Nandrolone-induced aggressive behavior is associated with alterations in extracellular glutamate homeostasis in mice.

Nandrolone decanoate (ND), an anabolic androgenic steroid (AAS), induces an aggressive phenotype by mechanisms involving glutamate-induced N-methyl-d-aspartate receptor (NMDAr) hyperexcitability. The astrocytic glutamate transporters remove excessive glutamate surrounding the synapse. However, the impact of supraphysiological doses of ND on glutamate transporters activity remains elusive. We investigated whether ND-induced aggressive behavior is interconnected with GLT-1 activity, glutamate levels and abnormal NMDAr responses. Two-month-old untreated male mice (CF1, n=20) were tested for baseline aggressive behavior in the resident-intruder test. Another group of mice (n=188) was injected with ND (15mg/kg) or vehicle for 4, 11 and 19days (short-, mid- and long-term endpoints, respectively) and was evaluated in the resident-intruder test. Each endpoint was assessed for GLT-1 expression and glutamate uptake activity in the frontoparietal cortex and hippocampal tissues. Only the long-term ND endpoint significantly decreased the latency to first attack and increased the number of attacks, which was associated with decreased GLT-1 expression and glutamate uptake activity in both brain areas. These alterations may affect extracellular glutamate levels and receptor excitability. Resident males were assessed for hippocampal glutamate levels via microdialysis both prior to, and following, the introduction of intruders. Long-term ND mice displayed significant increases in the microdialysate glutamate levels only after exposure to intruders. A single intraperitoneal dose of the NMDAr antagonists, memantine or MK-801, shortly before the intruder test decreased aggressive behavior. In summary, long-term ND-induced aggressive behavior is associated with decreased extracellular glutamate clearance and NMDAr hyperexcitability, emphasizing the role of this receptor in mediating aggression mechanisms.

Prolonged ethanol exposure alters glutamate uptake leading to astrogliosis and neuroinflammation in adult zebrafish brain.

High ethanol (EtOH) consumption is a serious condition that induces tremors, alcoholic psychosis, and delirium, being considered a public health problem worldwide. Prolonged EtOH exposure promotes neurodegeneration, affecting several neurotransmitter systems and transduction signaling pathways. Glutamate is the major excitatory amino acid in the central nervous system (CNS) and the extracellular glutamatergic tonus is controlled by glutamate transporters mostly located in astrocytes. Here, we explore the effects of prolonged EtOH exposure on the glutamatergic uptake system and its relationship with astroglial markers (GFAP and S100B), neuroinflammation (IL-1ß and TNF-α), and brain derived neurotrophic factor (BDNF) levels in the CNS of adult zebrafish. Animals were exposed to 0.5% EtOH for 7, 14, and 28 days continuously. Glutamate uptake was significantly decreased after 7 and 14 days of EtOH exposure, returning to baseline levels after 28 days of exposure. No alterations were observed in crucial enzymatic activities linked to glutamate uptake, like Na,K-ATPase or glutamine synthetase. Prolonged EtOH exposure increased GFAP, S100B, and TNF-α levels after 14 days. Additionally, increased BDNF mRNA levels were observed after 14 and 28 days of EtOH exposure, while BDNF protein levels increased only after 28 days. Collectively, our data show markedly brain astroglial, neuroinflammatory and neurotrofic responses after an initial impairment of glutamate uptake following prolonged EtOH exposure. This neuroplasticity event could play a key role in the modulatory effect of EtOH on glutamate uptake after 28 days of continuous exposure.

Coriandrum sativum Extract Prevents Alarm Substance-Induced Fear- and Anxiety-Like Responses in Adult Zebrafish.

Anxiety disorders appear to involve distinct neurobiological mechanisms and several medications are available against this mental health problem. However, pharmacological therapeutic approaches display undesirable side effects for patients, particularly when long-term therapy is required. Some evidences have suggested that Coriandrum sativum extract (CSE) provide sedative and anxiolytic effects. We investigate if CSE could attenuate anxiety-like behaviors induced by novelty and alarm substance exposures in zebrafish. Adult zebrafish were injected with vehicle, clonazepam, or CSE (25, 50 or 100 mg/kg) and submitted to novel tank test. At the end, saline or alarm substance was added and anxiety-like responses were recorded. Twenty-four hours after, fish were submitted to the light/dark test. Novelty associated with alarm substance exposure decreased distance traveled and total time mobile in novel tank, and CSE (at 50 and 100 mg/kg) prevented these alterations similarly to clonazepam. Alarm substance reduced the time spent in white compartment (p = 0.0193 as compared with vehicle group). Clonazepam and CSE prevented this anxiogenic effect of alarm substance. CSE presents anxiolytic effects against alarm substance-induced locomotor and anxiogenic responses similarly to clonazepam. These data corroborate with the use of this plant in traditional medicine and provides a putative new pharmacological intervention for anxiety disorders.

Memantine mediates astrocytic activity in response to excitotoxicity induced by PP2A inhibition.

Reduced activity of protein phosphatase 2 A (PP2A) is a common feature in Alzheimer's disease (AD) and non-AD tauopathies. The administration of okadaic acid (OKA), a potent PP2A and PP1 inhibitor, is a common research tool for inducing AD-like alterations such as tau hyperphosphorylation and cognitive decline. Recently, we showed that OKA increases cerebrospinal fluid (CSF) glutamate levels, which was strongly correlated with cognitive decline. Also, we demonstrated that memantine (MN), a glutamatergic NMDAR channel blocker, was capable of preventing the increase in CSF glutamate levels and cognitive decline. Here, we aimed to analyze whether the protective effects of MN involve intrinsic astrocytic properties, particularly related to glutamate uptake and astrocytic reactivity - indexed by the expression of S100B and glial fibrillary acidic protein (GFAP). Rats received intraperitoneal injections of MN or saline over 3 consecutive days before receiving intrahippocampal infusion of OKA or saline. Afterward, they were submitted to behavioral tasks and then, euthanatized for neurochemical analysis. Here, we showed that the neuroprotective effects of MN in response to OKA neurotoxicity involve astrocytic activation. MN decreased glutamate uptake in the hippocampus and increased the release of S100B protein in the CSF in response to OKA neurotoxicity, which indicates a possible neurons-astrocyte coupling protective mechanism. These findings shed light on astrocytes as potential targets for treating neurological disorders associated with decreased PP2A activity.

Weekly ethanol exposure alters dopaminergic parameters in zebrafish brain.

Binge drinking is defined as the infrequent consumption of excessive doses of alcohol in a short period of time. Zebrafish is a reliable model to investigate ethanol consumption impact on the CNS, including reward signaling like dopaminergic neurotransmission system. The aim of this study was to evaluate zebrafish brain dopaminergic parameters after intermittent weekly ethanol exposure (WEE), which mimics binge drinking. Thus, adult zebrafish were exposed to ethanol (1.4% v/v) for 30 min, once a week for three consecutive weeks. The groups were divided according to different time points after the third exposure and name as follow: immediately (WEEI), two days (WEE-2), and nine days (WEE-9) after last exposure to ethanol. Brain dopaminergic function was assessed by the activity of the dopamine transporters (DAT); monoamine oxidase (MAO) activity; dopamine and noradrenaline levels by chromatography. The WEE-I and WEE-2 groups presented a significant increase in DAT activity. The MAO activity was decreased for WEE-2 and WEE-9 groups. The WEE-2 and WEE-9 groups presented an increase in brain dopamine levels, while noradrenaline levels were not affected. Therefore, dopaminergic parameters are still altered two and nine days after the last ethanol exposure in this binge experimental model, resulting in a modulatory event in this neurotransmission pathway.

Memantine decreases neuronal degeneration in young rats submitted to LiCl-pilocarpine-induced status epilepticus.

Several works have demonstrated that status epilepticus (SE) induced-neurodegeneration appears to involve an overactivation of N-methyl-d-aspartate receptors and treatment with high-affinity NMDAR antagonists is neuroprotective against this brain damage. However, these compounds display undesirable side effects for patients since they block physiological NMDA receptor dependent-activity. In this context, memantine (MN), a well tolerable low-affinity NMDAR channel blocker, will be a promising alternative, since it does not compromise the physiological role of NMDA receptors on synaptic transmission. The aim of the present study was to investigate if MN could attenuate seizure severity and neuronal cell death caused by SE induced early in life. Wistar rats (15 days old; n = 6-8 per group) received memantine (20 mg/kg i.p.) in six different treatments: 6 and 3 h before SE onset; concomitant with pilocarpine; 15min and 1h after SE onset; and four consecutive administrations (15 min, 6 h, 12 h, and 18 h) after pilocarpine injection. Neurodegeneration was quantified by fluoro-jade C staining. Treatment with memantine increase latency to SE onset only in groups treated 3 h before or concomitant with pilocarpine. In CA1 hippocampal subfield, memantine significantly reduced neurodegeneration at the following times: 3 h prior SE-onset, concomitant with pilocarpine, and 15 min after pilocarpine injection. For amygdala and thalamus, all post-SE onset treatments were able to decrease neurodegeneration. In conclusion, the present study showed that MN was neuroprotective against SE-induced neuronal death and this neuroprotection appears to be time- and region-dependent.

Forebrain glutamate uptake and behavioral parameters are altered in adult zebrafish after the induction of Status Epilepticus by kainic acid.

The development of new antiepileptic drugs is a high-risk/high-cost research field, which is made even riskier if the behavioral epileptic seizure profile is the unique approach on which the development is based. In order to increase the effectiveness of the screening conducted in the zebrafish model of status epilepticus (SE), the evaluation of neurochemical markers of SE would be of great relevance. Epilepsy is associated with changes in the glutamatergic system, and glutamate uptake is one of the critical parameters of this process. Therefore, we evaluated the levels of glutamate uptake in the zebrafish brain and analyzed its correlation with the progression of behavioral changes in zebrafish at different times after the administration of kainic acid (5 mg/kg). The results showed that the zebrafish suffered with lethargy while swimming for up to 72 h after SE, had reduced levels of GFAP cells 12 h after SE, reduced levels of S100B up to 72 h after SE, and reduced levels of glutamate uptake in the forebrain between 3 h and 12 h after SE. The forebrain region of adult zebrafish after SE present similar changes to the neurochemical limbic alterations that are seen in rodent models of SE. This study demonstrated that there is a time window in which to use the KA zebrafish model of SE to explore some of the known neurochemical alterations that have been observed in rodent models of epilepsy and epileptic human patients.

Embryonic alcohol exposure promotes long-term effects on cerebral glutamate transport of adult zebrafish.

Ethanol is a widely consumed substance throughout the world. During development it can substantially damage the human fetus, whereas the developing brain is particularly vulnerable. The brain damage induced by prenatal alcohol exposure may lead to a variety of long-lasting behavioral and neurochemical problems. However, there are no data concerning the effects of developmental ethanol exposure on the glutamatergic system, where extracellular glutamate acts as signaling molecule. Here we investigated the effect of ethanol exposure for 2h (concentrations of 0.0%, 0.1%, 0.25%, 0.50%, and 1.00%) in embryos at 24h post-fertilization (hpf) by measuring the functionality of glutamate transporters in the brain of adult (4 months) zebrafish. However, ethanol 0.1%, 0.25% and 0.50% decreased transport of glutamate to 81.96%, 60.65% and 45.91% respectively, when compared with the control group. Interestingly, 1.00% was able to inhibit the transport activity to 68.85%. In response to the embryonic alcohol exposure, we found impairment in the function of cerebral glutamate transport in adult fish, contributing to long-term alteration in the homeostasis glutamatergic signaling.

A new device for step-down inhibitory avoidance task--effects of low and high frequency in a novel device for passive inhibitory avoidance task that avoids bioimpedance variations.

BACKGROUND: Step-down inhibitory avoidance task has been widely used to evaluate aversive memory, but crucial parameters inherent to traditional devices that may influence the behavior analysis (as stimulus frequency, animal's bioimpedance) are frequently neglected. NEW METHOD: We developed a new device for step-down inhibitory avoidance task by modifying the shape and distribution of the stainless steel bars in the box floor where the stimuli are applied. The bars are 2 mm wide, with rectangular shape, arranged in pairs at intervals of 1cm from the next pairs. Each pair makes an electrical dipole where the polarity inverts after each pulse. This device also presents a component that acquires and records the exact current received by the animal foot and precisely controls the frequency of stimulus applied during the entire experiment. RESULT: Different from conventional devices, this new apparatus increases the contact surface with bars and animal's paws, allowing the electric current pass through the animal's paws only, drastically reducing the influence of animal's bioimpedance. The analysis of recorded data showed that the current received by the animal was practically the same as applied, independent of the animal's body composition. Importantly, the aversive memory was observed at specific stimuli intensity and frequency (0.35 or 0.5 mA at 62 and 125 Hz but not at 0.20 mA or 20 Hz). Moreover, with this device it was possible to observe the well-known step-down inhibitory avoidance task memory impairment induced by guanosine. CONCLUSION: This new device offers a substantial improvement for behavioral analysis in step-down inhibitory avoidance task and allows us to precisely compare data from different animals with distinct body composition.

Effects of ethanol and acetaldehyde in zebrafish brain structures: an in vitro approach on glutamate uptake and on toxicity-related parameters.

Ethanol (EtOH) and its metabolite, acetaldehyde (ALD), induce deleterious effects on central nervous system (CNS). Here we investigate the in vitro toxicity of EtOH and ALD (concentrations of 0.25%, 0.5%, and 1%) in zebrafish brain structures [telencephalon (TE), opticum tectum (OT), and cerebellum (CE)] by measuring the functionality of glutamate transporters, MTT reduction, and extracellular LDH activity. Both molecules decreased the activity of the Na(+)-dependent glutamate transporters in all brain structures. The strongest glutamate uptake inhibition after EtOH exposure was 58% (TE-1%), and after ALD, 91% (CE-1%). The results of MTT assay and LDH released demonstrated that the actions of EtOH and its metabolite are concentration and structure-dependent, in which ALD was more toxic than EtOH. In summary, our findings demonstrate a differential toxicity in vitro of EtOH and ALD in zebrafish brain structures, which can involve changes on glutamatergic parameters. We suggest that this species may be an interesting model for assessing the toxicological actions of alcohol and its metabolite in CNS.