Anti-inflammatory effect of rosmarinic acid isolated from Blechnum brasiliense in adult zebrafish brain.

Neuroinflammation has been associated to neurodegenerative disease development, with evidence suggesting that high levels of proinflammatory cytokines promote neuronal dysfunction and death. Therefore, it is necessary to study new compounds that may be used as adjuvant treatments of neurodegenerative diseases by attenuating the inflammatory response in the central nervous system (CNS). The aim of this study was to utilize the lipopolysaccharide (LPS) induction model of neuroinflammation to evaluate the modulation of inflammation by rosmarinic acid (RA) isolated from Blechnum brasiliense in adult zebrafish. First, we investigated the toxicity and antioxidant properties of fractionated B. brasiliense extract (ethyl acetate fraction- EAF) and the isolated RA in zebrafish embryos. Next, we developed a model of neuroinflammation induction by intraperitoneal (i.p.) injection of LPS to observe the RA modulation of proinflammatory cytokines. The median lethal concentration (LC50) calculated was 185.2 ± 1.24 µg/mL for the ethyl acetate fraction (EAF) and 296.0 ± 1.27 µM for RA. The EAF showed free radical inhibition ranging from 23.09% to 63.44% at concentrations of 10-250 µg/mL. The RA presented a concentration-dependent response ranging from 18.24% to 47.63% at 10-250 µM. Furthermore, the RA reduced LPS induction of TNF-α and IL-1ß levels, with the greatest effect observed 6 h after LPS administration. Thus, the data suggested an anti-inflammatory effect of RA isolated from B. brasiliense and reinforced the utility of the new model of neuroinflammation to test the possible neuroprotective effects of novel drugs or compounds.

Acetyl-L-carnitine as a putative candidate for the treatment of stress-related psychiatric disorders: Novel evidence from a zebrafish model.

Stress-related psychiatric disorders are mental conditions that affect mood, cognition and behavior and arise because of the impact of prolonged stress on the central nervous system (CNS). Acetyl-L-carnitine (ALC) is an acetyl ester of L-carnitine that easily crosses the blood-brain barrier and was recently found to be decreased in patients with major depressive disorder. ALC plays a role in energy metabolism and is widely consumed as a nutritional supplement to improve physical performance. In this study, our objective was to evaluate the effects of ALC treatment (0.1 mg/L, 10 min) for 7 days on behavior and oxidative stress in zebrafish subjected to unpredictable chronic stress (UCS) protocol. Behavioral outcomes were assessed in the novel tank test, and parameters of oxidative status (lipid peroxidation and antioxidant defenses) were evaluated in the brain using colorimetric methods. According to our previous findings, UCS increased anxiety-like behavior and lipid peroxidation, while it decreased non-protein thiol levels and superoxide dismutase activity. However, ALC reversed the anxiety-like behavior and oxidative damage in stressed animals, while it was devoid of effect in control animals. Although our data reinforce the neuroprotective potential of ALC in the treatment of psychiatric disorders related to stress, further investigations are required to clarify its mechanisms of action and confirm its efficacy.

Teratogenic and anticonvulsant effects of zinc and copper valproate complexes in zebrafish.

Valproic acid (VPA) is an antiepileptic drug (AED) that has the broadest spectrum across all types of seizures and epileptic syndromes. Unfortunately, approximately 30% of epileptic patients are refractory to the classical AED. Metal ions have been frequently incorporated into pharmaceuticals for therapeutic or diagnostic purposes and research. In this preliminary study, we assess the embryo toxicity and the anticonvulsant activity of 4 novel metallodrugs, with Zn+2 and Cu+2, a derivative of valproic acid and the N-donor ligand in an adult zebrafish epileptic seizure model induced by pentylenetetrazole. The most toxic complex was [Cu(Valp)2Bipy], in which the LC50 was 0.22 µM at 48 h post fertilization (HPF) and 0.12 µM at 96 HPF, followed by [Zn(Valp)2Bipy] (LC50 = 10 µM). These same metallodrugs ([Cu(Valp)2Bipy] 10 mM/kg and [Zn(Valp)2Bipy] 30 mM and 100 mM/kg) displayed superior activity, thus reducing the seizure intensity by approximately 20 times compared to sodium valproate (175 mM/kg). Overall, [Cu(Valp)2Bipy] showed the best anticonvulsant effects. However, because of the toxicity of copper, [Zn(Valp)2Bipy] is considered the most promising anticonvulsant for future studies.

Environmental enrichment modulates the response to chronic stress in zebrafish.

Several studies have shown that manipulations to the housing environment modulate susceptibility to stress in laboratory animals, mainly in rodents. Environmental enrichment (EE) is one such manipulation that promotes neuroprotection and neurogenesis, besides affecting behaviors such as drug self-administration. Zebrafish are a popular and useful animal model for behavioral neuroscience studies; however, studies evaluating the impact of housing conditions in this species are scarce. In this study, we verified the effects of EE on behavioral (novel tank test) and biochemical [cortisol and reactive oxygen species (ROS)] parameters in zebrafish submitted to unpredictable chronic stress (UCS). Consistent with our previous findings, UCS increased anxiety-like behavior, cortisol and ROS levels in zebrafish. EE for 21 or 28 days attenuated the effects induced by UCS on behavior and cortisol, and prevented the effects on ROS levels. Our findings reinforce the idea that EE exerts neuromodulatory effects across species, reducing vulnerability to stress and its biochemical impact. Also, these results indicate that zebrafish is a suitable model animal to study the behavioral effects and neurobiological mechanisms related to EE.

Toxicological profile and acetylcholinesterase inhibitory potential of Palicourea deflexa, a source of ß-carboline alkaloids.

Palicourea genus is chemically and taxonomically close to Psychotria genus, a well-known source of neuroactive alkaloids. It has been previously reported the pharmacological potential of these alkaloids in some targets related to the neurodegenerative process. In this context, this study was carried out in order to evaluate the toxic effects and acetylcholinesterase (AChE) inhibitory potential of Palicourea deflexa fraction of total alkaloids (FTA). P. deflexa FTA was analyzed by means of HPLC-DAD and HRMS-ESI. We performed toxicological screening through Fish Embryo Toxicity (FET) test using zebrafish embryo and abnormal developmental phenotypes were recorded daily. For AChE inhibition, zebrafish brains were used as enzymatic source and formation of thiolate dianion of 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) was used to monitor acetylthiocholine hydrolysis. Lineaweaver-Burk double reciprocal plots were used to indicate mode of inhibition. Chemical analysis of the P. deflexa FTA allowed the identification of the main compound as harman-3-carboxylic acid. This fraction was evaluated in vivo for its toxicological effect. The zebrafish embryo test indicated that the FTA has a lethal concentration of 50% (LC50)=72.18µg/mL. Further, the FTA was evaluated for its AChE inhibitory profile, demonstrating an inhibitory concentration of 50% (IC50) of 50.65µg/mL. Lineaweaver-Burk double reciprocal plots indicated a mixed mode of inhibition. It is reported for the first time the toxicological and pharmacological profile of the alkaloid fraction of Palicourea deflexa in zebrafish models.

Chronic Treatment with a Clinically Relevant Dose of Methylphenidate Increases Glutamate Levels in Cerebrospinal Fluid and Impairs Glutamatergic Homeostasis in Prefrontal Cortex of Juvenile Rats.

The understanding of the consequences of chronic treatment with methylphenidate is very important since this psychostimulant is extensively prescribed to preschool age children, and little is known about the mechanisms underlying the persistent changes in behavior and neuronal function related with the use of methylphenidate. In this study, we initially investigate the effect of early chronic treatment with methylphenidate on amino acids profile in cerebrospinal fluid and prefrontal cortex of juvenile rats, as well as on glutamatergic homeostasis, Na(+),K(+)-ATPase function, and balance redox in prefrontal cortex of rats. Wistar rats at early age received intraperitoneal injections of methylphenidate (2.0 mg/kg) or an equivalent volume of 0.9% saline solution (controls), once a day, from the 15th to the 45th day of age. Twenty-four hours after the last injection, the animals were decapitated and the cerebrospinal fluid and prefrontal cortex were obtained. Results showed that methylphenidate altered amino acid profile in cerebrospinal fluid, increasing the levels of glutamate. Glutamate uptake was decreased by methylphenidate administration, but GLAST and GLT-1 were not altered by this treatment. In addition, the astrocyte marker GFAP was not altered by MPH. The activity and immunocontent of catalytic subunits (α1, α2, and α3) of Na(+),K(+)-ATPase were decreased in prefrontal cortex of rats subjected to methylphenidate treatment, as well as changes in α1 and α2 gene expression of catalytic α subunits of Na(+),K(+)-ATPase were also observed. CAT activity was increased and SOD/CAT ratio and sulfhydryl content were decreased in rat prefrontal cortex. Taken together, our results suggest that chronic treatment with methylphenidate at early age induces excitotoxicity, at least in part, due to inhibition of glutamate uptake probably caused by disturbances in the Na(+),K(+)-ATPase function and/or in protein damage observed in the prefrontal cortex.

Anticonvulsant properties of Euterpe oleracea in mice.

Açai (Euterpe oleracea Mart.), a highly consumed fruit in Amazon, is from a common palm with remarkable antioxidant properties. Because oxidative stress and seizures are intimately linked, this study investigated the potential neuroprotective and anticonvulsant effects of commercial clarified açai juice (EO). EO did not alter spontaneous locomotor activity. Four doses of EO were sufficient to increase latencies to both first myoclonic jerk and first generalized tonic-clonic seizure and significantly decrease the total duration of tonic-clonic seizures caused by pentylenetetrazol administration. Also, electrocortical alterations provoked by pentylenetetrazol were prevented, significantly decreasing amplitude of discharges and frequencies above 50 Hz. EO was also able to completely prevent lipid peroxidation in the cerebral cortex, showing a potent direct scavenging property. These results demonstrate for the first time that E. oleracea significantly protects against seizures and seizure-related oxidative stress, indicating an additional protection for humans who consume this fruit.

Evaluation of zinc effect on cadmium action in lipid peroxidation and metallothionein levels in the brain.

Cadmium (Cd) is a known hepato- and nephrotoxic pollutant and zinc (Zn) metalloproteins are important targets of Cd. Hence, the administration of Zn may mitigate Cd toxic effects. However, the interaction of Cd and Zn has been little investigated in the brain. Previously, we reported a protective effect of Zn on mortality caused by Cd in rats. Here, we tested whether the protective effect of Zn could be related to changes in brain Zn-proteins, metallothionein (MT) and δ-aminolevulinate dehydratse (δ-ALA-D). Male adult rats were daily administered for 10 days with Zn (2 mg kg-1), Cd (0.25 and 1 mg kg-1) and 0.25 mg kg-1 of Cd plus Zn and 1 mg kg-1 of Cd plus Zn. The body weight loss, food intake deprivation, and mortality occurred in 1 mg kg-1 of Cd, but Zn co-administration did mitigate these effects. The brain Zn content was not modified by treatment with Cd, whereas cerebral Cd levels increased in animals exposed to Cd. The administration of 0.25 mg kg-1 of Cd (with or without Zn) induced lipid peroxidation and decreased MT concentration, but 2 mg kg-1 of Zn and 1 mg kg-1 of Cd did not change these parameters. Brain δ-ALA-D was not modified by Cd and/or Zn treatments. Since the co-administration of Zn did not attenuate the changes induced by Cd in the brain, our results suggest that the protective effect of Zn on impairments caused by Cd in animal status is weakly related to a cerebral interaction of these metals.

Anxiolytic effects of diphenyl diselenide on adult zebrafish in a novelty paradigm.

Anxiety-related disorders are frequently observed in the population. Because the available pharmacotherapies for anxiety can cause side effects, new anxiolytic compounds have been screened using behavioral tasks. For example, diphenyl diselenide (PhSe)2, a simple organoselenium compound with neuroprotective effects, has demonstrated anxiolytic effects in rodents. However, this compound has not yet been tested in a novelty-based paradigm in non-mammalian animal models. In this study, we assessed the potential anxiolytic effects of (PhSe)2 on the behavior of adult zebrafish under novelty-induced stress. The animals were pretreated with 0.1, 0.25, 0.5, and 1µM (PhSe)2 in the aquarium water for 30min. The fish were then exposed to a novel tank, and their behavior was quantified during a 6-min trial. (PhSe)2 treatment altered fish behavior in a concentration-dependent manner. At 0.01 and 0.25µM, (PhSe)2 did not elicit effects on fish behavior. At 0.5µM, moderate behavioral side effects (e.g., lethargy and short episodic immobility) were noted. At the highest concentration tested (1µM), dramatic side effects were observed, such as burst behavior and longer periods of immobility. The results were confirmed by spatiotemporal analysis of each group. Occupancy plot data showed dispersed homebase formation in the 0.25µM (PhSe)2-treated group compared with the control group (treated with 0.04% DMSO). Furthermore, animals treated with 0.25µM (PhSe)2 showed a reduction in latency to enter the top and spent more time in the upper area of the tank. These data suggest that (PhSe)2 may induce an anxiolytic-like effect in situations of anxiety evoked by novelty.

Topographical analysis of reactive zinc in the central nervous system of adult zebrafish (Danio rerio).

Reactive zinc (Zn) is crucial for neuronal signaling and is largely distributed within presynaptic vesicles of some axon terminals of distinct vertebrates. However, the distribution of reactive Zn throughout the central nervous system (CNS) is not fully explored. We performed a topographical study of CNS structures containing reactive Zn in the adult zebrafish (Danio rerio). Slices of CNS from zebrafish were stained by Neo-Timm and/or cresyl violet. The Zn specificity of Neo-Timm was evaluated with Zn chelants, N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), sodium diethyldithiocarbamate (DEDTC), Zn sulfide washing solution, and hydrochloric acid (HCl). Unfixed slices were also immersed in the fluorescent Zn probe (zinpyr-1). Yellow-to-brown-to-black granules were revealed by Neo-Timm in the zebrafish CNS. Telencephalon exhibited slightly stained regions, while rhombencephalic structures showed high levels of staining. Although stained granules were found on the cell bodies, rhombencephalic structures showed a neuropil staining profile. The TPEN produced a mild reduction in Neo-Timm staining, while HCl and mainly DEDTC abolished the staining, indicating a large Zn content. This result was also confirmed by the application of a Zn probe. The present topographical study revealed reactive Zn throughout the CNS in adult zebrafish that should be considered in future investigation of Zn in the brain on a larger scale.

Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization.

Pentylenetetrazole (PTZ) is a common convulsant agent used in animal models to investigate the mechanisms of seizures. Although adult zebrafish have been recently used to study epileptic seizures, a thorough characterization of the PTZ-induced seizures in this animal model is missing. The goal of this study was to perform a detailed temporal behavior profile characterization of PTZ-induced seizure in adult zebrafish. The behavioral profile during 20 min of PTZ immersion (5, 7.5, 10, and 15 mM) was characterized by stages defined as scores: (0) short swim, (1) increased swimming activity and high frequency of opercular movement, (2) erratic movements, (3) circular movements, (4) clonic seizure-like behavior, (5) fall to the bottom of the tank and tonic seizure-like behavior, (6) death. Animals exposed to distinct PTZ concentrations presented different seizure profiles, intensities and latencies to reach all scores. Only animals immersed into 15 mM PTZ showed an increased time to return to the normal behavior (score 0), after exposure. Total mortality rate at 10 and 15 mM were 33% and 50%, respectively. Considering all behavioral parameters, 5, 7.5, 10, and 15 mM PTZ, induced seizures with low, intermediate, and high severity, respectively. Pretreatment with diazepam (DZP) significantly attenuated seizure severity. Finally, the brain PTZ levels in adult zebrafish immersed into the chemoconvulsant solution at 5 and 10 mM were comparable to those described for the rodent model, with a peak after a 20-min of exposure. The PTZ brain levels observed after 2.5-min PTZ exposure and after 60-min removal from exposure were similar. Altogether, our results showed a detailed temporal behavioral characterization of a PTZ epileptic seizure model in adult zebrafish. These behavioral analyses and the simple method for PTZ quantification could be considered as important tools for future investigations and translational research.

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.

Impairment of the organization of locomotor and exploratory behaviors in bile duct-ligated rats.

Hepatic encephalopathy (HE) arises from acute or chronic liver diseases and leads to several problems, including motor impairment. Animal models of chronic liver disease have extensively investigated the mechanisms of this disease. Impairment of locomotor activity has been described in different rat models. However, these studies are controversial and the majority has primarily analyzed activity parameters. Therefore, the aim of the present study was to evaluate locomotor and exploratory behavior in bile duct-ligated (BDL) rats to explore the spatial and temporal structure of behavior. Adult female Wistar rats underwent common bile duct ligation (BDL rats) or the manipulation of common bile duct without ligation (control rats). Six weeks after surgery, control and BDL rats underwent open-field, plus-maze and foot-fault behavioral tasks. The BDL rats developed chronic liver failure and exhibited a decrease in total distance traveled, increased total immobility time, smaller number of rearings, longer periods in the home base area and decreased percentage of time in the center zone of the arena, when compared to the control rats. Moreover, the performance of the BDL rats was not different from the control rats for the elevated plus-maze and foot-fault tasks. Therefore, the BDL rats demonstrated disturbed spontaneous locomotor and exploratory activities as a consequence of altered spatio-temporal organization of behavior.

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.

Hyperhomocysteinemia reduces glutamate uptake in parietal cortex of rats.

In the present study we evaluated the effect of acute and chronic homocysteine administrations on glutamate uptake in parietal cortex of rats. The immunocontent of glial glutamate transporter (GLAST) and sodium-dependent glutamate/aspartate transporter (GLT-1) in the same cerebral structure was also investigated. For acute treatment, neonate or young rats received a single injection of homocysteine or saline (control) and were sacrificed 1, 8, 12 h, 7 or 30 days later. For chronic treatment, homocysteine was administered to rats twice a day at 8 h interval from their 6th to their 28th days old; controls and treated rats were sacrificed 12 h, 1, 7 or 30 days after the last injection. Results show that acute hyperhomocysteinemia caused a reduction on glutamate uptake in parietal cortex of neonate and young rats, and that 12h after homocysteine administration the glutamate uptake returned to normal levels in young rats, but not in neonate. Chronic hyperhomocysteinemia reduced glutamate uptake, and GLAST and GLT-1 immunocontent. According to our results, it seems reasonable to postulate that the reduction on glutamate uptake in cerebral cortex of rats caused by homocysteine may be mediated by the reduction of GLAST and GLT-1 immunocontent, leading to increased extracellular glutamate concentrations, promoting excitotoxicity.

Gestational and postnatal malnutrition affects sensitivity of young rats to picrotoxin and quinolinic acid and uptake of GABA by cortical and hippocampal slices.

It is widely known that a complex interaction between excitatory and inhibitory systems is required to support the adequate functioning of the brain and that significant alterations induced by early protein restriction are complex, involving many systems. Based on such assumptions, we investigated the effects of maternal protein restriction during pregnancy and lactation followed by offspring protein restriction on some GABAergic and glutamatergic parameters, which mediate inhibitory and excitatory transmission, respectively. The sensitivity of young malnourished rats to convulsant actions of the GABA(A) receptor antagonist picrotoxin (PCT; s.c.) and to N-methyl-d-aspartate (NMDA) receptor agonist quinolinic acid (QA; i.c.v) and also gamma-amino-n-butyric acid (GABA) and glutamate uptake by cortical and hippocampal slices were evaluated in P25 old rats. Early protein malnutrition induced higher sensitivity to picrotoxin, which could be associated with the observed higher GABA uptake by cortical, and hippocampal slices in malnourished rats. In contrast, we observed lower sensitivity to quinolinic acid in spite of unaltered glutamate uptake by the same cerebral structures. Picrotoxin enhanced GABA uptake in hippocampus in well- and malnourished rats; however, it did not affect cortical GABA uptake. Our data corroborate our previous report, showing that malnutrition depresses the glutamatergic activity, and point to altered modulation of GABAergic neurotransmission. Such findings allow us to speculate that malnutrition may affect the excitatory and inhibitory interaction.