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In vitro mitochondrial failure and oxidative stress mimic biochemical features of Alzheimer disease
No full textPrimary cortical neurons exposed to the mitochondrial toxin NaN3 (0.1–3 mM) were submitted to oxidative stress with H2O2 (30–150 μM), to mimic conditions observed in neurodegenerative disorders. The effects of such treatment on a series of parameters useful in characterizing neuronal damage were investigated: (i) the basal release of glutamate, evaluated as 3H-d-Aspartate efflux, was sharply, concentration-dependently, increased; (ii) the phosphorylation status of intracellular markers known to be involved in the neurodegenerative processes, in particular in Alzheimer disease: tau and GSK3β were increased, as well as the protein level of β-secretase (BACE1) and p35/25 evaluated by Western blotting, while (iii) the cell metabolic activity, measured with the MTT method, was reduced, in a concentration- and time-dependent manner. The latter effect, as well as tau hyperphosphorylation, was prevented both by a mixture of antioxidant drugs (100 μM ascorbic acid, 10 μM trolox, 100 μM glutathione) and by the anti-Alzheimer drug, memantine, 20 μM. Since it is well known that hippocampal cholinergic neurons are particularly affected in Alzheimer disease, the effects of NaN3 and H2O2 were also studied in electrically stimulated rat hippocampal slices, evaluating the 3H-Choline efflux, as an index of acetylcholine release. The neurotoxic treatment depressed the neurosecretory function and the mixture of antioxidant drugs, as well as memantine, were able to restore it. The neuronal damage induced by the in vitro protocol adopted in the present work displays peculiarities of neurodegenerative disorders, e.g. Alzheimer disease, underlining the role of mitochondrial failure and oxidative stress, which appear to occur upstream the neurodegenerative process; such protocol could be utilized to test the efficacy of neuroprotective treatments
Mechanisms of sodium azide-induced changes in intracellular calcium concentration in rat primary cortical neurons
No full textAn intracellular calcium ([Ca2+]i) increase is involved in sodium azide (NaN3)-induced neurotoxicity, an in vitro model of brain ischemia. In this study the questions of possible additional sources of calcium influx, besides glutamate receptor activation, and of the time-course of NaN3 effects have been addressed by measuring [Ca2+]i in rat primary cortical cultures with the FURA-2 method. Basal [Ca2+]i of neuronal populations was concentration-dependently increased 30 min, but not 24 hours, after a 10-min NaN3 (3-30 mM) treatment; conversely, the net increase induced by electrical stimulation (10 Hz, 10 sec) was consistently reduced. All the above effects depended on glutamate release and consequent NMDA receptor activation, since the NMDA antagonist MK-801 (1 μM) prevented them, and the spontaneous efflux of [3H]D-aspartate from superfused neurons was concentration-dependently increased by NaN3. In single neuronal cells, NaN3 application progressively and concentration-dependently increased [Ca2+]i (to 177 ± 5 % and 249 ± 7 % of the controls, 4 and 12 min after a 10 mM-treatment, respectively). EGTA (5 mM) pretreatment reduced the effect of 10 mM NaN3 (to 118 ± 5 % at 4 min, and to 148 ± 10 % at 12 min, respectively), while 1 μM cyclosporin A did not. Both MK-801 and CNQX (a non-NMDA glutamate antagonist, 10 μM) prevented NaN3 effect at 4 min (to 147 ± 8 % and 153 ± 5 %, respectively), but not at 12 min after NaN3 treatment. Conversely, 10 μM verapamil and 0.1 μM omega-conotoxin (L- and N-type calcium channel blockers, respectively) significantly attenuated NaN3 effects at 12 min (to 198 ± 8 % and 164 ± 5 %, respectively), but not at 4 min; the P/Q-type calcium channel blocker, agatoxin, 0.3 μM, was ineffective. These findings show that the predominant source of calcium increase induced by NaN3 is extracellular, involving glutamate receptor activation in a first step and calcium channel (mainly of the N-type) opening in a second step
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Evidence for an in vivo and in vitro modulation of endogenous cortical GABA release by α-glycerylphosphorylcholine
No full textThe effects of alpha-glycerylphosphorylcholine (alpha-GPC) on endogenous cortical GABA release were studied both in vivo and in vitro. In freely moving rats, equipped with epidural cups, alpha-GPC (30-300 mg/kg i.p.) increased GABA release. This effect was potentiated by atropine, both systematically administered (5 mg/kg i.p.) and locally applied (1.4 microM), but not by mecamylamine (4 mg/kg i.p.). The alpha-GPC-induced increase in GABA release was abolished in rats pretreated with the alpha 1 receptor antagonist prazosin (14 micrograms/kg i.p.). In cortical slices alpha-GPC (0.4 mM) increased the spontaneous GABA efflux. This effect was abolished by tetrodotoxin (0.5 microM) and prazosin (1 microM), but not by atropine (0.15 microM) or mecamylamine (2.5 microM). These results indicate that the facilitatory response by alpha-GPC on GABA release does not depend on a direct activation of either muscarinic or nicotinic receptors, but suggest the involvement of the noradrenergic system
Sodium Azide Induced Neuronal Damage In Vitro: Evidence for Non-Apoptotic Cell Death
No full textThe features of neuronal damage induced by
the mitochondrial toxin NaN3 were investigated in rat
primary cortical neuron cultures. Cell viability (MTT colorimetric
determination) and transmembrane mitochondrial
potential (J-C1 fluorescence) were concentration-dependently
reduced 24 h after NaN3; neither nuclear
fragmentation by DAPI, nor Annexin V positivity by flow
cytometry were detected, ruling out the occurrence of
apoptosis. The loss in cell viability (to 54 ± 2%) observed
24 h after a 10-min treatment with 3 mM NaN3 was prevented
by the NMDA glutamate receptor antagonist
MK801 (1 lM), by the antioxidants trolox (100 lM) and
acetyl-L-carnitine (1 mM) and by the nitric oxide synthase
inhibitor, L-NAME (100 lM), but not by the guanylylcyclase
inhibitor ODQ, 10 lM. The mitochondrial
dysfunction induced by NaN3 provides a common platform
for investigating the mechanisms of both ischemic and
degenerative neuronal injury, useful for screening potential
protective agents against neuronal death
Serotonin modulation of cell excitability and of [3-H]GABA and [3-H]D-aspartate efflux in primary cultures of rat cortical neurons
No full textThe effects of 5-hydroxytryptamine (5-HT) on neuronal excitability, evaluated as depolarisation-induced firing rate, and on amino acid release, measured as electrically-evoked [3H]GABA and [3H]-D-aspartate efflux, were investigated in rat primary cortical neuronal cultures.
5-HT displayed a concentration-dependent, bimodal effect on neuronal excitability: at 3-10 μM it increased excitability through 5-HT2A receptors, and was blocked by the selective 5-HT2A antagonist MDL 100907, whereas at 30-100 μM it reduced excitability through 5-HT1A receptors, and was, in turn, blocked by the selective 5-HT1A antagonist WAY 100135.
The electrically-evoked [3H]GABA efflux was concentration-dependently inhibited by 5-HT (pEC50 = 4.74) and such inhibition was prevented by WAY 100135, but not by Gr 55562, a selective 5-HT1D/B receptor antagonist
Conversely, 5-HT concentration-dependently increased stimulus-evoked [3H]D-aspartate efflux (pEC50 = 4.71). The increase was facilitated by methiothepin and was reversed into inhibition by ICS 205930, a selective 5-HT3 receptor antagonist. In the presence of ICS 205930, the inhibition induced by 5-HT was prevented by the selective 5-HT1D/B receptor antagonist Gr 55562, but not by WAY 100135.
These findings suggest that 5-HT inhibits GABA release through 5-HT1A receptors and exerts a dual modulation on glutamate release, mostly facilitatory (through 5-HT3 receptors) but also inhibitory (through 5-HT1D/B receptors), leading to a prevalently positive modulation of the excitatory signal by amino acid neurotransmitter containing neurons
Effects of nociceptin/orphanin FQ and endomorphin-1 on glutamate and GABA release, intracellular [Ca2+] and cell excitability in primary cultures of rat cortical neurons
No full textThe effects of nociceptin/orphanin FQ (N/OFQ) and endomorphin-1 (EM-1) on glutamate and GABA release, intracellular calcium, neuronal excitability and glutamate current were investigated in rat primary cortical neuronal cultures. Through their specific receptors N/OFQ and EM-1 (0.02-1 microM) inhibited the electrically evoked outflow of [3H]D-aspartate at most to -50% and that of [3H]GABA to -30%. In addition, at 1 microM, both peptides induced a decrease of the firing rate caused by electrical depolarization. N/OFQ 1-10 microM did not influence either the electrically evoked calcium influx or the glutamate-evoked currents, whereas EM-1 1 microM significantly inhibited them. Thus, in cortical neurons in culture, both N/OFQ and EM-1 inhibited the secretory process and neuronal excitability but EM-1 also affected calcium influx and cell body responsiveness to glutamate. Consequently, EM-1 appeared to dampen this excitatory signal more then N/OFQ did
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