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.

Contributions of Zebrafish Studies to the Behavioural Consequences of Early Alcohol Exposure: A Systematic Review.

BACKGROUND: The consequences of mild to severe exposure to alcohol during brain development is still a matter of debate and scientific investigation. The long-term behavioural effects of ethanol exposure have been related to impaired social skills and cognition. Zebrafish have become a suitable animal model to investigate the effects of early ethanol exposure because it is very feasible to promote drug delivery during early development. OBJECTIVE: The goal of the current report is to review existing behavioural studies addressing the impact of early alcohol exposure using zebrafish to determine whether these models resemble the behavioural effects of early alcohol exposure in humans. METHODS: A comprehensive search of biomedical databases was performed using the operation order: "ZEBRAFISH AND BEHAV* AND (ETHANOL OR ALCOHOL)". The eligibility of studies was determined using the PICOS strategy, contemplating the population as zebrafish, intervention as exposure to ethanol, comparison with a non-exposed control animal, and outcomes as behavioural parameters. RESULTS: The systematic search revealed 29 scientific articles as eligible. The zebrafish is presented as a versatile animal model that is useful to study FASD short and long-term behaviour impairments, such as anxiety, impaired sociability, aggressiveness, learning problems, memory impairment, seizure susceptibility, sleep disorders, motivational problems, and addiction. CONCLUSION: This systematic review further promotes the use of zebrafish as a model system to study the pathophysiological and behavioural consequences of early alcohol exposure (PROSPERO CRD42020215072).

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.

Toxicological evaluation of naringin-loaded nanocapsules in vitro and in vivo.

Naringin is a flavonoid widely known for its pharmacological properties, such as: anti-inflammatory and antioxidant ones, being an ally to avoid oxidative damage. Although naringin is an active easily found in citrus fruits, it has low bioavailability, biodistribution and also undergoes biotransformation in naringenin, limiting the described effects. The use of nanocapsules as drug carriers may increase solubility, improve biodistribution, impede the biotransformation thereof, and thus could improve the performance of naringin for use in treating neurological diseases. Therefore, the objective of this work is to produce a nanocapsule containing naringin, validate an analytical method by RP-HPLC to determination of the drug in nanoparticle and evaluate the toxicity. To that end, the blank nanocapsules (NB, without the drug) or naringin-loaded nanocapsules (NN) at the concentration of 2 mg/mL were prepared by interfacial deposition of the preformed polymer and the quantification of naringin by HPLC. Toxicity of the formulations was evaluated in vitro in rat hippocampal slices and in vivo models with C. elegans and Danio rerio (zebrafish). The analytical parameters evaluated (linearity, limit of detection and quantification, specificity, precision, accuracy and robustness) indicated adequate method to assay of naringin in nanocapsules by HPLC. There was no indication of toxicity by the nanocapsules in the evaluated biological assays.

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.

The influence of temperature on adult zebrafish sensitivity to pentylenetetrazole.

Pentylenetetrazole (PTZ) is one of the most valuable drugs used to induce seizure-like state in zebrafish especially considering the pharmacological screening for anticonvulsants and the study of basic mechanisms of epilepsy. Here, the effect of gender, weight and changes in temperature on latency to adult zebrafish reach classical seizure states induced by PTZ (10mM) was evaluated. Gender and weight (200-250mg versus 400-500mg) did not affect the profile of response to PTZ. When water temperature was changed from 22 to 30°C the lower temperature increased the latency time to reach seizure states and the higher temperature significantly decreased it, in comparison to the control group maintained at 26°C. The blockage of kainate receptors by DNQX (10µM) were unable to prevent the increased susceptibility of adult zebrafish exposed to hyperthermia and PTZ-induced seizures. The NMDA block by MK-801 (2.5µM) prevented the additive effect of hyperthermia on PTZ effects in adult zebrafish. This report emphasize that PTZ model in adult zebrafish exhibits no confounder factors from gender and weight, but water temperature is able to directly affect the response to PTZ, especially through a mechanism related to NMDA receptors.

Copper at low levels impairs memory of adult zebrafish (Danio rerio) and affects swimming performance of larvae.

Metal contamination at low levels is an important issue because it usually produces health and environmental effects, either positive or deleterious. Contamination of surface waters with copper (Cu) is a worldwide event, usually originated by mining, agricultural, industrial, commercial, and residential activities. Water quality criteria for Cu are variable among countries but allowed limits are generally in the µg/L range, which can disrupt several functions in the early life-stages of fish species. Behavioral and biochemical alterations after Cu exposure have also been described at concentrations close to the allowed limits. Aiming to search for the effects of Cu in the range of the allowed limits, larvae and adult zebrafish (Danio rerio) were exposed to different concentrations of dissolved Cu (nominally: 0, 5, 9, 20 and 60µg/L; measured: 0.4, 5.7, 7.2 16.6 and 42.3µg/L, respectively) for 96h. Larvae swimming and body length, and adult behavior and biochemical biomarkers (activity of glutathione-related enzymes in gills, muscle, and brain) were assessed after Cu exposure. Several effects were observed in fish exposed to 9µg/L nominal Cu, including increased larvae swimming distance and velocity, abolishment of adult inhibitory avoidance memory, and decreased glutathione S-transferase (GST) activity in gills of adult fish. At the highest Cu concentration tested (nominally: 60µg/L), body length of larvae, spatial memory of adults, and gill GST activity were decreased. Social behavior (aggressiveness and conspecific interaction), and glutathione reductase (GR) activity were not affected in adult zebrafish. Exposure to Cu, at concentrations close to the water quality criteria for this metal in fresh water, was able to alter larvae swimming performance and to induce detrimental effects on the behavior of adult zebrafish, thus indicating the need for further studies to reevaluate the currently allowed limits for Cu in fresh water.

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.

Evaluation of age-dependent response to NMDA receptor antagonism in zebrafish.

Imbalances in glutamatergic signaling have been proposed as the cause of several neurological disturbances. The use of MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, to mimic features of these neurological disorders is effective both in mammals and in fish. However, the variability of the subunits comprising the NMDA receptor during development alters the pharmacokinetic properties of the receptor and leads to different responses to this drug. Here, we evaluated the locomotor response of zebrafish to MK-801 (1, 5, and 20 µM) through the development (30 days postfertilization [dpf] to 2 years postfertilization [ypf]). The NMDA receptor subunit gene expression was also analyzed through the development (7 dpf to 2 ypf). Zebrafish displayed an age-related response to MK-801 with a higher response at 60 and 120 dpf. The magnitude of hyperlocomotion promoted by MK-801 seems to be less powerful for zebrafish in relation to rodents. The verification of expression levels in zebrafish NMDA receptor subunits shows that NR1.1 had a slight reduction throughout the development, while the NR2 subunits, especially NR2A.2 and NR2C.1, vary their expression levels according to the stage of development. The time-specific locomotor response to MK-801 through the development could be a consequence of differential NMDA receptor subunit expression. This result of developmental response to MK-801 is a crucial component in the consolidation of zebrafish as a suitable model to study glutamatergic neurotransmission in early phases.

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.

Maternal caffeine exposure alters neuromotor development and hippocampus acetylcholinesterase activity in rat offspring.

The objective of this study was to evaluate the effects of maternal caffeine intake on the neuromotor development of rat offspring and on acetylcholine degradation and acetylcholinesterase (AChE) expression in the hippocampus of 14-day-old infant rats. Rat dams were treated with caffeine (0.3g/L) throughout gestation and lactation until the pups were 14 days old. The pups were divided into three groups: (1) control, (2) caffeine, and (3) washout caffeine. The washout group received a caffeine solution until the seventh postnatal day (P7). Righting reflex (RR) and negative geotaxis (NG) were assessed to evaluate postural parameters as an index of neuromotor reflexes. An open-field (OF) test was conducted to assess locomotor and exploratory activities as well as anxiety-like behaviors. Caffeine treatment increased both RR and NG latency times. In the OF test, the caffeine group had fewer outer crossings and reduced locomotion compared to control, while the washout group showed increased inner crossings in relation to the other groups and fewer rearings only in comparison to the control group. We found decreased AChE activity in the caffeine group compared to the other groups, with no alteration in AChE transcriptional regulation. Chronic maternal exposure to caffeine promotes important alterations in neuromotor development. These results highlight the ability of maternal caffeine intake to interfere with cholinergic neurotransmission during brain development.

Hyperglycemia induces memory impairment linked to increased acetylcholinesterase activity in zebrafish (Danio rerio).

Diabetes mellitus, which causes hyperglycemia, affects the central nervous system and can impairs cognitive functions, such as memory. The aim of this study was to investigate the effects of hyperglycemia on memory as well as on the activity of acethylcholinesterase. Hyperglycemia was induced in adult zebrafish by immersion in glucose 111mM by 14 days. The animals were divided in 4 groups: control, glucose-treated, glucose-washout 7-days and glucose-washout 14-days. We evaluated the performance in inhibitory avoidance task and locomotor activity. We also determined acethylcholinesterase activity and gene expression from whole brain. In order to counteract the effect of hyperglycemia underlined by effects on acethylcholinesterase activity, we treated the animals with galantamine (0.05ng/g), an inhibitor of this enzyme. Also we evaluated the gene expression of insulin receptor and glucose transporter from zebrafish brain. The hyperglycemia promoted memory deficit in adult zebrafish, which can be explained by increased AChE activity. The ache mRNA levels from zebrafish brain were decrease in 111mM glucose group and returned to normal levels after 7 days of glucose withdrawal. Insulin receptors (insra-1, insra-2, insrb-1 and insrb-2) and glut-3 mRNA levels were not significantly changed. Our results also demonstrated that galantamine was able to reverse the memory deficit caused by hyperglycemia, demonstrating that these effects involve modulation of AChE activity. These data suggest that the memory impairment induced by hyperglycemia is underlined by the cholinergic dysfunction caused by the mechanisms involving the control of acetylcholinesterase function and gene expression.