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Melatonin is rhythmically synthesized and released by the pineal gland and, in some species, retina during the night and regulates many physiological and behavioral processes in birds and mammals. Chick diencephalic astrocytes express two melatonin receptor subtypes in vitro, and melatonin plays a role in regulating metabolic activity. We examined the role of glial cells in circadian function and asked if melatonin modulated glial functions within the retina and the brain. Calcium waves were potentiated by physiological concentrations of melatonin. Melatonin increased resting calcium levels and reduced gap junctional coupling among astrocytes at these same concentrations. Both mouse and chick diencephalic and telencephalic astrocytes express melatonin receptor protein. Nanomolar melatonin modulated astrocytic calcium waves of the mouse and chick diencephalon but not waves of the telencephalon. Mammalian intercellular calcium waves spread farther than avian calcium waves, and the nature of the spread of the waves differed between telencephalic and diencephalic mammalian astrocytes. These differences in propagation were abolished by melatonin. Using northern analysis, we identified period2, period3, cryptochrome1, cryptochrome2, clock, melanopsin and peropsin within chick diencephalic astrocytes. The clock genes cry1 and, per2 were expressed rhythmically in a LD cycle, but metabolic activity was not rhythmic. When cells were placed in constant darkness and rhythmically administrated melatonin, a robust rhythm in glucose uptake was induced without a coordinated clock gene rhythm, suggesting rhythmic clock gene expression and metabolic activity are separable processes. Melatonin affected visual function as assessed by electroretinogram. Circadian rhythms of a- and b-wave implicit times and amplitudes were observed. Melatonin (1 mg/kg and 100 ng/kg) decreased a- and b-wave amplitudes greater during the night than during the day and it increased a- and b-wave implicit times while 1 ng/kg melatonin had little to no effect over the saline controls. These data indicate that melatonin modulates glial intercellular communication, affects metabolic activity in astrocytes, and may play a role in regulating a day and night functional shift in the retina, at least partially through Müller glial cells. Thus, melatonin can regulate glia function and thereby, affect outputs of the vertebrate biological clock.

Chronic exposure to manganese (Mn) causes the neurodegenerative movement disorder, manganism. A mouse model was developed to elucidate mechanisms involved in the etiology and progression of injury. Twelve-week old female C57Bl/6J mice were exposed to MnCl2 (100 mg/kg/day) by oral gavage daily for 8 weeks. After the experiment striatal dopamine (DA) content was decreased with the manifestation of hypoactivity. A distinct population of neurons was vulnerable to the effects of Mn, including enkephalin (ENK)-positive projection neurons, interneurons expressing neuronal nitric oxide synthetase (nNOS/NOS1), and choline acetyltransferase (ChAT)-expressing interneurons. Activation of surrounding astrocytes occurred with expression of inducible nitric oxide synthase (iNOS/NOS2) and production of nitric oxide (NO)/peroxynitrite (ONOO-). Activated astrocytes were detected primarily near the microvasculature in both the striatum and globus pallidus (GP). It is suggested that Mn exposure may damage the blood-brain barrier (BBB) and induce astrocytosis and NOS2 expression, subsequent NO production may cause the death of adjacent neurons. This hypothesis was also tested in an in vitro co-culture model. Differentiated pheochromocytoma cells (PC12 cells) were co-cultured with primary astrocytes and exposed to Mn and inflammatory cytokines. Mn and cytokines induced NOS2 expression and NO production in astrocytes, which correlated with apoptosis of PC12 cells. Apoptosis of PC12 cells was prevented by overexpression of a phosphorylation-deficient mutant of IκBα that inhibited NOS2 expression in astrocytes. It is concluded that Mn-and cytokine-dependent apoptosis in PC12 cells requires astrocyte-derived NO and nuclear factor κB (NF-κB)-dependent expression of NOS2. To explore possible means of interdicting this inflammatory process in astrocytes, a noval pharmacologic ligands of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, 1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl) methane (DIM-C-pPhCF3) were used in the same co-culture system. DIM-C-pPhCF3 protected PC12 cells from apoptosis through inhibition of NOS2 expression in astrocytes after Mn and cytokines exposure. By contrast, the PPARγ antagonist, 2-chloro-5-nitrobenzanilide (GW9622), had the opposite effect, increasing both NO production in astrocytes and neuronal injury. It is concluded that PPARγ is involved in the regulation of NOS2 expression in astrocytes and that agonists of PPARγ may represent a potential treatment method for Mn neurotoxicity.

Melatonin is rhythmically synthesized and released by the pineal gland and, in some species, retina during the night and regulates many physiological and behavioral processes in birds and mammals. Chick diencephalic astrocytes express two melatonin receptor subtypes in vitro, and melatonin plays a role in regulating metabolic activity. We examined the role of glial cells in circadian function and asked if melatonin modulated glial functions within the retina and the brain. Calcium waves were potentiated by physiological concentrations of melatonin. Melatonin increased resting calcium levels and reduced gap junctional coupling among astrocytes at these same concentrations. Both mouse and chick diencephalic and telencephalic astrocytes express melatonin receptor protein. Nanomolar melatonin modulated astrocytic calcium waves of the mouse and chick diencephalon but not waves of the telencephalon. Mammalian intercellular calcium waves spread farther than avian calcium waves, and the nature of the spread of the waves differed between telencephalic and diencephalic mammalian astrocytes. These differences in propagation were abolished by melatonin. Using northern analysis, we identified period2, period3, cryptochrome1, cryptochrome2, clock, melanopsin and peropsin within chick diencephalic astrocytes. The clock genes cry1 and, per2 were expressed rhythmically in a LD cycle, but metabolic activity was not rhythmic. When cells were placed in constant darkness and rhythmically administrated melatonin, a robust rhythm in glucose uptake was induced without a coordinated clock gene rhythm, suggesting rhythmic clock gene expression and metabolic activity are separable processes. Melatonin affected visual function as assessed by electroretinogram. Circadian rhythms of a- and b-wave implicit times and amplitudes were observed. Melatonin (1 mg/kg and 100 ng/kg) decreased a- and b-wave amplitudes greater during the night than during the day and it increased a- and b-wave implicit times while 1 ng/kg melatonin had little to no effect over the saline controls. These data indicate that melatonin modulates glial intercellular communication, affects metabolic activity in astrocytes, and may play a role in regulating a day and night functional shift in the retina, at least partially through Müller glial cells. Thus, melatonin can regulate glia function and thereby, affect outputs of the vertebrate biological clock.

Chronic exposure to manganese (Mn) causes the neurodegenerative movement disorder, manganism. A mouse model was developed to elucidate mechanisms involved in the etiology and progression of injury. Twelve-week old female C57Bl/6J mice were exposed to MnCl2 (100 mg/kg/day) by oral gavage daily for 8 weeks. After the experiment striatal dopamine (DA) content was decreased with the manifestation of hypoactivity. A distinct population of neurons was vulnerable to the effects of Mn, including enkephalin (ENK)-positive projection neurons, interneurons expressing neuronal nitric oxide synthetase (nNOS/NOS1), and choline acetyltransferase (ChAT)-expressing interneurons. Activation of surrounding astrocytes occurred with expression of inducible nitric oxide synthase (iNOS/NOS2) and production of nitric oxide (NO)/peroxynitrite (ONOO-). Activated astrocytes were detected primarily near the microvasculature in both the striatum and globus pallidus (GP). It is suggested that Mn exposure may damage the blood-brain barrier (BBB) and induce astrocytosis and NOS2 expression, subsequent NO production may cause the death of adjacent neurons. This hypothesis was also tested in an in vitro co-culture model. Differentiated pheochromocytoma cells (PC12 cells) were co-cultured with primary astrocytes and exposed to Mn and inflammatory cytokines. Mn and cytokines induced NOS2 expression and NO production in astrocytes, which correlated with apoptosis of PC12 cells. Apoptosis of PC12 cells was prevented by overexpression of a phosphorylation-deficient mutant of IκBα that inhibited NOS2 expression in astrocytes. It is concluded that Mn-and cytokine-dependent apoptosis in PC12 cells requires astrocyte-derived NO and nuclear factor κB (NF-κB)-dependent expression of NOS2. To explore possible means of interdicting this inflammatory process in astrocytes, a noval pharmacologic ligands of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, 1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl) methane (DIM-C-pPhCF3) were used in the same co-culture system. DIM-C-pPhCF3 protected PC12 cells from apoptosis through inhibition of NOS2 expression in astrocytes after Mn and cytokines exposure. By contrast, the PPARγ antagonist, 2-chloro-5-nitrobenzanilide (GW9622), had the opposite effect, increasing both NO production in astrocytes and neuronal injury. It is concluded that PPARγ is involved in the regulation of NOS2 expression in astrocytes and that agonists of PPARγ may represent a potential treatment method for Mn neurotoxicity.

Les astrocytes périsynaptiques participent activement, au côté des neurones, dans le traitement de l’information cérébrale. Une propriété essentielle des astrocytes est d’exprimer un niveau élevé de protéines appelées connexines (Cxs), et formant les sous-unités des jonctions communicantes. Étonnamment, bien qu’il ait été suggéré très tôt que la Cx30 astrocytaire soit impliquée dans des processus cognitifs, son rôle exact dans la neurophysiologie demeure cependant encore mal connu. Nous avons récemment révélé que la Cx30, via une fonction non-canal inédite, contrôle la force et la plasticité de la transmission synaptique glutamatergique de l’hippocampe en régulant les niveaux synaptiques de glutamate par le biais du transport astrocytaire du glutamate. Cependant, les mécanismes moléculaire et cellulaire impliqués dans ce contrôle, ainsi que sa régulation dynamique par l’activité neuronale et son impact in vivo dans un contexte physiologique restaient inconnus. Dans le cadre de cette problématique, j’ai démontré durant ma thèse que: 1) La Cx30 induit la maturation morphologique des astrocytes de l’hippocampe par l’intermédiaire de la modulation d’une voie de signalisation dépendante de la laminine et régulant la polarisation cellulaire ; 2) l’expression de la Cx30, sa localisation perisynaptique, ainsi que ses fonctions sont modulées par l’activité neuronale ; 3) Le contrôle de la couverture astrocytaire des synapses du noyau supraoptique de l’hypothalamus par la Cx30 fixe les niveaux plasmatiques de base de la neurohormone ocytocine et ainsi favorise la mise en place de comportements sociaux adaptés. Dans l’ensemble, ces résultats éclairent les régulations des Cxs astrocytaires par l’activité neuronale et leur rôle dans le développement postnatal des réseaux neurogliaux, ainsi que dans le contrôle des interactions structurelles astrocytes-synapses à l’origine de processus comportementaux. / Perisynaptic astrocytes are active partners of neurons in cerebral information processing. A key property of astrocytes is to express high levels of the gap junction forming proteins, the connexins (Cxs). Strikingly, astroglial Cx30 was suggested early on to be involved in cognitive processes; however, its specific role in neurophysiology has yet been unexplored. We recently reveal that Cx30, through an unconventional non-channel function, controls hippocampal glutamatergic synaptic strength and plasticity by directly setting synaptic glutamate levels through astroglial glutamate clearance. Yet the cellular and molecular mechanisms involved in such control, its dynamic regulation by activity and its impact in vivo in a physiological context were unknown. To answer these questions, I demonstrated during my PhD that: 1) Cx30 drives the morphological maturation of hippocampal astrocytes via the modulation of a laminin signaling pathway regulating cell polarization; 2) Cx30 expression, perisynaptic localization and functions are modulated by neuronal activity; 3) Cx30-mediated control of astrocyte synapse coverage in the supraoptic nucleus of the hypothalamus sets basal plasmatic level of the neurohormone oxytocin and hence promotes appropriate oxytocin-based social abilities. Taken together, these data shed new light on astroglial Cxs activity-dependent regulations and roles in the postnatal development of neuroglial networks, as well as in astrocyte-synapse structural interactions mediating behavioral processes.

Astrocytes, the most abundant glial cell type in the brain, undergo a number of roles in brain physiology; among them, the energetic support of neurons is the best characterized. Contained within astrocytes is the brain’s obligate energy store, glycogen. Through glycogenolysis, glycogen, a storage form of glucose, is converted to pyruvate that is further reduced to lactate and transferred to neurons as an energy source via MCTs. Glycogen is a multi-branched polysaccharide synthesized from the glucose uptaken in astrocytes. It has been shown that glycogen accumulates with age and contributes to the physiological ageing process in the brain. In this study, we compared glycogen distribution between young adults and geriatric mice to understand the energy consumption of synaptic terminals during ageing using computational tools. We segmented and densely reconstructed neuropil and glycogen granules within six (three 4 month old old and three 24 month old) volumes of Layer 1 somatosensory cortex mice brains from FIB-SEM stacks, using a combination of semi-automated and manual tools, ilastik and TrakEM2. Finally, the 3D visualization software, Blender, was used to analyze the dataset using the DBSCAN and KDTree Nearest neighbor algorithms to study the distribution of glycogen granules compared to synapses, using a plugin that was developed for this purpose. The Nearest Neighbors and clustering results of 6 datasets show that glycogen clusters around excitatory synapses more than inhibitory synapses and that, in general, glycogen is found around axonal boutons more than dendritic spines. There was no significant accumulation of glycogen with ageing within our admittedly small dataset. However, there was a homogenization of glycogen distribution with age and that is consistent with published literature. We conclude that glycogen distribution in the brain is not a random process but follows a function distribution.

Neuroinflammation occurs in Alzheimer’s disease (AD) brain, and plays a role in neurodegeneration. The main aim of this study was to determine how treatments with exogenous apolipoprotein E (ApoE2, E3 and E4 isoforms), a genetic risk factor for AD, affects the amyloid-β (Aβ) induced inflammatory response in vitro in astrocytes. Recombinant, lipid-free ApoE4 was found not to affect Aβ-induced inflammation in rat astrocytes, while ApoE2 showed a protective effect. Mouse cells expressing human ApoE isoforms, which have similar lipidation and modification to native human ApoE, showed ApoE4 promoting inflammation, and no ApoE2 protective effect upon Aβ treatment. A Protein/DNA array was used to screen 345 transcription factors in rat astrocytes treated with Aβ and/or ApoE isoforms, in order to determine which contribute to the observed ApoE2 protection. Some candidates were validated by Western Blot or EMSA and/or by inhibition or activation. The findings suggest ApoE isoforms differentially regulate Aβ-induced inflammation, and multiple signalling pathways are involved in the process.

The structure and function of brain show sexual dimorphism in vertebrates. Brain sexual differentiation is resulted from the neural development. The neural development of brain is determined by genetic regulation and also influenced by external environmental factors(ex: temperature¡B neurotransmitter ). Serotonin (5-hydroxytrptamine, 5-HT) function as a neurotransmitter and/or neuromodulator in the central nervous system. Serotonin plays a important role in the development of the central nervous system via serotonin receptor¡CAstrocyte has the role of neural supporting and neural protection in CNS. Astrocyte has the important role of brain development. In the present study, the influence of temperature on the proliferation of astrocyte was investigated.These results show that the proliferation of astrocyte are varied with the temperature. Serotonin is not involved in the proliferation of astrocyte .Wherease,but has an effect on the ratio 5-HT+-cell in the astrocyte culture via the 5-HT1A receptor.

Neuroinflammation occurs in Alzheimer’s disease (AD) brain, and plays a role in neurodegeneration. The main aim of this study was to determine how treatments with exogenous apolipoprotein E (ApoE2, E3 and E4 isoforms), a genetic risk factor for AD, affects the amyloid-β (Aβ) induced inflammatory response in vitro in astrocytes. Recombinant, lipid-free ApoE4 was found not to affect Aβ-induced inflammation in rat astrocytes, while ApoE2 showed a protective effect. Mouse cells expressing human ApoE isoforms, which have similar lipidation and modification to native human ApoE, showed ApoE4 promoting inflammation, and no ApoE2 protective effect upon Aβ treatment. A Protein/DNA array was used to screen 345 transcription factors in rat astrocytes treated with Aβ and/or ApoE isoforms, in order to determine which contribute to the observed ApoE2 protection. Some candidates were validated by Western Blot or EMSA and/or by inhibition or activation. The findings suggest ApoE isoforms differentially regulate Aβ-induced inflammation, and multiple signalling pathways are involved in the process.