Low Temperature Delays the Effects of Ischemia in Bergmann Glia and in Cerebellar Tissue Swelling.

Cerebral ischemia results in oxygen and glucose deprivation that most commonly occurs after a reduction or interruption in the blood supply to the brain. The consequences of cerebral ischemia are complex and involve the loss of metabolic ATP, excessive K+ and glutamate accumulation in the extracellular space, electrolyte imbalance, and brain edema formation. So far, several treatments have been proposed to alleviate ischemic damage, yet few are effective. Here, we focused on the neuroprotective role of lowering the temperature in ischemia mimicked by an episode of oxygen and glucose deprivation (OGD) in mouse cerebellar slices. Our results suggest that lowering the temperature of the extracellular 'milieu' delays both the increases in [K+]e and tissue swelling, two dreaded consequences of cerebellar ischemia. Moreover, radial glial cells (Bergmann glia) display morphological changes and membrane depolarizations that are markedly impeded by lowering the temperature. Overall, in this model of cerebellar ischemia, hypothermia reduces the deleterious homeostatic changes regulated by Bergmann glia.

Are quinoa proteins a promising alternative to be applied in plant-based emulsion gel formulation?

Emulsion gels are structured emulsion systems that behave as soft solid-like materials. Emulsion gels are commonly used in food-product design both as fat replacers and as delivery carriers of bioactive compounds. Different plant-derived proteins like soy, chia, and oat have been used in emulsion gel formulation to substitute fat in meat products and to deliver some vegetable dyes or extracts. Quinoa protein isolates have been scarcely applied in emulsion gel formulation although they seem to be a promising alternative as emulsion stabilizers. Quinoa protein isolates have a high protein content with a well-balanced amino acid profile and show good emulsifying and gelling capabilities. Unlike quinoa starch, quinoa protein isolates do not require any chemical modification before being used. The present article reviews the state of the art in food emulsion gels stabilized with vegetable proteins and highlights the potential uses of quinoa proteins in emulsion gel formulation.

Dopaminergic neuromodulation of prefrontal cortex activity requires the NMDA receptor coagonist d-serine.

Prefrontal control of cognitive functions critically depends upon glutamatergic transmission and N-methyl D-aspartate (NMDA) receptors, the activity of which is regulated by dopamine. Yet whether the NMDA receptor coagonist d-serine is implicated in the dopamine-glutamate dialogue in the prefrontal cortex (PFC) and other brain areas remains unexplored. Here, using electrophysiological recordings, we show that d-serine is required for the fine-tuning of glutamatergic neurotransmission, neuronal excitability, and synaptic plasticity in the PFC through the actions of dopamine at D1 and D3 receptors. Using in vivo microdialysis, we show that D1 and D3 receptors exert a respective facilitatory and inhibitory influence on extracellular levels and activity of d-serine in the PFC, with actions expressed primarily via the cAMP/protein kinase A (PKA) signaling cascade. Further, using functional magnetic resonance imaging (fMRI) and behavioral assessment, we show that d-serine is required for the potentiation of cognition by D3R blockade as revealed in a test of novel object recognition memory. Collectively, these results unveil a key role for d-serine in the dopaminergic neuromodulation of glutamatergic transmission and PFC activity, findings with clear relevance to the pathogenesis and treatment of diverse brain disorders involving alterations in dopamine-glutamate cross-talk.

Regulation of GluA1 phosphorylation by d-amphetamine and methylphenidate in the cerebellum.

Prescription stimulants, such as d-amphetamine or methylphenidate are used to treat suffering from attention-deficit hyperactivity disorder (ADHD). They potently release dopamine (DA) and norepinephrine (NE) and cause phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 in the striatum. Whether other brain regions are also affected remains elusive. Here, we demonstrate that d-amphetamine and methylphenidate increase phosphorylation at Ser845 (pS845-GluA1) in the membrane fraction of mouse cerebellum homogenate. We identify Bergmann glial cells as the source of pS845-GluA1 and demonstrate a requirement for intact NE release. Consequently, d-amphetamine-induced pS845-GluA1 was prevented by ß1-adenoreceptor antagonist, whereas the blockade of DA D1 receptor had no effect. Together, these results indicate that NE regulates GluA1 phosphorylation in Bergmann glial cells in response to prescription stimulants.

Functional properties of amaranth, quinoa and chia proteins and the biological activities of their hydrolyzates.

Amaranth, quinoa and chia are non-conventional sources of proteins whose interest has increased in recent years due to their excellent nutritional value. Vegetable proteins can be used as food ingredients to replace animal proteins in human diet. The present article provides a comprehensive analysis of amaranth, quinoa and chia proteins and focuses on their solubility, superficial, gelling and textural properties as well as on the biological activities of enzymatic hydrolyzates.

A Light-Controlled Allosteric Modulator Unveils a Role for mGlu4 Receptors During Early Stages of Ischemia in the Rodent Cerebellar Cortex.

Metabotropic glutamate receptors (mGlus) are G Protein coupled-receptors that modulate synaptic transmission and plasticity in the central nervous system. Some act as autoreceptors to control neurotransmitter release at excitatory synapses and have become attractive targets for drug therapy to treat certain neurological disorders. However, the high degree of sequence conservation around the glutamate binding site makes the development of subtype-specific orthosteric ligands difficult to achieve. This problem can be circumvented by designing molecules that target specific less well conserved allosteric sites. One such allosteric drug, the photo-switchable compound OptoGluNAM4.1, has been recently employed to reversibly inhibit the activity of metabotropic glutamate 4 (mGlu4) receptors in cell cultures and in vivo. We studied OptoGluNAM4.1 as a negative modulator of neurotransmission in rodent cerebellar slices at the parallel fiber - Purkinje cell synapse. Our data show that OptoGluNAM4.1 antagonizes pharmacological activation of mGlu4 receptors in a fully reversible and photo-controllable manner. In addition, for the first time, this new allosteric modulator allowed us to demonstrate that, in brain slices from the rodent cerebellar cortex, mGlu4 receptors are endogenously activated in excitotoxic conditions, such as the early phases of simulated cerebellar ischemia, which is associated with elevated levels of extracellular glutamate. These findings support OptoGluNAM4.1 as a promising new tool for unraveling the role of mGlu4 receptors in the central nervous system in physio-pathological conditions.

Cerebellar synapse properties and cerebellum-dependent motor and non-motor performance in Dp71-null mice.

Recent emphasis has been placed on the role that cerebellar dysfunctions could have in the genesis of cognitive deficits in Duchenne muscular dystrophy (DMD). However, relevant genotype-phenotype analyses are missing to define whether cerebellar defects underlie the severe cases of intellectual deficiency that have been associated with genetic loss of the smallest product of the dmd gene, the Dp71 dystrophin. To determine for the first time whether Dp71 loss could affect cerebellar physiology and functions, we have used patch-clamp electrophysiological recordings in acute cerebellar slices and a cerebellum-dependent behavioral test battery addressing cerebellum-dependent motor and non-motor functions in Dp71-null transgenic mice. We found that Dp71 deficiency selectively enhances excitatory transmission at glutamatergic synapses formed by climbing fibers (CFs) on Purkinje neurons, but not at those formed by parallel fibers. Altered basal neurotransmission at CFs was associated with impairments in synaptic plasticity and clustering of the scaffolding postsynaptic density protein PSD-95. At the behavioral level, Dp71-null mice showed some improvements in motor coordination and were unimpaired for muscle force, static and dynamic equilibrium, motivation in high-motor demand and synchronization learning. Dp71-null mice displayed altered strategies in goal-oriented navigation tasks, however, suggesting a deficit in the cerebellum-dependent processing of the procedural components of spatial learning, which could contribute to the visuospatial deficits identified in this model. In all, the observed deficits suggest that Dp71 loss alters cerebellar synapse function and cerebellum-dependent navigation strategies without being detrimental for motor functions.

Amaranth, quinoa and chia protein isolates: Physicochemical and structural properties.

An increasing use of vegetable protein is required to support the production of protein-rich foods which can replace animal proteins in the human diet. Amaranth, chia and quinoa seeds contain proteins which have biological and functional properties that provide nutritional benefits due to their reasonably well-balanced aminoacid content. This review analyses these vegetable proteins and focuses on recent research on protein classification and isolation as well as structural characterization by means of fluorescence spectroscopy, surface hydrophobicity and differential scanning calorimetry. Isolation procedures have a profound influence on the structural properties of the proteins and, therefore, on their in vitro digestibility. The present article provides a comprehensive overview of the properties and characterization of these proteins.

Oxygen and Glucose Deprivation Induces Bergmann Glia Membrane Depolarization and Ca2+ Rises Mainly Mediated by K+ and ATP Increases in the Extracellular Space.

During brain ischemia, intense energy deficiency induces a complex succession of events including pump failure, acidosis and exacerbated glutamate release. In the cerebellum, glutamate is the principal mediator of Purkinje neuron anoxic depolarization during episodes of oxygen and glucose deprivation (OGD). Here, the impact of OGD is studied in Bergmann glia, specialized astrocytes closely associated to Purkinje neurons. Patch clamp experiments reveal that during OGD Bergmann glial cells develop a large depolarizing current that is not mediated by glutamate and purinergic receptors but is mainly due to the accumulation of K+ in the extracellular space. Furthermore, we also found that increases in the intracellular Ca2+ concentration appear in Bergmann glia processes several minutes following OGD. These elevations require, in an early phase, Ca2+ mobilization from internal stores via P2Y receptor activation, and, over longer periods, Ca2+ entry through store-operated calcium channels. Our results suggest that increases of K+ and ATP concentrations in the extracellular space are primordial mediators of the OGD effects on Bergmann glia. In the cerebellum, glial responses to energy deprivation-triggering events are therefore highly likely to follow largely distinct rules from those of their neuronal counterparts.

Physicochemical study of mixed systems composed by bovine caseinate and the galactomannan from Gleditsia amorphoides.

Model systems formed by sodium caseinate (NaCAS) and espina corona gum (ECG) were studied. There was no evidence of attractive interactions between NaCAS and ECG macromolecules. Aqueous mixtures of NaCAS and ECG phase-separate segregatively over a wide range of concentrations. According to the images obtained by confocal laser scanning microscopy, NaCAS particles form larger protein aggregates when ECG is present in the system. An increase in the hydrodynamic diameter of NaCAS particles, as a result of ECG addition, was also observed by light scattering in diluted systems. A depletion-flocculation phenomenon, in which ECG is excluded from NaCAS surface, is proposed to occur in the concentrated mixed systems, resulting in NaCAS aggregation. ECG raises the viscosity of NaCAS dispersions without affecting the Newtonian flow behaviour of NaCAS. These results contribute to improve the knowledge of a barely-studied hydrocolloid which may be useful in the development of innovative food systems.

Purinergic signaling in the cerebellum: Bergmann glial cells express functional ionotropic P2X7 receptors.

Astrocytes constitute active networks of intercommunicating cells that support the metabolism and the development of neurons and affect synaptic functions via multiple pathways. ATP is one of the major neurotransmitters mediating signaling between neurons and astrocytes. Potentially acting through both purinergic metabotropic P2Y receptors (P2YRs) and ionotropic P2X receptors (P2XRs), up until now ATP has only been shown to activate P2YRs in Bergmann cells, the radial glia of the cerebellar cortex that envelopes Purkinje cell afferent synapses. In this study, using multiple experimental approaches in acute cerebellar slices we demonstrate the existence of functional P2XRs on Bergmann cells. In particular, we show here that Bergmann cells express uniquely P2X7R subtypes: (i) immunohistochemical analysis revealed the presence of P2X7Rs on Bergmann cell processes, (ii) in whole cell recordings P2XR pharmacological agonists induced depolarizing currents that were blocked by specific antagonists of P2X7Rs, and could not be elicited in slices from P2X7R-deficient mice and finally, (iii) calcium imaging experiments revealed two distinct calcium signals triggered by application of exogenous ATP: a transient signal deriving from release of calcium from intracellular stores, and a persistent one following activation of P2X7Rs. Our data thus reveal a new pathway by which extracellular ATP may affect glial cell function, thus broadening our knowledge on purinergic signaling in the cerebellum.

Role of the vesicular transporter VGLUT3 in retrograde release of glutamate by cerebellar Purkinje cells.

In the cerebellum, retrograde release of glutamate (Glu) by Purkinje cells (PCs) participates in the control of presynaptic neurotransmitter release responsible for the late component of depolarization-induced suppression of excitation (DSE), as well as for depolarization-induced potentiation of inhibition (DPI). It might also participate in the depolarization-induced slow current (DISC) in PCs, although this contribution was later challenged. We also know that both DPI and DISC are soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent processes, although the molecular nature of the vesicular transporter was not determined. In PCs, VGLUT3 is the only known vesicular glutamate transporter identified and is expressed during the same developmental frame as when DPI, DISC, and the Glu-dependent component of DSE are observed. We therefore tested the hypothesis that all these processes depend on the presence of VGLUT3 by comparing the Glu-dependent component of DSE, DPI, and DISC in nearly mature (2- to 3-wk-old) wild-type and VGLUT3 knockout mice. Our data demonstrate that, in nearly mature mice, the slow component of DSE occurs through vesicular release of Glu that involves VGLUT3. This Glu-dependent component of DSE is no longer present in fully mature mice. This study also establishes that, in nearly mature mice, DPI also depends on the presence of VGLUT3, whereas this is not the case for DISC. Finally, the unusually large basal paired-pulse facilitation observed in nearly mature VGLUT3(-/-) mice but not in adult ones suggests that some basal retrograde release of Glu occurs during development and contributes to basal concentrations of extracellular Glu.

Neural progenitor cell death is induced by extracellular ATP via ligation of P2X7 receptor.

Neural progenitor cells (NPCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes, and have been used to treat several animal models of CNS disorders. In the present study, we show that the P2X7 purinergic receptor (P2X7R) is present on NPCs. In NPCs, P2X7R activation by the agonists extracellular ATP or benzoyl ATP triggers opening of a non-selective cationic channel. Prolonged activation of P2X7R with these nucleotides leads to caspase independent death of NPCs. P2X7R ligation induces NPC lysis/necrosis demonstrated by cell membrane disruption accompanied with loss of mitochondrial membrane potential. In most cells that express P2X7R, sustained stimulation with ATP leads to the formation of a non-selective pore allowing the entry of solutes up to 900 Da, which are reportedly involved in P2X7R-mediated cell lysis. Surprisingly, activation of P2X7R in NPCs causes cell death in the absence of pore formation. Our data support the notion that high levels of extracellular ATP in inflammatory CNS lesions may delay the successful graft of NPCs used to replace cells and repair CNS damage.

Impairments in motor coordination without major changes in cerebellar plasticity in the Tc1 mouse model of Down syndrome.

Down syndrome (DS) is a genetic disorder arising from the presence of a third copy of human chromosome 21 (Hsa21). Recently, O'Doherty et al. [An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes. Science 309 (2005) 2033-2037] generated a trans-species aneuploid mouse line (Tc1) that carries an almost complete Hsa21. The Tc1 mouse is the most complete animal model for DS currently available. Tc1 mice show many features that relate to human DS, including alterations in memory, synaptic plasticity, cerebellar neuronal number, heart development and mandible size. Because motor deficits are one of the most frequently occurring features of DS, we have undertaken a detailed analysis of motor behaviour in cerebellum-dependent learning tasks that require high motor coordination and balance. In addition, basic electrophysiological properties of cerebellar circuitry and synaptic plasticity have been investigated. Our results reveal that, compared with controls, Tc1 mice exhibit a higher spontaneous locomotor activity, a reduced ability to habituate to their environments, a different gait and major deficits on several measures of motor coordination and balance in the rota rod and static rod tests. Moreover, cerebellar long-term depression is essentially normal in Tc1 mice, with only a slight difference in time course. Our observations provide further evidence that support the validity of the Tc1 mouse as a model for DS, which will help us to provide insights into the causal factors responsible for motor deficits observed in persons with DS.

Tonic activation of NMDA receptors by ambient glutamate of non-synaptic origin in the rat hippocampus.

In several neuronal types of the CNS, glutamate and GABA receptors mediate a persistent current which reflects the presence of a low concentration of transmitters in the extracellular space. Here, we further characterize the tonic current mediated by ambient glutamate in rat hippocampal slices. A tonic current of small amplitude (53.99 +/- 6.48 pA at +40 mV) with the voltage dependency and the pharmacology of NMDA receptors (NMDARs) was detected in virtually all pyramidal cells of the CA1 and subiculum areas. Manipulations aiming at increasing D-serine or glycine extracellular concentrations failed to modify this current indicating that the glycine binding sites of the NMDARs mediating the tonic current were saturated. In contrast, non-transportable inhibitors of glutamate transporters increased the amplitude of this tonic current, indicating that the extracellular concentration of glutamate primarily regulates its magnitude. Neither AMPA/kainate receptors nor metabotropic glutamate receptors contributed significantly to this tonic excitation of pyramidal neurons. In the presence of glutamate transporter inhibitors, however, a significant proportion of the tonic conductance was mediated by AMPA receptors. The tonic current was unaffected when inhibiting vesicular release of transmitters from neurons but was increased upon inhibition of the enzyme converting glutamate in glutamine in glial cells. These observations indicate that ambient glutamate is mainly of glial origin. Finally, experiments with the use-dependent antagonist MK801 indicated that NMDARs mediating the tonic conductance are probably extra-synaptic NMDARs.

Group I metabotropic glutamate receptors inhibit GABA release at interneuron-Purkinje cell synapses through endocannabinoid production.

Actions of endocannabinoids in the cerebellum can be demonstrated following distinct stimulation protocols in Purkinje cells. First, depolarization-induced elevations of intracellular Ca2+ lead to the suppression of neurotransmitter release from both inhibitory and excitatory afferents. In another case, postsynaptic group I metabotropic glutamate receptors (mGluRs) trigger a strong inhibition of the glutamatergic inputs from parallel and climbing fibers. Both pathways involve endocannabinoids retrogradely acting on type 1 cannabinoid receptors (CB1Rs) at presynaptic terminals. Here, we show that group I mGluR activation also depresses GABAergic transmission at the synapses between molecular layer interneurons and Purkinje cells. Using paired recordings, we found that application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine reduced the evoked IPSCs in Purkinje cells. This effect was independent of postsynaptic Ca2+ increases and was completely blocked by a CB1R antagonist. Experiments performed with the GTP-analogues GDP-betaS and GTP-gammaS provided evidence that endocannabinoids released after G-protein activation can also inhibit GABAergic inputs onto nearby, unstimulated Purkinje cells. Block of the enzymes DAG lipase or phospholipase C reduced the group I mGluR-dependent inhibition, suggesting that 2-arachidonyl glycerol could act as retrograde messenger. Finally, group I mGluR activation by brief bursts of activity of the parallel fibers induced a short-lived depression of spontaneous IPSCs via presynaptic CB1Rs. Our results reveal a mechanism with potential physiological importance, by which glutamatergic synapses induce an endocannabinoid-mediated inhibition of the GABAergic inputs onto Purkinje cells.

Presynaptic ryanodine-sensitive calcium stores contribute to evoked neurotransmitter release at the basket cell-Purkinje cell synapse.

Presynaptic terminals of cerebellar basket cells are known to contain ryanodine-sensitive calcium stores (RyCSs); recently, it has been shown that these stores control the frequency of miniature synaptic currents in the absence of presynaptic spiking. Here, using paired recordings of basket cell-Purkinje cell synapses, we show that blocking the RyCSs with high concentration of ryanodine decreases the mean amplitude of evoked IPSCs to 70% of the control value. The paired-pulse ratio and failure rate increase, indicating that the reduction stems from a decreased probability of evoked neurotransmitter release. Various control experiments eliminate the possibility of an indirect effect of ryanodine via activation of postsynaptic receptors. Prolonged application of cyclopiazonic acid, a blocker of the endoplasmic reticulum calcium pump, totally abolishes the ryanodine action. Our results indicate that calcium released from presynaptic RyCSs enhances the amplitude of evoked GABAergic synaptic currents. The precise mechanism by which calcium released from internal stores affect action potential-dependent release is unknown; however, our results suggest that these stores do not provide additional calcium for each presynaptic action potential; rather, they appear to enhance depolarization-induced calcium signals indirectly, perhaps by increasing the basal level of cytosolic calcium concentration in the vicinity of release sites.