Functioning and Gene Expression of Adenosine A1 Receptor During Zebrafish (Danio rerio) Development.

The A1 adenosine receptor is the most widely expressed P1 receptor in vertebrates, performing inhibitory tone of the nervous system. Increased levels of adenosine are crucial to promote tissue protection in threatening situations, such as convulsion and hypoxia. Zebrafish is an established model organism for studies on health and disease. In this study, we evaluated the functionality of A1 adenosine receptor through development of zebrafish (6-7-day-, 3-, 8-, and 24-month-old), assessing: (I) the effects of the agonist N6-cyclopenthyladenosine (CPA) over locomotor parameters, (II) the anticonvulsant properties of CPA and adenosine per se in the pentylenetetrazol-induced seizure, and (III) the gene expression of adora1b through development. CPA promoted decreased distance traveled in the highest concentrations/doses tested (larvae: 75 to 500 µM; adults: 20 mg.kg-1), altered mean velocity (larvae: 50-500 µM; adults: 20 mg.kg-1) and time in the bottom zone of apparatus (adults: decrease in 20 mg.kg-1). Adenosine increased the latency of the larvae to reach stage II at 5 and 10 µM. CPA anticonvulsant effect against convulsive stage II was reached at 75 µM, although it decreased basal locomotor activity in larvae. For adults, CPA 10 mg.kg-1 was effective as anticonvulsant without locomotory effects. Adenosine had minor anticonvulsant effects in the concentration tested (larvae: 5 and 10 µM). The level of gene expression of adora1b was stable in brain from adult animals (8- and 24-month-old animals). These results suggest that zebrafish has similar responses to CPA as mammals. To avoid confounding factors, such as locomotor effects, during any brain function investigation using A1 adenosine receptor as a target, the concentration below 75 µM or below the dose of 20 mg.kg-1 of CPA is ideal for zebrafish at larval and adult stages, respectively.

What do we know about the sleep effects of caffeine used to treat apnoea of prematurity? A systematic review of the literature.

OBJECTIVE: Scientific scrutiny has proved the safety and benefits of caffeine to treat apnoea of prematurity (AOP). However, there is no consensus on the effects of this treatment on sleep, especially considering the key role of adenosine and early brain development for sleep maturation. We systematically reviewed studies with sleep as a primary and/or secondary outcome or any mention of sleep parameters in the context of caffeine treatment for AOP. METHODS: We performed a systematic search of PubMed, Web of Science and the Virtual Health Library from inception to 7 September 2022 to identify studies investigating the short- and long-term effects of caffeine to treat AOP on sleep parameters. We used the PIC strategy considering preterm infants as the Population, caffeine for apnoea as the Intervention and no or other intervention other than caffeine as the Comparison. We registered the protocol on PROSPERO (CRD42021282536). RESULTS: Of 4019 studies, we deemed 20, including randomised controlled trials and follow-up and observational studies, to be eligible for our systematic review. The analysed sleep parameters, the evaluation phase and the instruments for sleep assessment varied considerably among the studies. The main findings can be summarised as follows: (i) most of the eligible studies in this systematic review indicate that caffeine used to treat AOP seems to have no effect on key sleep parameters and (ii) the effects on sleep when caffeine is administered earlier, at higher doses or for longer periods than the most common protocol have not been investigated. There is a possible correlation between the caffeine concentration and period of exposure and negative sleep quality, but the sleep assessment protocols used in the included studies did not have high-quality standards and could not provide good evidence. CONCLUSIONS AND IMPLICATIONS: Sleep quality is an important determinant of health, and better investments in research with adequate sleep assessment tools are necessary to guarantee the ideal management of children who were born preterm.

Anticonvulsant Role of Adenosine is Blunted During Alcohol Withdrawal Syndrome in an Adult Zebrafish Model.

Alcohol (ethanol) dependence and related disorders are life-threatening conditions and source of suffering for the user, family members and society. Alcohol withdrawal syndrome (AWS) is a little-known dynamic process associated with a high frequency of relapses. A state of hyperglutamatergic neurotransmission and imbalanced GABAergic function is related to an increased susceptibility to seizures during alcohol withdrawal. Adenosine signaling display an important role in endogenous response to decrease seizure and related damages. Here, an intermittent alcohol exposure regimen (1 h daily of 0.5% ethanol solution) for 16 days or 8 days of the same ethanol exposure regimen followed by 1 or 8 days of ethanol withdrawal was used to assess adenosine signaling in the context of seizure susceptibility using adult zebrafish. In both abstainer groups, a sub-convulsant dose of pentylenetetrazol (2.5 mM) was able to increase the frequency of animals reaching a clonic seizure-like state, while continuous-treated animals had no seizure, as did control animals. The total brain mRNA expression of A1 adenosine receptor was decreased in animals with 1 day of ethanol withdrawal. The agonism of A1 adenosine receptor induced an anticonvulsant effect in animals with 1 day of ethanol withdrawal after the injection of the specific agonist (N6-cyclopentyladenosine, 10 mg.Kg- 1; i.p.). These findings reinforce A1 adenosine receptor as a key target in acute alcohol withdrawal syndrome and zebrafish as an excellent platform to study biological mechanism of AWS.

Participation of ecto-5'-nucleotidase in the inflammatory response in an adult zebrafish (Danio rerio) model.

The ecto-5'-nucleotidase is an important source of adenosine in the extracellular medium. Adenosine modulation appears early in evolution and performs several biological functions, including a role as an anti-inflammatory molecule. Here, we evaluate the activity and mRNA expression of ecto-5'-nucleotidase in response to lipopolysaccharide (LPS) using zebrafish as a model. Adult zebrafish were injected with LPS (10 µg/g). White blood cell differential counts, inflammatory markers, and ecto-5'-nucleotidase activity and expression in the encephalon, kidney, heart, and intestine were evaluated at 2, 12, and 24 h post-injection (hpi). At 2 hpi of LPS, an increase in neutrophils and monocytes in peripheral blood was observed, which was accompanied by increased tnf-α expression in the heart, kidney, and encephalon, and increased cox-2 expression in the intestine and kidney. At 12 hpi, monocytes remained elevated in the peripheral blood, while tnf-α expression was also increased in the intestine. At 24 hpi, the white blood cell differential count no longer differed from that of the control, whereas tnf-α expression remained elevated in the encephalon but reduced in the kidney compared with the controls. AMP hydrolysis in LPS-treated animals was increased in the heart at 24 hpi [72 %; p = 0.029] without affecting ecto-5'-nucleotidase gene expression. These data indicate that, in most tissues studied, inflammation does not affect ecto-5'-nucleotidase activity, whereas in the heart, a delayed increase in ecto-5'-nucleotidase activity could be related to tissue repair.

Inhibition of ecto-5'-nucleotidase and adenosine deaminase is able to reverse long-term behavioural effects of early ethanol exposure in zebrafish (Danio rerio).

The behavioural impacts of prenatal exposure to ethanol include a lower IQ, learning problems, anxiety and conduct disorders. Several components of the neurochemical network could contribute to the long-lasting effects of ethanol embryonic exposure. Adenosine is an important neuromodulator, that has been indicated to be affected by acute and chronic exposure to ethanol. Here, embryos of zebrafish exposed to 1% ethanol during the developmental stages of gastrula/segmentation or pharyngula exhibited anxiolytic effect, increased aggressiveness, and decreased social interaction. The exposure during pharyngula stage was able to affect all behavioural parameters analysed at 3 months-post fertilization (mpf), while the treatment during gastrula stage affected the anxiety and social interaction parameters. The aggressiveness was the only behavioural effect of early ethanol exposure that lasted to 12 mpf. The use of a specific inhibitor of adenosine production, the inhibitor of ecto-5'-nucleotidase (AMPCP/150 mg/kg), and the specific inhibitor of adenosine degradation, the inhibitor of adenosine deaminase, EHNA (100 mg/kg) did not affect the effects over anxiety. However, AMPCP at 3 mpf, but not EHNA, reversed aggressive parameters. AMPCP also recovered the social interaction parameter at 3 mpf in animals treated in both stages, while EHNA recovered this parameter just in those animals treated with ethanol during the gastrula stage. These results suggest that long-lasting behavioural effects of ethanol can be modulated by intervention on ecto-5'-nucleotidase and adenosine deaminase activities.

Persistent increase in ecto­5'­nucleotidase activity from encephala of adult zebrafish exposed to ethanol during early development.

Prenatal alcohol exposure causes alterations to the brain and can lead to numerous cognitive and behavioral outcomes. Long-lasting effects of early ethanol exposure have been registered in glutamatergic and dopaminergic systems. The purinergic system has been registered as an additional target of ethanol exposure. The objective of this research was to evaluate if the ecto­5'­nucleotidase and adenosine deaminase activities and gene expression of adult zebrafish exposed to 1% ethanol during early development could be part of the long-lasting targets of ethanol. Zebrafish embryos were exposed to 1% ethanol in two distinct developmental phases: gastrula/segmentation (5-24 h post-fertilization) or pharyngula (24-48 h post-fertilization). At the end of three months, after checking for morphological outcomes, the evaluation of enzymatic activity and gene expression was performed. Exposure to ethanol did not promote gross morphological defects; however, a significant decrease in the body length was observed (17% in the gastrula and 22% in the pharyngula stage, p < 0.0001). Ethanol exposure during the gastrula/segmentation stage promoted an increase in ecto­5'­nucleotidase activity (39.5%) when compared to the control/saline group (p < 0.0001). The ecto­5'­nucleotidase gene expression and the deamination of adenosine exerted by ecto and cytosolic adenosine deaminase were not affected by exposure to ethanol in both developmental stages. HPLC experiments did not identify differences in adenosine concentration on the whole encephala of adult animals exposed to ethanol during the gastrula stage or on control animals (p > 0.05). Although the mechanism underlying these findings requires further investigation, these results indicate that ethanol exposure during restricted periods of brain development can have long-term consequences on ecto­5'­nucleotidase activity, which could have an impact on subtle sequels of ethanol early exposure.

Early exposure to ethanol is able to affect the memory of adult zebrafish: Possible role of adenosine.

Ethanol is one of the most widely consumed drugs in the world, and the effects of ethanol during early development include morphological and cognitive problems. The regulation of adenosine levels is essential for the proper function of major neurotransmitter systems in the brain, particularly glutamate and dopamine; thus, the investigation of the relation of adenosine and memory after early ethanol exposure becomes relevant. Embryos of zebrafish were exposed to 1% ethanol during two distinct developmental stages: gastrula/segmentation or pharyngula. The evaluation of memory, morphology, and locomotor parameters was performed when fish were 3 months old. The effect of ecto-5'-nucleotidase and adenosine deaminase inhibition on the consequences of ethanol exposure with regard to memory formation was observed. Morphological evaluation showed decreases in body length and the relative telencephalic and cerebellar areas in ethanol exposed animals. The locomotor parameters evaluated were not affected by ethanol. In the inhibitory avoidance paradigm, ethanol exposure during the gastrula/segmentation and pharyngula stages decreased zebrafish memory retention. When ethanol was given in the pharyngula stage, the inhibition of ecto-5'-nucleotidase in the acquisition phase of memory tests was able to revert the effects of ethanol on the memory of adults. These findings suggest that the increased adenosine levels caused by ethanol could alter the neuromodulation of important components of memory formation, such as neurotransmitters. The adjustment of adenosine levels through ecto-5'-nucleotidase inhibition appears to be effective at restoring normal adenosine levels and the acquisition of memory in animals exposed to ethanol during the pharyngula stage.

Transient Disruption of Adenosine Signaling During Embryogenesis Triggers a Pro-epileptic Phenotype in Adult Zebrafish.

Adenosinergic signaling has important effects on brain function, anatomy, and physiology in both late and early stages of development. Exposure to caffeine, a non-specific blocker of adenosine receptor, has been indicated as a developmental risk factor. Disruption of adenosinergic signaling during early stages of development can change the normal neural network formation and possibly lead to an increase in susceptibility to seizures. In this work, morpholinos (MO) temporarily blocked the translation of adenosine receptor transcripts, adora1, adora2aa, and adora2ab, during the embryonic phase of zebrafish. It was observed that the block of adora2aa and adora2aa + adora2ab transcripts increased the mortality rate and caused high rate of malformations. To test the susceptibility of MO adora1, MO adora2aa, MO adora2ab, and MO adora2aa + adora2ab animals to seizure, pentylenetetrazole (10 mM) was used as a convulsant agent in larval and adult stages of zebrafish development. Although no MO promoted significant differences in latency time to reach the seizures stages in 7-day-old larvae, during the adult stage, all MO animals showed a decrease in the latency time to reach stages III, IV, and V of seizure. These results indicated that transient interventions in the adenosinergic signaling through high affinity adenosine receptors during embryonic development promote strong outcomes on survival and morphology. Additionally, long-term effects on neural development can lead to permanent impairment on neural signaling resulting in increased susceptibility to seizure.

Hyperglycemia alters E-NTPDases, ecto-5'-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio).

Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment.

Reprint of "Caffeine protects against memory loss induced by high and non-anxiolytic dose of cannabidiol in adult zebrafish (Danio rerio)".

Cannabidiol (CBD) has been investigated in a wide spectrum of clinical approaches due to its psychopharmacological properties. CBD has low affinity for cannabinoid neuroreceptors and agonistic properties to 5-HT receptors. An interaction between cannabinoid and purinergic receptor systems has been proposed. The purpose of this study is to evaluate CBD properties on memory behavioral and locomotor parameters and the effects of pre-treatment of adenosine receptor blockers on CBD impacts on memory using adult zebrafish. CBD (0.1, 0.5, 5, and 10mg/kg) was tested in the avoidance inhibitory paradigm and anxiety task. We analyzed the effect of a long-term caffeine pre-treatment (~20mg/L - four months). Also, acute block of adenosine receptors was performed in co-administration with CBD exposure in the memory assessment. CBD promoted an inverted U-shaped dose-response curve in the anxiety task; in the memory assessment, CBD in the dose of 5mg/Kg promoted the strongest effects without interfering with social and aggressive behavior. Caffeine treatment was able to prevent CBD (5mg/kg) effects on memory when CBD was given after the training session. CBD effects on memory were partially prevented by co-treatment with a specific A2A adenosine receptor antagonist when given prior to or after the training session, while CBD effects after the training session were fully prevented by adenosine A1 receptor antagonist. These results indicated that zebrafish have responses to CBD anxiolytic properties that are comparable to other animal models, and high doses changed memory retention in a way dependent on adenosine.

Caffeine protects against memory loss induced by high and non-anxiolytic dose of cannabidiol in adult zebrafish (Danio rerio).

Cannabidiol (CBD) has been investigated in a wide spectrum of clinical approaches due to its psychopharmacological properties. CBD has low affinity for cannabinoid neuroreceptors and agonistic properties to 5-HT receptors. An interaction between cannabinoid and purinergic receptor systems has been proposed. The purpose of this study is to evaluate CBD properties on memory behavioral and locomotor parameters and the effects of pre-treatment of adenosine receptor blockers on CBD impacts on memory using adult zebrafish. CBD (0.1, 0.5, 5, and 10mg/kg) was tested in the avoidance inhibitory paradigm and anxiety task. We analyzed the effect of a long-term caffeine pre-treatment (~20mg/L - four months). Also, acute block of adenosine receptors was performed in co-administration with CBD exposure in the memory assessment. CBD promoted an inverted U-shaped dose-response curve in the anxiety task; in the memory assessment, CBD in the dose of 5mg/Kg promoted the strongest effects without interfering with social and aggressive behavior. Caffeine treatment was able to prevent CBD (5mg/kg) effects on memory when CBD was given after the training session. CBD effects on memory were partially prevented by co-treatment with a specific A2A adenosine receptor antagonist when given prior to or after the training session, while CBD effects after the training session were fully prevented by adenosine A1 receptor antagonist. These results indicated that zebrafish have responses to CBD anxiolytic properties that are comparable to other animal models, and high doses changed memory retention in a way dependent on adenosine.

Contributions from extracellular sources of adenosine to the ethanol toxicity in zebrafish larvae.

The effects of ethanol exposure on extracellular adenosine sources in zebrafish were evaluated. In the acute treatment, the embryos were exposed to 2% ethanol on day 1 post-fertilization (dpf). In the chronic treatment, the exposure was continued for 2h/day up to 6 dpf. Ecto-5'-nucleotidase activity was assessed by colorimetric method and gene expression determined by RT-qPCR in 7 dpf zebrafish. Body length, ocular distance and surface area of the eyes were registered in animals acutely exposed to ethanol and pretreated with AOPCP (5-500 nM), an ecto-5'-nucleotidase inhibitor, or dipyridamole (10-100 µM), a blocker of nucleoside transport. Both ethanol exposures promoted increased ecto-5'-nucleotidase activity, impaired locomotion and morphology. Ecto-5'-nucleotidase expression was not affected. AOPCP promoted mild prevention of morphological defects caused by acute treatment, while dipyridamole worsened these defects. Early ethanol exposure altered adenosinergic tonus, especially through nucleoside transporters, contributing to morphological defects produced by ethanol in zebrafish.

Role of adenosine signaling on pentylenetetrazole-induced seizures in zebrafish.

Adenosine is a well-known endogenous modulator of neuronal excitability with anticonvulsant properties. Thus, the modulation exerted by adenosine might be an effective tool to control seizures. In this study, we investigated the effects of drugs that are able to modulate adenosinergic signaling on pentylenetetrazole (PTZ)-induced seizures in adult zebrafish. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) decreased the latency to the onset of the tonic-clonic seizure stage. The adenosine A1 receptor agonist cyclopentyladenosine (CPA) increased the latency to reach the tonic-clonic seizure stage. Both the adenosine A2A receptor agonist and antagonist, CGS 21680 and ZM 241385, respectively, did not promote changes in seizure parameters. Pretreatment with the ecto-5'nucleotidase inhibitor adenosine 5'-(α,ß-methylene) diphosphate (AMPCP) decreased the latency to the onset of the tonic-clonic seizure stage. However, when pretreated with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), or with the nucleoside transporter (NT) inhibitors, dipyridamole and S-(4-Nitrobenzyl)-6-thioinosine (NBTI), animals showed longer latency to reach the tonic-clonic seizure status. Finally, our molecular analysis of the c-fos gene expression corroborates these behavioral results. Our findings indicate that the activation of adenosine A1 receptors is an important mechanism to control the development of seizures in zebrafish. Furthermore, the actions of ecto-5'-nucleotidase, ADA, and NTs are directly involved in the control of extracellular adenosine levels and have an important role in the development of seizure episodes in zebrafish.

Arginine exposure alters ectonucleotidase activities and morphology of zebrafish larvae (Danio rerio).

Hyperargininemia is an inborn error of metabolism (IEM) characterized by tissue accumulation of arginine (Arg). Mental retardation and other neurological features are common symptoms in hyperargininemic patients. Considering purinergic signaling has a crucial role from the early stages of development and underlying mechanisms of this disease are poorly established, we investigated the effect of Arg administration on locomotor activity, morphological alterations, and extracellular nucleotide hydrolysis in larvae and adult zebrafish. We showed that 0.1 mM Arg was unable to promote changes in locomotor activity. In addition, 7-day-post-fertilization (dpf) larvae treated with Arg demonstrated a decreased body size. Arg exposure (0.1 mM) promoted an increase in ATP, ADP, and AMP hydrolysis when compared to control group. These findings demonstrated that Arg might affect morphological parameters and ectonucleotidase activities in zebrafish larvae, suggesting that purinergic system is a target for neurotoxic effects induced by Arg.

A ketogenic diet did not prevent effects on the ectonucleotidases pathway promoted by lithium-pilocarpine-induced status epilepticus in rat hippocampus.

A Ketogenic Diet (KD) mimics the anticonvulsant effects of fasting, which are known to suppress seizures. The purinergic system has been investigated in the matter of epilepsy development, especially the nucleoside adenosine, which has been considered a natural brain anticonvulsant. During epileptic seizures, extracellular adenosine concentration rises rapidly to micromolar levels. Adenosine can exert its anticonvulsant functions, after its release by nucleoside bidirectional transport, or by production through the sequential catabolism of ATP by ectonucleotidases, such as E-NTPDases (ectonucleoside triphosphate diphosphohydrolases) and ecto-5'-nucleotidase. Here, we have investigated the effect of a ketogenic diet on the nucleotide hydrolysis and NTPDases expression in the lithium-pilocarpine (Li-Pilo) model of epilepsy. For the induction of Status Epileticus (SE), 21-day-old female Wistar rats received an i.p. injection of lithium chloride (127 mg/kg) and 18-19 h later an i.p. injection of pilocarpine hydrochloride (60 mg/kg). The control groups received an injection of saline. After induction of SE, the control and Li-Pilo groups received standard or ketogenic diets for 6 weeks. The lithium-pilocarpine exposure affected the ATP (a decrease of between 8 % and 16 %) and ADP (an increase of between 18 % and 22 %) hydrolysis in both groups whereas the diet did not impact the nucleotide hydrolysis. NTPDase2 and 3 mRNA expressions decreased in the Li-Pilo group (41 % and 42 %). This data highlights the participation of the purinergic system in the pathophysiology of this model of epilepsy, since nucleotide hydrolysis and NTPDase expressions were altered by Li-Pilo exposure, with no significant effects of the ketogenic diet. However, the interaction between purinergic signaling and a ketogenic diet on epilepsy still needs to be better elucidated.

Early exposure to caffeine affects gene expression of adenosine receptors, DARPP-32 and BDNF without affecting sensibility and morphology of developing zebrafish (Danio rerio).

Adenosine receptors are the most important biochemical targets of caffeine, a common trimethylxanthine found in food and beverages. Adenosine plays modulatory action during the development through adenosine receptors and their intracellular pathways activation. In this study, we aimed to evaluate if caffeine gave to zebrafish in the very first steps of development is able to affect its direct targets, through the adenosine receptors mRNA expression evaluation, and latter indirect targets, through evaluation of the pattern of dopamine and cAMP-regulated phosphoprotein and brain-derived neurotrophic factor (BDNF) mRNA expression. Here, we demonstrate that zebrafish express adenosine receptor subtypes (A1, A2A1, A2A2 and A2B) since 24h post-fertilization (hpf) and that caffeine exposure is able to affect the expression of these receptors. Caffeine exposure from 1 hpf is able to increase A1 expression at 72-96 hpf and A2A1 expression at 72 hpf. No alterations occurred in A2A2 and A2B expression after caffeine treatment. DARPP-32, a phosphoprotein involved in adenosine intracellular pathway is also expressed since 24 hpf and early exposure to caffeine increased DARPP-32 expression at 168 hpf. We also evaluate the expression of BDNF as one of the targets of adenosine intracellular pathway activation. BDNF was also expressed since 24 hpf and caffeine treatment increased its expression at 48 and 72 hpf. No morphological alterations induced by caffeine treatment were registered by the check of general body features and total body length. Assessment of tactile sensibility also demonstrated no alterations by caffeine treatment. Altogether, these results suggest that caffeine is able to affect expression of its cellular targets since early phases of development in zebrafish without affect visible features. The up-regulation of direct and indirect targets of caffeine presents as a compensatory mechanism of maintenance of adenosinergic modulation during the developmental phase.

Neonatal morphine exposure alters E-NTPDase activity and gene expression pattern in spinal cord and cerebral cortex of rats.

The neonate opioid system has been frequently investigated, and studies have shown that exposure to drugs in early life can have implications for nervous system development. It has been proposed that adenosine is involved in opioid antinociception, and ATP is involved in central and peripheral mechanisms of nociception. Extracellular nucleotides can be hydrolyzed by E-NTPDases and ecto-5'nucleotidase, which present the functions of removing ATP and generating adenosine. In this study, we evaluated ATP, ADP, and AMP hydrolysis in synaptosomes from spinal cord and cerebral cortex of rats at postnatal day 16 after repeated morphine exposure in early life (postnatal day 8 to 14). Additionally, we evaluated E-NTPDase (1, 2 and 3) and ecto-5'nucleotidase gene expression by semi-quantitative RT-PCR analysis. We observed an increase in ATP hydrolysis in the cerebral cortex, and a decrease in ADP hydrolysis in spinal cord. Expression levels of E-NTPDase 1 decreased in cerebral cortex and increased in spinal cord. Our findings highlight the importance of the purinergic system in young rats submitted to repeated morphine exposure by showing that in the neonatal period such exposure is capable of affecting the control system for nucleotide levels, which can promote changes in modulation or transmission of painful stimuli.

Iron leads to memory impairment that is associated with a decrease in acetylcholinesterase pathways.

Increasing evidence indicates that excessive iron in selective regions of the brain may be involved in the etiology of neurodegenerative disorders. Accordingly, increased levels of iron have been described in brain regions of patients in Parkinson's and Alzheimer's diseases. We have characterized neonatal iron loading in rodents as a novel experimental model that mimics the brain iron accumulation observed in patients with neurodegenerative diseases and produces severe cognitive impairment in the adulthood. In the present study we have investigated the involvement of the cholinergic system on iron-induced memory impairment. The effects of a single administration of the acetylcholinesterase (AChE) inhibitor galantamine or the muscarinic receptor agonist oxotremorine on iron-induced memory deficits in rats were examined. Male Wistar rats received vehicle or iron (10.0 mg/kg) orally at postnatal days 12 to 14. At the age of 2-3 months, animals were trained in a novel object recognition task. Iron-treated rats showed long-term impairments in recognition memory. The impairing effect was reversed by systemic administration of galantamine (1 mg/kg) immediately after training. In addition, iron-treated rats that received oxotremorine (0.5 mg/kg) showed enhanced memory retention. Rats given iron showed a decreased AChE activity in the striatum when compared to controls. The results suggest that, at least in part, iron-induced cognitive deficits are related to a dysfunction of cholinergic neural transmission in the brain. These findings might have implications for the development of novel therapeutic strategies aimed at ameliorating cognitive decline associated with neurodegenerative disorders.

Effects of arsenic (As) exposure on the antioxidant status of gills of the zebrafish Danio rerio (Cyprinidae).

In fishes, arsenic (As) is absorbed via the gills and is capable of causing disturbance to the antioxidant system. The objective of present study was to evaluate antioxidant responses after As exposure in gills of zebrafish (Danio rerio, Cyprinidae). Fish were exposed for 48 h to three concentration of As, including the highest As concentration allowed by current Brazilian legislation (10 microg As/L). A control group was exposed to tap water (pH 8.0; 26 degrees C; 7.20 mg O(2)/L). As exposure resulted in (1) an increase (p<0.05) of glutathione (GSH) levels after exposure to 10 and 100 microg As/L, (2) an increase of the glutamate cysteine ligase (GCL) activity in the same concentrations (p<0.05), (3) no significant differences in terms of glutathione reductase, glutathione-S-transferase and catalase activities; (4) a significantly lower (p<0.05) oxygen consumption after exposure to 100 microg As/L; (4) no differences in terms of oxygen reactive species generation and lipid peroxidation content (p>0,05). In the gills, only inorganic As was detected. Overall, it can be concluded that As affected the antioxidant responses increasing GCL activity and GSH levels, even at concentration considered safe by Brazilian legislation.

Peripheral nucleotide hydrolysis in rats submitted to a model of electroconvulsive therapy.

Electroconvulsive therapy (ECT) is an efficacious and safe method for the treatment of mood disorders. Its utilization is accompanied by a myriad of biochemical and cellular changes, which are far from fully understood. The present work investigates in rat serum the effects of seizures induced by electroconvulsive shocks (ECS), an animal model of ECT, on enzymes that hydrolyze ATP, ADP and AMP to adenosine. Two different models of ECS were used, consisting in the application of one or eight ECS sessions, and respectively named acute or chronic. Serum samples were collected at several time points after the single shock in the acute and after the eighth and last shock in the chronic model. A single shock produced a sudden and short-lived inhibition of enzymatic activity (P<0.01 for ADP and AMP), whereas in the chronic model significant increases were noticed starting as early as 12 h after the last shock, remaining significantly elevated until the last measurement 7 days later for ATP and ADP. Analysis of hydrolysis was assessed at the selected time point of 7 days in cerebrospinal fluid samples, also demonstrating a significant activation in the chronic model (P<0.0001 for ATP and ADP). These results support the idea that adenosine nucleotides may be involved in the biochemical mechanisms underlying longer lasting therapeutic effects associated with ECT, and suggest that peripheral markers can possibly contribute to the evaluation of activity in the central nervous system.