died by suicide, matched with non-schizophrenia suicide victims (n=13) and non-suicide controls (n=33). The density of receptor proteins was evaluated by immunodetection techniques, and their relative mRNA expression was quantified by quantitative real-time polymerase chain reaction. RESULTS: In schizophrenia, the densities of A2A (90+/-6%, n=24) and D2-like receptors (95+/-5%, n=22) did not differ from those in controls (100%). Antipsychotic treatment did not induce changes in the protein expression. In contrast, the immunodensity of CB1 receptors was significantly decreased (71+/-7%, n=11; p<0.05) in antipsychotic-treated subjects with schizophrenia but not in drug-free subjects (104+/-13%, n=11). The relative mRNA amounts encoding for A2A, D2, and CB1 receptors were similar in brains of drug-free, antipsychotic-treated subjects with schizophrenia and controls. CONCLUSIONS: The findings suggest that antipsychotics induce down-regulation of CB1 receptors in brain. Since A2A, D2, and CB1 receptors coexpress on brain GABAergic neurons and reductions in markers of GABA neurotransmission have been identified in schizophrenia, a lower density of CB1 receptor induced by antipsychotics could represent an adaptative mechanism that reduces the endocannabinoid-mediated suppression of GABA release, contributing to the normalization of cognitive functions in the disorder.

Expression of the GABAA receptor associated protein Gec1 is circadian and dependent upon the cellular clock machinery in GnRH secreting GnV-3 cells.

The timely regulation of gonadotropin-releasing hormone (GnRH) secretion requires a GABAergic signal. We hypothesized that GEC1, a protein promoting the transport of GABA(A) receptors, could represent a circadian effector in GnRH neurons. First, we demonstrated that gec1 is co-expressed with the GABA(A) receptor in hypothalamic rat GnRH neurons. We also confirmed that the clock genes per1, cry1 and bmal1 are expressed and oscillate in GnRH secreting GnV-3 cells. Then we could show that gec1 is expressed in GnV-3 cells, and oscillates in a manner temporally related to the oscillations of the clock transcription factors. Furthermore, we could demonstrate that these oscillations depend upon Per1 expression. Finally, we observed that GABA(A) receptor levels at the GnV-3 cell membrane are timely modulated following serum shock. Together, these data demonstrate that gec1 expression is dependent upon the circadian clock machinery in GnRH-expressing neurons, and suggest for the first time that the level of GABA(A) receptor at the cell membrane may be under timely regulation. Overall, they provide a potential mechanism for the circadian regulation of GnRH secretion by GABA, and may also be relevant to the general understanding of circadian rhythms.