Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis.

Changes in social behavior are associated with brain disorders, including mood disorders, stress, schizophrenia, Alzheimer's disease, and autism spectrum disorders (ASD). Autism is a complex neurodevelopmental disorder characterized by deficits in social interaction, impaired communication, anxiety, hyperactivity, and the presence of restricted interests. Zebrafish is one of the most social vertebrates used as a model in biomedical research, contributing to an understanding of the mechanisms that underlie social behavior. Valproic acid (VPA) is used as an anti-epileptic drug and mood stabilizer; however, prenatal VPA exposure in humans has been associated with an increased incidence of autism and it can also affect fetal brain development. Therefore, we conducted a behavioral screening at different periods of zebrafish development at 6, 30, 70, and 120dpf (days postfertilization) after VPA exposure in the early development stage to investigate social behavior, locomotion, aggression, and anxiety. VPA (48µM) exposure during the first 48hpf (hours postfertilization) did not promote changes on survival, morphology, and hatching rate at 24hpf, 48hpf, and 72hpf. The behavioral patterns suggest that VPA exposure induces changes in locomotor activity and anxiety at different developmental periods in zebrafish. Furthermore, a social interaction deficit is present at 70dpf and 120dpf. VPA exposure did not affect aggression in the adult stage at 70dpf and 120dpf. This is the first study that demonstrated zebrafish exposed to VPA during the first 48h of development exhibit deficits in social interaction, anxiety, and hyperactivity at different developmental periods.

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.

Effect of ketogenic diet on nucleotide hydrolysis and hepatic enzymes in blood serum of rats in a lithium-pilocarpine-induced status epilepticus.

The ketogenic diet (KD) is a high-fat and low-carbohydrate diet, used for treating refractory epilepsy in children. We have previously shown alterations in nucleotidase activities from the central nervous system and blood serum of rats submitted to different models of epilepsy. In this study we investigated the effect of KD on nucleotidase activities in the blood serum, as well if KD has any influence in the activity of liver enzymes such as alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities in Wistar rats submitted to the lithium-pilocarpine model of epilepsy. At 21 days of age, rats received an injection of lithium chloride and, 18-19 h later, they received an injection of pilocarpine hydrochloride for status epilepticus induction. The results reported herein show that seizures induced by lithium-pilocarpine elicit a significant increase in ATP hydrolysis and alkaline phosphatase activity, as well as a decrease in ADP hydrolysis and aspartate aminotransferase activity. The KD is a rigorous regimen that can be associated with hepatic damage, as shown herein by the elevated activities of liver enzymes and 5'-nucleotidase in blood serum. Further studies are necessary to investigate the mechanism of inhibition of lithium on nucleotidases in blood serum.

Pentylenetetrazol kindling alters adenine and guanine nucleotide catabolism in rat hippocampal slices and cerebrospinal fluid.

Pentylenetetrazol (PTZ) is commonly used as a convulsant drug. The enhanced seizure susceptibility induced by kindling is probably attributable to plastic changes in the synaptic efficacy. Adenosine and guanosine act both as important neuromodulators and neuroprotectors with mostly inhibitory effects on neuronal activity. Adenosine and guanosine can be released per se or generated from released nucleotides (ATP, ADP, AMP, GTP, GDP, and GMP) that are metabolized and rapidly converted to adenosine and guanosine. The aim of this study was to evaluate nucleotide hydrolysis by ecto- and soluble nucleotidases (hippocampal slices and CSF, respectively) after PTZ-kindling (stages 3, 4, or 5 seizures) or saline treatment in rats. Additionally, the levels of purines in rat cerebrospinal fluid (CSF), as well as ecto-NTPDases (1, 2, 3, 5, 6 and 8) and ecto- 5'-nucleotidase expression were determined. Ecto-enzyme assays demonstrated that ATP, AMP, GDP, and GMP hydrolysis enhanced when compared with controls. In addition, there was an increase of ADP, GDP, and GMP hydrolysis by soluble nucleotidases in PTZ-kindling rats compared to control group. The HPLC analysis showed a marked increase in PTZ-kindled CSF concentrations of GTP, ADP, and uric acid, but GDP, AMP, and hypoxanthine concentrations were decreased. Such alterations indicate that the modulatory role of purines in CNS could be affected by PTZ-kindling. However, the physiological significance of these findings remains to be elucidated.

Lipopolysaccharide alters nucleotidase activities from lymphocytes and serum of rats.

ATP exerts a proinflammatory role and induces cytokine release by acting at P2X(7) receptors. The product of ATP hydrolysis is the nucleoside adenosine, an important immunomodulator. The main source of extracellular adenosine is the hydrolysis of extracellular ATP by a group of ecto-enzymes: ENTPDase family, NPP family and ecto-5'-nucleotidase. Considering the role of ATP and adenosine in inflammatory processes, we investigated the effect of lipopolysaccharide on ectonucleotidases activities and expression in lymphocytes from mesenteric lymph nodes and serum of rats, in order to better understand the involvement of extracellular nucleotide hydrolysis in an endotoxemia model. We observed significant changes on nucleotidase activities from lymphocytes and serum of rats after in vitro and in vivo exposure to LPS. In vitro results have shown an increase on nucleotide hydrolysis in lymphocytes and a decrease on the enzyme activity of NPP in blood serum. In vivo, we observed an increase on nucleotide hydrolysis in lymphocytes and a decrease in the hydrolysis of all nucleotides tested in blood serum. After 24 and 48 h of LPS treatment, there was a reduction in NTPDase1, 2, 3 and ecto-5'-nucleotidase transcripts. These results suggest that there is a time-dependent enhancement of extracellular nucleotides metabolism in lymphocytes and blood serum after the induction of an endotoxemic model. The changes observed suggest that these enzymes can act in the regulation of extracellular nucleosides and nucleotides in a model able to trigger inflammatory process.

Ontogenetic profile of ectonucleotidase activities from brain synaptosomes of pilocarpine-treated rats.

Adenosine, a well-known neuromodulator, can act as an endogenous anticonvulsant via the activation of adenosine A1 receptors. This adenine nucleoside can be produced in the synaptic cleft by the ectonucleotidase cascade, which includes the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5'-nucleotidase. It has been previously reported that ectonucleotidase activities are increased in female adult rats submitted to the pilocarpine model of epilepsy. Several studies have suggested that the immature brain is less vulnerable to morphologic and physiologic alterations after status epilepticus (SE). Here, we evaluate the ectonucleotidase activities of synaptosomes from the hippocampus and cerebral cortex of male and female rats at different ages (7-9, 14-16 and 27-30-day old) submitted to the pilocarpine model of epilepsy. Our results show that ATP and ADP hydrolysis in the hippocampus and cerebral cortex were not altered by the pilocarpine treatment in female and male rats at 7-9, 14-16 and 27-30 days. There were no changes in AMP hydrolysis in female and male rats submitted to the model at different ages, but a significant increase in AMP hydrolysis (71%) was observed in synaptosomes from the cerebral cortex of male rats at 27-30 days. Pilocarpine-treated male rats (60-70-day old) presented an enhancement in ectonucleotidase activities in the synaptosomes of the cerebral cortex (33, 40 and 64% for ATP, ADP and AMP hydrolysis, respectively) and hippocampus (55, 98 and 101% for ATP, ADP and AMP hydrolysis, respectively). These findings highlight differences between the purinergic system of young and adult rats submitted to the pilocarpine model of epilepsy.

Unique Ca(2+)-activated ATPase in the nervous ganglia of Phyllocaulis soleiformis (Mollusca).

Nucleotide-metabolizing enzymes play important roles in the regulation of intracellular and extracellular nucleotide levels. We studied ATPase activity in the nervous ganglia of Phyllocaulis soleiformis, a terrestrial slug. The ATPase was divalent cation-dependent, with a maximal rate for ATP hydrolysis at pH 6.0 and 7.2 in the presence of Ca(2+) (5 mM). Mg(2+)-ATPase activity was only 26% of the activity observed in the presence of Ca(2+) (5 mM). ZnCl2 (10 mM) produced a significant inhibition of 70%. Ca(2+)-ATPase activity was insensitive to the classical ATPase inhibitors ouabain, N-ethylmaleimide, orthovanadate and sodium azide. Levamisole, an inhibitor of alkaline phosphatase, was ineffective. Among nucleotides, ATP was the best substrate. The apparent K(m) ((ATP)) for Ca(2+)-ATPase was 348+/-84 microM ATP and the V(max) was 829+/-114 nmol Pi min(-1) mg(-1) protein. The P. soleiformis ganglial ATPase does not appear to fit clearly into any of the previously described types of Ca(2+)-ATPases.