Renato D. Dias

Characterization of an ATP diphosphohydrolase (EC 3.6.1.5) in synaptosomes from cerebral cortex of adult rats.

Data from the literature have demonstrated that synaptosomal preparations from various sources can hydrolyze externally added ATP. Various authors characterized this activity as an ecto-ATPase. In the present report, we demonstrate that synaptosomal preparations obtained from the cerebral cortex of rats show ATPase activity that could not be dissociated from ADPase activity, suggesting that an ATP-diphosphohydrolase is involved in ATP and ADP hydrolysis. Furthermore, the ATP and ADP hydrolysis could not be attributed to associations of enzymes that could mimic an ATP-diphosphohydrolase because none of the following activities were detected in our assay conditions inorganic pyrophosphatase, adenylate kinase, or nonspecific phosphatases. A possible association between an ATPase and an ADPase was excluded on the basis of both the kinetics and much additional data on inhibitors, ion dependence, pH, etc. The present results demonstrate that in synaptosomal preparations from cerebral cortex an ATP-diphosphohydrolase is involved, at least in part, in ATP and ADP hydrolysis.

Beta-endorphin causes retrograde amnesia and is released from the rat brain by various forms of training and stimulation

The endogenous opiate peptide, beta-endorphin (0.4, 1.0, 2.0, and 10.0 μg/kg) was injected IP into rats immediately after training in a shuttle avoidance task, and its effect on memory retention was evaluated in test sessions carried out 24 h later. The drug was found to cause retrograde amnesia, the ED50 being 1.0 μg/kg. Beta-endorphin immunoreactivity was measured in the hypothalamus and rest of the brain of rats submitted to training, or test sessions of shuttle avoidance learning, pseudoconditioning in the shuttle-box, tones alone, or foot-shocks alone. After training in any of the four paradigms, there was a marked (46–60%) depletion of beta-endorphin immunoreactivity in the rest of the brain. No changes were detected in the hypothalamus or after test sessions. The loss of beta-endorphin immunoreactivity may be attributed to release of this substance caused by the stimuli used for training. From the present findings, as well as previous observations on the memory-facilitating influence of the opiate receptor antagonist, naloxone, it is concluded that there is a physiological amnesic mechanism mediated by beta-endorphin (and perhaps other opoid peptides as well), which is triggered by the non-associative factors present in the various forms of learning.

ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) in human blood platelets

Human platelets contain an ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) that is Ca(2+) dependent, hydrolyses ATP and ADP and also GTP, ITP, CTP, GDP, IDP, CDP. The enzyme does not hydrolyse AMP, p-nitrophenylphosphate, inorganic phosphate or glucose-6-phosphate. Contaminant activities were ruled out because the enzyme was not inhibited by 2 μg/d ouabain, 1.0 μM levamisole, 10 μM ApSA or 1.0 mM azide. The enzyme was sensitive to 100 μM orthovanadate, 100μMApSA and 10 mM azide, reagents that have been described as inhibitors of some other apyrases. A strong inhibition by 1.0 mM NEM was observed, indicating that sulphydryl groups are involved in the enzyme activity. The parallel behaviour of ATPase and ADPase activities and the competition plot presented suggest that ATP and ADP hydrolysis occurs at the same active site. ATP diphosphohydrolase from human platelets may be involved in the modulation of nucleotide concentration in the circulation and thus in vascular tonus.

Differences in Spatio-Temporal Behavior of Zebrafish in the Open Tank Paradigm after a Short-Period Confinement into Dark and Bright Environments

The open tank paradigm, also known as novel tank diving test, is a protocol used to evaluate the zebrafish behavior. Several characteristics have been described for this species, including scototaxis, which is the natural preference for dark environments in detriment of bright ones. However, there is no evidence regarding the influence of “natural stimuli” in zebrafish subjected to novelty-based paradigms. In this report, we evaluated the spatio-temporal exploratory activity of the short-fin zebrafish phenotype in the open tank after a short-period confinement into dark/bright environments. A total of 44 animals were individually confined during a 10-min single session into one of three environments: black-painted, white-painted, and transparent cylinders (dark, bright, and transparent groups). Fish were further subjected to the novel tank test and their exploratory profile was recorded during a 15-min trial. The results demonstrated that zebrafish increased their vertical exploratory activity during the first 6-min, where the bright group spent more time and travelled a higher distance in the top area. Interestingly, all behavioral parameters measured for the dark group were similar to the transparent one. These data were confirmed by automated analysis of track and occupancy plots and also demonstrated that zebrafish display a classical homebase formation in the bottom area of the tank. A detailed spatio-temporal study of zebrafish exploratory behavior and the construction of representative ethograms showed that the experimental groups presented significant differences in the first 3-min vs. last 3-min of test. Although the main factors involved in these behavioral responses still remain ambiguous and require further investigation, the current report describes an alternative methodological approach for assessing the zebrafish behavior after a forced exposure to different environments. Additionally, the analysis of ethologically-relevant patterns across time could be a potential phenotyping tool to evaluate the zebrafish exploratory profile in the open tank task.

Effect of various behavioral training and testing procedures on brain β-endorphin-like immunoreactivity and the possible role of β-endorphin in behavioral regulation

Beta-Endorphin-like immunoreactivity is reduced in the rat diencephalon after the animals are exposed for the first time to any of the following behavioral situations: 50 tones (habituation), 50 tone-footshock shuttle avoidance trials, one step-down inhibitory avoidance trial, simple exposure to the avoidance apparatus with no footshocks, or inescapable shock. The effect is not observed when animals are exposed to any of these situations for a second time. The reduction of brain beta-endorphin-like immunoreactivity is attributable to release and subsequent metabolism of the substance, and correlates with the novelty inherent in the diverse training or test situations. The role of beta-endorphin in behavior is discussed in the light of these and previous results which showed that it causes both retrograde amnesia and a facilitation of retrieval. The substance would appear to serve an adaptive function when animals are exposed to a new experience, by inducing a temporary forgetting of the experience together with (or leading to) a state of alertness or preparedness for what may happen next.

Effect of ACTH, epinephrine, β-endorphin, naloxone, and of the combination of naloxone or β-endorphin with ACTH or epinephrine on memory consolidation

Abstract The effect on retention of the post-training intraperitoneal administration of ACTH1–24 (0.2 or 2.0 μg/kg), epinephrine HC1 (5.0 or 50.0 μg/kg), human β-endorphin (0.1 or 1.0 μg/kg), naloxone (0.4 mg/kg), and of the combination of naloxine or β-endorphin with ACTH or epinephrine was studied in two different but closely related step-down inhibitory avoidance tasks in rats: task 1 (5 cm high 25 × 25 cm platform; 0.5 mA continuous footshock) and task 2 (7 × 25 cm platform, 0.3 mA discontinuous footshock). In task 1, saline control animals showed good retention in a test session carried out 24 hr later; β-endorphin, ACTH and epinephrine caused amnesia; β-endorphin potentiated the amnesic effect of ACTH and epinephrine; and naloxone caused memory facilitation and reversed the amnesic effect of ACTH and epinephrine. In task 2, control animals showed poor retention; β-endorphin caused amnesia at the dose of 0.1 but not 1.0 μg/kg; the other three drugs caused memory facilitation; naloxone potentiated the facilitatory effect of ACTH and epinephrine; and β-endorphin reversed it and transformed it into a deep amnesia. These findings suggest that an opioid-mediated amnesic mechanism modulates the effect of ACTH and epinephrine on memory consolidation, either by dampening that effect when training parameters tend to make it facilitatory, or by enhancing it when training conditions tend to make it amnesic. On the basis of these and previous data it seems likely that the amnesic effect of ACTH and epinephrine could be mediated by endogenous β-endorphin release.

Behavioral effects of taurine pretreatment in zebrafish acutely exposed to ethanol.

Taurine (TAU) is an amino sulfonic acid that plays protective roles against neurochemical impairments induced by ethanol (EtOH). Mounting evidence shows the applicability of zebrafish for evaluating locomotor parameters and anxiety-like behavioral phenotypes after EtOH exposure in a large scale manner. In this study, we assess the effects of TAU pretreatment on the behavior of zebrafish in the open tank after acute 1% EtOH (v/v) exposure (20 and 60 min of duration) and on brain alcohol contents. The exposure for 20 min exerted significant anxiolytic effects, which were prevented by 42, 150, and 400 mg/L TAU. Conversely, the 60-min condition induced depressant/sedative effects, in which the changes on vertical activity were associated to modifications on the exploratory profile. Although all TAU concentrations kept locomotor parameters at basal levels, 150 mg/L TAU, did not prevent the impairment on vertical activity of EtOH[60]. Despite the higher brain EtOH content detected in the 60-min exposure, 42, 150, and 400 mg/L TAU attenuated the increase of alcohol content in EtOH[60] group. In conclusion, our data suggest that both protocols of acute EtOH exposure induce significant changes in the spatio-temporal behavior of zebrafish and that TAU may exert a preventive role by antagonizing the effects induced by EtOH possibly due to its neuromodulatory role and also by decreasing brain EtOH levels. The hormetic dose-response of TAU on vertical exploration suggests a complex interaction between TAU and EtOH in the central nervous system.

Characterization of an ATP diphosphohydrolase activity (APYRASE, EC 3.6.1.5) in rat blood platelets

In the present report we describe an apyrase (ATP diphosphohydrolase, EC 3.6.1.5) in rat blood platelets. The enzyme hydrolyses almost identically quite different nucleoside di- and triphosphates. The calcium dependence and pH requirement were the same for the hydrolysis of ATP and ADP and the apparent Km values were similar for both Ca2+-ATP and Ca2+-ADP as substrates. Ca2+-ATP and Ca2+-ADP hydrolysis could not be attributed to the combined action of different enzymes because adenylate kinase, inorganic pyrophosphatase and nonspecific phosphatases were not detected under our assay conditions. The Ca2+-ATPase and Ca2+-ADPase activity was insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors, thus excluding these enzymes as contaminants. The results demonstrate that rat blood platelets contain an ATP diphosphohydrolase involved in the hydrolysis of ATP and ADP which are vasoactive and platelet active adenine nucleotides.

Differential effect of posttraining naloxone, β-endorphin, leu-enkephalin and electroconvulsive shock administration upon memory of an open-field habituation and of a water-finding task ☆

Rats were trained and tested in an open field for habituation of rearing responses, for a water-finding task, or for both tasks simultaneously. Training-test interval was 24 hr. The water-finding task consisted of locating a metal tube in one of the walls of the box, which was attached to a water bottle on the outside; animals were water deprived between training and testing. Retention was estimated by measuring the latency to lick from the tube on the test session. Rats learned this task either with or without water deprivation, also prior to training. Habituation learning (reduction of the number of rearings between the training and test session) occurred either simultaneously with the water-finding task or in animals trained without the water tube, so that they could not learn the water-finding task. As happens with many other tasks, training in the open field was followed by a large decrease of hypothalamic beta-endorphin immunoreactivity, attributable to a release of this substance. Posttraining IP naloxone (1.6 mg/kg) administration facilitated, and posttraining beta-endorphin (2.0 micrograms/kg), leu-enkephalin (5.0 micrograms/kg), or electroconvulsive shock (15 mA, 60 Hz, 2 sec) depressed the retention of habituation; this occurred regardless of whether the animals were trained and/or tested with or without water deprivation, and whether the task was acquired alone or simultaneously with the water-finding task. By contrast, none of these treatments had any effect on retention of the water finding task, acquired either with or without prior water deprivation. Thus, habituation was, and water-finding was not, sensitive to posttraining treatments known to affect endogenous opioids: the opioids themselves, their antagonist, naloxone, and electroconvulsive shock which releases brain opioids and causes naloxone-reversible retrograde amnesia. Learning of the water-finding task was merely incidental to exploration of the open field; it took place even when the animals were trained without the water tube. This suggests that the posttraining treatments that affect endogenous opioid function affect memory only of the task(s) that actually cause the release of brain beta-endorphin (in this case, probably habituation), and not of others that may occur simultaneously but are merely incidental (water-finding). A feature apparently common to the former is that they must directly involve either the recognition of novelty, or the initiation of an interaction with a new environment, or perhaps the habituation of such interaction.

Influence on memory of posttraining or pre-test injections of ACTH, vasopressin, epinephrine, and β-endorphin, and their interaction with naloxone

The intraperitoneal (i.p.) injection of ACTH 1-24 (0.2 microgram/kg), lysine--vasopressin (10.0 micrograms/kg) or epinephrine HCl (5.0 micrograms/kg) shortly after training or prior to testing caused memory facilitation of a step-down inhibitory avoidance task in rats, acquired with low intensity training footshocks (0.3 mA, 60 Hz). Naloxone HCl (0.4 mg/kg) potentiated their posttraining effect, but antagonized their pre-test effect. Naloxone on its own caused retrograde memory facilitation but had no effect on the test session. Posttraining human beta-endorphin (1.0 microgram/kg) was amnestic, and its pre-test administration enhanced retention. Both effects were naloxone-reversible. Neither the pre-test facilitation caused by beta-endorphin nor those caused by any of the other drugs (which are possible releasers of endogenous beta-endorphin) were observed in animals in which the influence of endogenous opioids was prevented at the posttraining period by the administration of naloxone. These results are compatible with, and considerably strengthen, the previously advanced hypothesis that learning of this task, and possibly others, depends on a state induced by beta-endorphin after training, and that it would normally be dissociated because this peptide is normally not released during test sessions. In addition, the posttraining facilitation caused by ACTH, vasopressin, and epinephrine stands out as an effect separate from, and in fact normally hindered by, posttraining beta-endorphin release.