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Summary:Purpose: N-methyl D-aspartate (NMDA) preconditioning has been used to prevent cellular death induced by glutamate or NMDA in cultured neurons. Quinolinic acid (QA)-induced seizures are used to average NMDA receptors-evoked neurotoxicity in animal models. The purpose of this study was to investigate the potential neuroprotective effects of NMDA preconditioning against QA-induced seizures and hippocampal damage in vivo.Methods: Mice were pretreated with nonconvulsant doses of NMDA for different times before i.c.v. QA infusion and observed for the occurrence of seizures. Hippocampal slices from mice were assayed to measure cellular viability.Results: NMDA preconditioning presented 53% protection against QA-induced seizures, as well as QA-induced cellular death in the hippocampus. The NMDA receptor antagonist, MK-801, prevented the protection evoked by NMDA preconditioning. The adenosine A 1 receptor antagonist, CPT, prevented the protection evoked by NMDA preconditioning against QA-induced seizures, but not against QA-induced hippocampal cellular damage. The adenosine A 1 receptor agonist, CPA, did not mimic the NMDA preconditioning-evoked protective effects.Conclusions: These results suggest that in vivo preconditioning with subtoxic doses of NMDA protected mice against seizures and cellular hippocampal death elicited by QA, probably through mechanisms involving NMDA receptors operating with adenosine A 1 receptors.

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.

Synaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by ‘crowding’ in the vesicle membrane from stable interaction modules.

Large dense-core vesicles (LDcVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRnAs (miRnAs). isolation of LDcVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against Rnase A. this protocol can be useful in research on the biological functions of LDcVs. Neurons communicate by neurotransmission, which involves vesicle fusion and neurotransmitter release 1,2. Synaptic vesicles and large dense-core vesicles (LDCVs) participate in different functions in the nervous system 3,4. Synaptic vesicles in the presynapse store classical neurotransmitters (e.g., glutamate, acetylcholine, GABA, and glycine), and mainly activate ion channels in the postsynaptic terminals, thereby transmitting electrical signals to the postsynapse. In contrast, LDCVs contain amines, neuropeptides, and other hormones that mainly stimulate G-protein-coupled receptors (GPCRs) to modulate synaptic responses 3-5. LDCVs are minor components, amounting to only 1~2% when compared to synaptic vesicles in the central nervous system 6-8 , but they are highly abundant in specialized brain regions (such as the hypothalamus), in dopaminergic neurons and in sympathetic neurons of the peripheral nervous system 6. Chromaffin cells are modified sympathetic ganglia of the sympathetic nervous system 7,8. Chromaffin cells are enriched with LDCVs, also called chromaffin granules, and therefore constitute a widely-used model system for in-depth study of LDCVs 9. LDCVs of chromaffin cells contain catecholamines (i.e., adrenaline, noradrenaline, and dopamine), peptides (e.g., encephalin and neuropeptide Y), and proteins (chromogranins, secretogranins) 10. Recently, we reported that LDCVs store microRNAs (miRNAs), which are released by vesicle fusion in response to stimulation, and miR-375 is the most abundant miRNA in LDCVs 11,12. A protocol to isolate LDCV from bovine adrenal medulla was reported in the 1950s 13 , then modified 14 to include step density gradient centrifugation in the late 1960s. This protocol was widely used in many studies and formed the basis for establishing the proteomics of LDCVs 15 , and for identifying their content including amines, peptides, and proteins released by LDCV exocytosis 10. However, the purity of LDCVs isolate...

Essential omega-3 polyunsaturated fatty acids (omega3) are crucial to brain development and function, being relevant for behavioral performance. In the present study we examined the influence of dietary omega3 in the development of the glutamatergic system and on behavior parameters in rats. Female rats received isocaloric diets, either with omega3 (omega3 group) or a omega3 deficient diet (D group). In ontogeny experiments of their litters, hippocampal immunocontent of ionotropic NMDA and AMPA glutamatergic receptors subunits (NR2 A\B and GluR1, respectively) and the alpha isoform of the calcium-calmodulin protein kinase type II (alphaCaMKII) were evaluated. Additionally, hippocampal [(3)H]glutamate binding and uptake were assessed. Behavioral performance was evaluated when the litters were adult (60 days old), through the open-field, plus-maze, inhibitory avoidance and flinch-jump tasks. The D group showed decreased immunocontent of all proteins analyzed at 02 days of life (P2) in comparison with the omega3 group, although the difference disappeared at 21 days of life (except for alphaCaMKII, which content normalized at 60 days old). The same pattern was found for [(3)H]glutamate binding, whereas [(3)H]glutamate uptake was not affected. The D group also showed memory deficits in the inhibitory avoidance, increased in the exploratory pattern in open-field, and anxiety-like behavior in plus-maze. Taken together, our results suggest that dietary omega3 content is relevant for glutamatergic system development and for behavioral performance in adulthood. The putative correlation among the neurochemical and behavioral alterations caused by dietary omega3 deficiency is discussed.

Accumulating evidences indicate that endogenous modulators of excitatory synapses in the mammalian brain are potential targets for treating neuropsychiatric disorders. Indeed, glutamatergic and adenosinergic neurotransmissions were recently highlighted as potential targets for developing innovative anxiolytic drugs. Accordingly, it has been shown that guanine-based purines are able to modulate both adenosinergic and glutamatergic systems in mammalian central nervous system. Here, we aimed to investigate the potential anxiolytic-like effects of guanosine and its effects on the adenosinergic and glutamatergic systems. Acute/systemic guanosine administration (7.5 mg/kg) induced robust anxiolytic-like effects in three classical anxiety-related paradigms (elevated plus maze, light/dark box, and round open field tasks). These guanosine effects were correlated with an enhancement of adenosine and a decrement of glutamate levels in the cerebrospinal fluid. Additionally, pre-administration of caffeine (10 mg/kg), an unspecific adenosine receptors' antagonist, completely abolished the behavioral and partially prevented the neuromodulatory effects exerted by guanosine. Although the hippocampal glutamate uptake was not modulated by guanosine (both ex vivo and in vitro protocols), the synaptosomal K-stimulated glutamate release in vitro was decreased by guanosine (100 μM) and by the specific adenosine A receptor agonist, 2-chloro-N -cyclopentyladenosine (CCPA, 100 nM). Moreover, the specific adenosine A receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM) fully reversed the inhibitory guanosine effect in the glutamate release. The pharmacological modulation of A receptors has shown no effect in any of the evaluated parameters. In summary, the guanosine anxiolytic-like effects seem closely related to the modulation of adenosinergic (A receptors) and glutamatergic systems.