Ângelo R. Tomé

Morphological field emission-SEM study of the effect of six phosphoric acid etching agents on human dentin

OBJECTIVES This study evaluated the effects of six phosphoric acid-etching agents on dentin, the independent variables being two acid concentrations (10% and 32%-37%) and three thickener conditions (no thickener, silica, and polymer). The tested hypothesis was that the use of different etchants with similar concentrations of phosphoric acid would result in similar depths of dentin demineralization. METHODS Thirty dentin disks were obtained from extracted human teeth by microtome sectioning. The dentin surfaces were etched with one of the etching agents, fixed, dehydrated and dried. The specimens were observed using a FE-SEM. The mean deepest demineralization of intertubular dentin was measured from fracture surfaces of the disks. These values were analyzed by ANOVA and Duncans Test. The morphological appearance of the dentin surfaces was compared using the following observation criteria: 1) Presence of a cuff of peritubular dentin; 2) Relative thickness of the layer containing residual collagen or smear layer particles; and 3) Formation of a submicron hiatus at the bottom of the exposed collagen network. The pH of each of the etching agents was measured. A correlation analysis was made of the pH vs. the depth of dentin demineralization. RESULTS Silica-thickened etchants did not demineralize dentin as deeply as did polymer-thickened etchants and unthickened etchants. High magnifications revealed three distinct zones within the demineralized dentin layer; an upper porous zone of residual smear layer or denatured collagen and residual silica particles (in groups etched with silica-thickened etchants), an intermediate area with randomly oriented collagen fibers, and a lower zone with submicron hiatus, few collagen fibers, and scattered hydroxyapatite inclusions. This hiatus was observable in all the specimens etched with the polymer-thickened etchants, in 90% of the specimens etched with the unthickened phosphoric acid liquids, and in 60% of the specimens etched with the silica-thickened gels. SIGNIFICANCE The results obtained suggest that similar concentrations of phosphoric acid etchants containing distinct thickeners result in different demineralization depths as well as different morphology of etched dentin.

Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress

Significance Epidemiological studies show that individuals exposed to repeated stress, a major trigger of depression, increase their caffeine intake, which correlates inversely with the incidence of depression. However, the mechanism underlying this protective effect is unknown. We used an animal model of chronic unpredictable stress (CUS) to show that caffeine prevents the maladaptive changes caused by CUS in a manner mimicked by the selective blockade of adenosine A2A receptors (A2AR). CUS enhanced A2AR in synapses, and the selective elimination of neuronal A2AR abrogated CUS modifications. Moreover, A2AR blockade also afforded a therapeutic benefit, paving the way to consider A2AR blockers as a strategy to manage the negative impact of chronic stress on mood and memory. The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function.

Electrophysiological and Immunocytochemical Evidence for P2X Purinergic Receptors in Pancreatic β Cells

Objectives: Glucose-induced insulin secretion from pancreatic &bgr; cells is modulated by several hormones and transmitters, namely adenosine triphosphate (ATP) via purinergic receptors. Although P2Y receptors are well documented in &bgr; cells, the presence of P2X receptors remains elusive. We present the first electrophysiological evidence for the presence of P2X receptors in single &bgr; cells of different species. Methods: Ionic currents were recorded from voltage-clamped &bgr; cells near their resting potential using the perforated (nystatin) whole-cell patch-clamp configuration. Receptors were detected by immunocytochemistry. Results: When bathed in substimulatory (2 mM) glucose, mouse &bgr; cells, isolated from islets displaying immunochemical colocalization of P2X1 or P2X3 receptors and insulin, developed large (~250 pA/pF), rapidly activating, and then biexponentially decaying (&tgr;1, ~20 milliseconds/&tgr;2, ~1 second) inward currents on exposure to micromolar concentrations of ATP and &agr;,&bgr;-methylene ATP. The ATP also evoked inward currents (100-300 pA/pF) from porcine and human &bgr; cells, albeit with a slower and more complex inactivation pattern. Conclusions: The ATP-gated ion channels are present in pancreatic &bgr; cells from different species. Specifically, mouse &bgr; cells express rapidly desensitizing P2X1 and P2X3 receptors. Paracrine or neural activation of these receptors may contribute to the initial outburst of glucose- or acetylcholine-evoked insulin release, thus enhancing the islet secretory response.

Clinically relevant concentrations of ketamine mainly affect long-term potentiation rather than basal excitatory synaptic transmission and do not change paired-pulse facilitation in mouse hippocampal slices

Ketamine, an analgesic/anesthetic drug, is increasingly popular in clinical practice due to its analgesic properties and importance for emergency procedures. The impact of ketamine on basal excitatory synaptic transmission and synaptic plasticity are not yet fully understood. Therefore we investigated the effects of different concentrations of ketamine on basal excitatory synaptic transmission and on two forms of synaptic plasticity: paired-pulse facilitation (PPF) and long-term potentiation (LTP). Evoked field excitatory postsynaptic potentials (fEPSP) were recorded in Schaffer fiber - CA1 pyramid synapses of mouse hippocampal slices and the initial slope of the fEPSP was measured to estimate the percentage of inhibition of the basal synaptic transmission. Presynaptic volley amplitude, PPF and LTP induction and maintenance were also calculated. For basal synaptic transmission and PPF increasing concentrations of ketamine (1, 3, 10, 30, 100, 200, 300 and 600μM) were applied to each slice and for LTP individual slices were used for each concentration (3, 10, 30 or 100μM). Clinically relevant concentrations of ketamine decreased LTP in a concentration-dependent manner without changing PPF, whereas basal excitatory synaptic transmission and presynaptic volley amplitude was affected only with high concentrations of ketamine (300 and 600μM). These results allow dissociating the blockade of LTP from a reduced synaptic input in the action of clinically relevant concentrations of ketamine in the CA1 region of the mouse hippocampus. Moreover, this work shows that the effects of ketamine on LTP and on basal synaptic transmission are dependent of the concentration used.

Functional interaction between pre-synaptic α6β2-containing nicotinic and adenosine A2A receptors in the control of dopamine release in the rat striatum

Pre‐synaptic nicotinic ACh receptors (nAChRs) and adenosine A2A receptors (A2ARs) are involved in the control of dopamine release and are putative therapeutic targets in Parkinsons disease and addiction. Since A2ARs have been reported to interact with nAChRs, here we aimed at mapping the possible functional interaction between A2ARs and nAChRs in rat striatal dopaminergic terminals.

Differential contribution of syntaxin 1 and SNAP-25 to secretion in noradrenergic and adrenergic chromaffin cells

We used botulinum neurotoxins (BoNT) to examine whether differences in the secretory activity of noradrenergic and adrenergic chromaffin cells are related to differences in the exocytotic machinery of these two types of bovine adrenal medulla cells. Cleavage of syntaxin and SNAP-25 by BoNT/C1 decreased in a dose-dependent way the release of both noradrenaline and adrenaline, but noradrenaline release was more sensitive to BoNT/C1. Cleavage of SNAP-25 by BoNT/A also had a larger inhibitory effect on noradrenaline release than on adrenaline release. Neither BoNT/C1 nor BoNT/A affected the intracellular Ca2+ responses induced by K+-depolarisation, and the extent of the inhibition of K+-evoked catecholamine release by selective blockers of voltage-gated Ca2+ channels was not affected by BoNT/C1. Therefore, our data do not support the hypothesis of a regulatory effect of syntaxin or SNAP-25 on the activity of Ca2+ channels. The lower sensitivity of adrenaline release to BoNT was not due to a reduced ability of the toxins to enter or to cleave their protein targets in adrenergic cells, since immunoblot analysis showed the cleavage of a larger fraction of syntaxin 1A in adrenergic cells, as compared to the cleavage in noradrenergic cells. The immunoblot analysis also showed larger amounts of syntaxin 1A in noradrenergic chromaffin cells than in adrenergic cells. Thus, in spite of a greater cleavage of syntaxin 1A in adrenergic cells by BoNT/C1, adrenaline release was less sensitive to BoNT/C1, suggesting that the release process in noradrenergic cells might be more dependent on syntaxin 1A and SNAP-25, as compared to adrenergic cells.

Functional distribution of Ca2+-coupled P2 purinergic receptors among adrenergic and noradrenergic bovine adrenal chromaffin cells

BackgroundAdrenal chromaffin cells mediate acute responses to stress through the release of epinephrine. Chromaffin cell function is regulated by several receptors, present both in adrenergic (AD) and noradrenergic (NA) cells. Extracellular ATP exerts excitatory and inhibitory actions on chromaffin cells via ionotropic (P2X) and metabotropic (P2Y) receptors. We have taken advantage of the actions of the purinergic agonists ATP and UTP on cytosolic free Ca2+ concentration ([Ca2+]i) to determine whether P2X and P2Y receptors might be asymmetrically distributed among AD and NA chromaffin cells.ResultsThe [Ca2+]i and the [Na+]i were recorded from immunolabeled bovine chromaffin cells by single-cell fluorescence imaging. Among the ATP-sensitive cells ~40% did not yield [Ca2+]i responses to ATP in the absence of extracellular Ca2+ (Ca2+o), indicating that they expressed P2X receptors and did not express Ca2+- mobilizing P2Y receptors; the remainder expressed Ca2+-mobilizing P2Y receptors. Relative to AD-cells approximately twice as many NA-cells expressed P2X receptors while not expressing Ca2+- mobilizing P2Y receptors, as indicated by the proportion of cells lacking [Ca2+]i responses and exhibiting [Na+]i responses to ATP in the absence and presence of Ca2+o, respectively. The density of P2X receptors in NA-cells appeared to be 30–50% larger, as suggested by comparing the average size of the [Na+]i and [Ca2+]i responses to ATP. Conversely, approximately twice as many AD-cells expressed Ca2+-mobilizing P2Y receptors, and they appeared to exhibit a higher (~20%) receptor density. UTP raised the [Ca2+]i in a fraction of the cells and did not raise the [Na+]i in any of the cells tested, confirming its specificity as a P2Y agonist. The cell density of UTP-sensitive P2Y receptors did not appear to vary among AD- and NA-cells.ConclusionAlthough neither of the major purinoceptor types can be ascribed to a particular cell phenotype, P2X and Ca2+-mobilizing P2Y receptors are preferentially located to noradrenergic and adrenergic chromaffin cells, respectively. ATP might, in addition to an UTP-sensitive P2Y receptor, activate an UTP-insensitive P2Y receptor subtype. A model for a short-loop feedback interaction is presented whereby locally released ATP acts upon P2Y receptors in adrenergic cells, inhibiting Ca2+ influx and contributing to terminate evoked epinephrine secretion.

Treatment with A2A receptor antagonist KW6002 and caffeine intake regulate microglia reactivity and protect retina against transient ischemic damage

Transient retinal ischemia is a major complication of retinal degenerative diseases and contributes to visual impairment and blindness. Evidences indicate that microglia-mediated neuroinflammation has a key role in the neurodegenerative process, prompting the hypothesis that the control of microglia reactivity may afford neuroprotection to the retina against the damage induced by ischemia–reperfusion (I–R). The available therapeutic strategies for retinal degenerative diseases have limited potential, but the blockade of adenosine A2A receptor (A2AR) emerges as candidate strategy. Therefore, we evaluated the therapeutic potential of a selective A2AR antagonist (KW6002) against the damage elicited by I–R. The administration of KW6002 after I–R injury reduced microglia reactivity and inflammatory response and afforded protection to the retina. Moreover, we tested the ability of caffeine, an adenosine receptor antagonist, in mediating protection to the retina in the I–R injury model. We demonstrated that caffeine administration dually regulated microglia reactivity and cell death in the transient retinal ischemic model, depending on the reperfusion time. At 24 h of reperfusion, caffeine increased microglial reactivity, inflammatory response and cell death elicited by I–R. However, at 7 days of reperfusion, caffeine administration decreased microglia reactivity and reduced the levels of proinflammatory cytokines and cell death. Together, these results provide a novel evidence for the use of adenosine A2AR antagonists as potential therapy for retinal ischemic diseases and demonstrate the effect of caffeine on the regulation of microglia-mediated neuroinflammation in the transient ischemic model.

The Effects of Different Concentrations of the α2-Adrenoceptor Agonist Medetomidine on Basal Excitatory Synaptic Transmission and Synaptic Plasticity in Hippocampal Slices of Adult Mice.

BACKGROUND:&agr;2-Adrenoceptor agonists are used frequently in human and veterinary anesthesia as sedative/analgesic drugs. However, they can impair cognition. Little is known about the concentration-dependent effects of &agr;2-adrenoceptor agonists on synaptic plasticity, the neurophysiological basis of learning and memory. Therefore, we investigated the effects of different concentrations of medetomidine, an &agr;2-adrenoceptor agonist, on basal excitatory synaptic transmission and on 2 forms of synaptic plasticity: paired-pulse facilitation (PPF) and long-term potentiation (LTP). METHODS:Evoked field excitatory postsynaptic potentials were recorded in Schaffer fibers-CA1 pyramidal cell synapses of mouse hippocampal slices, and the initial field excitatory postsynaptic potentials slope was measured. For basal synaptic transmission and PPF, increasing concentrations of medetomidine (1–200 &mgr;M) were applied to each slice. For LTP experiments, individual slices were used for each tested concentration of medetomidine (0.1–0.4 &mgr;M), where LTP induction and LTP maintenance were measured. RESULTS:The lower tested concentrations of medetomidine decreased LTP in a concentration-dependent manner, whereas greater concentrations were required to decrease fiber volley amplitude and basal excitatory synaptic transmission. PPF was only affected by the greatest concentration (200 &mgr;M). CONCLUSIONS:Medetomidine decreased LTP in the mouse hippocampus, in accordance with the ability of medetomidine to induce memory deficits.