Bruno Lobão-Soares

The elevated T-maze task as an animal model to simultaneously investigate the effects of drugs on long-term memory and anxiety in mice.

The elevated T-maze (ETM) is an apparatus derived from the elevated plus-maze test, which is used to evaluate anxiety. Because anxiety is a biasing factor in models of memory, this study proposed the ETM as a task for the simultaneous assessment of memory and anxiety in mice. The ETM consists of one enclosed and two open arms. The procedure is based on the avoidance of open spaces learned during training session, in which mice were exposed to the enclosed arm as many times as needed to stay 300s. In the test session, memory is assessed by re-exposing the mouse to the enclosed arm and the latency to enter an open arm was recorded. The anxiolytic diazepam (DZP; 1 or 2mg/kg) and the amnestic biperiden (BPR; 0.5, 1 or 3mg/kg) were injected at three distinct times: pre-training, post-training, and pre-test. Pre-training administration of BPR 1 and DZP 2 increased the number of trials needed to reach the avoidance criterion, suggesting a passive avoidance learning impairment. However, BPR induced hyperlocomotion, which could bias the interpretation of any BPR-induced effects during the training session. Pre-training injection of BPR did not affect the spontaneous increase in the latency to enter an open arm between trials, while DZP reduced latencies in the first three trials suggesting anxiolysis. In the test session, pre-training injection of BPR 1 and DZP 2 reduced latencies to enter an open arm, indicating memory impairment. Post-training and pre-test injection of DZP or BPR did not affect memory. In conclusion, the proposed ETM task is practical for the detection of the anxiolytic and amnesic effects of drugs.

Nitroprusside single-dose prevents the psychosis-like behavior induced by ketamine in rats for up to one week

Recently, we found a rapid and long-lasting improvement of symptoms in schizophrenic patients on antipsychotics after a single four-hour infusion of sodium nitroprusside (SNP), a nitric oxide (NO) donor with a short half-life. This improvement persisted for up to 4weeks. Because these patients remained on antipsychotics after infusion of SNP was finished, the question arises about whether this improvement was due to SNP itself. We have now investigated whether SNP, alone, can produce preventive antipsychotic effects in rats treated with ketamine (KET). 56 adult rats divided into 7 groups were infused with SNP 4mg/kg, KET 25mg/kg, or saline as follows: group1 - saline, group2 - SNP, group3 - KET, group4 - KET 12h after SNP, group5 - KET 1day after SNP, group6 - KET 2days after SNP, and group7 - KET 1week after SNP. The animals were filmed in an open field arena for 30min and the videos were later analyzed by ANY-Maze software to measure activity and stereotypy. SNP significantly prevented the emergence of hyperactivity induced by KET when it was administered for up to 1week before KET, and prevented the emergence of stereotypies when it was administered for up to 1day before KET. These findings in rats, which have an even faster metabolic rate than humans, suggest that the long-lasting effects observed in our clinical trial with SNP in humans could have been due to SNP itself, and indicate for the first time that SNP may present preventive antipsychotic effects.

Glucose-dependent insulinotropic peptide receptor expression in the hippocampus and neocortex of mesial temporal lobe epilepsy patients and rats undergoing pilocarpine induced status epilepticus.

The glucose-dependent insulinotropic peptide receptor (GIPR) has been implicated with neuroplasticity and may be related to epilepsy. GIPR expression was analyzed by immunohistochemistry in the hippocampus (HIP) and neocortex (Cx) of rats undergoing pilocarpine induced status epilepticus (Pilo-SE), and in three young male patients with left mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS) treated surgically. A combined GIPR immunohistochemistry and Fluoro-Jade staining was carried out to investigate the association between the GIPR expression and neuronal degeneration induced by Pilo-SE. GIPR was expressed in the cytoplasm of neurons from the HIP CA subfields, dentate gyrus (DG) and Cx of animals and human samples. The GIPR expression after the Pilo-SE induction increases significantly in the HIP after 1h and 5 days, but not after 12h or 50 days. In the Cx, the GIPR expression increases after 1h, 12h and 5 days, but not 50 days after the Pilo-SE. The expression of GIPR 12h after Pilo-SE was inversely proportional to the Fluoro-Jade staining intensity. In the human tissue, GIPR expression patterns were similar to those observed in chronic Pilo-SE animals. No Fluoro-Jade stained cells were observed in the human sample. GIPR is expressed in human HIP and Cx. There was a time and region dependent increase of GIPR expression in the HIP and Cx after Pilo-SE that was inversely associated to neuronal degeneration.

Beta2 oscillations (23-30 Hz) in the mouse hippocampus during novel object recognition.

The oscillatory activity of hippocampal neuronal networks is believed to play a role in memory acquisition and consolidation. Particular focus has been given to characterising theta (4–12 Hz), gamma (40–100 Hz) and ripple (150–250 Hz) oscillations. Beyond these well‐described network states, few studies have investigated hippocampal beta2 (23–30 Hz) activity in vivo and its link to behaviour. A previous sudy showed that the exploration of novel environments may lead to the appearance of beta2 oscillations in the mouse hippocampus. In the present study we characterised hippocampal beta2 oscillations in mice during an object recognition task. We found prominent bursts of beta2 oscillations in the beginning of novel exploration sessions (four new objects), which could be readily observed by spectral analysis and visual inspection of local field potentials. Beta2 modulated hippocampal but not neocortical neurons and its power decreased along the session. We also found increased beta2 power in the beginning of a second exploration session performed 24 h later in a slightly modified environment (two new, two familiar objects), but to a lesser extent than in the first session. However, the increase in beta2 power in the second exploration session became similar to the first session when we pharmacologically impaired object recognition in a new set of experiments performed 1 week later. Our results suggest that hippocampal beta2 activity is associated with a dynamic network state tuned for novelty detection and which may allow new learning to occur.

D2 dopamine receptor regulation of learning, sleep and plasticity.

Dopamine and sleep have been independently linked with hippocampus-dependent learning. Since D2 dopaminergic transmission is required for the occurrence of rapid-eye-movement (REM) sleep, it is possible that dopamine affects learning by way of changes in post-acquisition REM sleep. To investigate this hypothesis, we first assessed whether D2 dopaminergic modulation in mice affects novel object preference, a hippocampus-dependent task. Animals trained in the dark period, when sleep is reduced, did not improve significantly in performance when tested 24h after training. In contrast, animals trained in the sleep-rich light period showed significant learning after 24h. When injected with the D2 inverse agonist haloperidol immediately after the exploration of novel objects, animals trained in the light period showed reduced novelty preference upon retesting 24h later. Next we investigated whether haloperidol affected the protein levels of plasticity factors shown to be up-regulated in an experience-dependent manner during REM sleep. Haloperidol decreased post-exploration hippocampal protein levels at 3h, 6h and 12h for phosphorylated Ca(2+)/calmodulin-dependent protein kinase II, at 6h for Zif-268; and at 12h for the brain-derived neurotrophic factor. Electrophysiological and kinematic recordings showed a significant decrease in the amount of REM sleep following haloperidol injection, while slow-wave sleep remained unaltered. Importantly, REM sleep decrease across animals was strongly correlated with deficits in novelty preference (Rho=0.56, p=0.012). Altogether, the results suggest that the dopaminergic regulation of REM sleep affects learning by modulating post-training levels of calcium-dependent plasticity factors.

Sodium nitroprusside, a nitric oxide donor for novel treatment of schizophrenia, may also modulate dopaminergic systems

“Schizophrenia is arguably the worst disease affecting mankind, even AIDS not excepted”. Since this statement in 1988 (Editors, 1988), schizophrenia still remains a major challenge to medicine, with up to 60% of patients not responding adequately to treatment despite the relatively large arsenal of antipsychotics currently available. By modulating the nitric oxide (NO) pathway, a new paradigm for schizophrenia treatment apparently involving modulation of nitric oxide (NO) has been proposed (Oliveira et al., 2011).

Lithium and valproate prevent methylphenidate-induced mania-like behaviors in the hole board test

Manic bipolar is diagnosed by psychomotor agitation, increased goal-directed activity, insomnia, grandiosity, excessive speech, and risky behavior. Animal studies aimed to modeling mania are commonly based in psychostimulants-induced hyperlocomotion. The exploration of other behaviors related with mania is mandatory to investigate this phase of bipolar disorder in animals. In this study, the hole board apparatus was suggested for evaluating mania-like behaviors induced by the psychostimulant methylphenidate. The treatment with methylphenidate (10mg/kg, ip) increased locomotion in the open field test. The pretreatment with lithium (50mg/kg, ip) and valproate (400mg/kg, ip) significantly prevented the hyperlocomotion. In the hole-board test, methylphenidate increased interactions with the central and peripheral holes and the exploration of central areas. Lithium was more effective than valproate in preventing all the behavioral manifestations induced by the psychostimulant. These findings were discussed based on the ability of methylphenidate-treated mice mimicking two symptoms of mania in the hole board test: goal-directed action and risk-taking behavior. In conclusion, the results point to a new approach to study mania through the hole board apparatus. The hole board test appears to be a sensitive assay to detect the efficacy of antimanic drugs.

Object recognition impairment and rescue by a dopamine D2 antagonist in hyperdopaminergic mice

Genetically-modified mice without the dopamine transporter (DAT) are hyperdopaminergic, and serve as models for studies of addiction, mania and hyperactive disorders. Here we investigated the capacity for object recognition in mildly hyperdopaminergic mice heterozygous for DAT (DAT +/-), with synaptic dopaminergic levels situated between those shown by DAT -/- homozygous and wild-type (WT) mice. We used a classical dopamine D2 antagonist, haloperidol, to modulate the levels of dopaminergic transmission in a dose-dependent manner, before or after exploring novel objects. In comparison with WT mice, DAT +/- mice showed a deficit in object recognition upon subsequent testing 24h later. This deficit was compensated by a single 0.05mg/kg haloperidol injection 30min before training. In all mice, a 0.3mg/kg haloperidol injected immediately after training impaired object recognition. The results indicate that a mild enhancement of dopaminergic levels can be detrimental to object recognition, and that this deficit can be rescued by a low dose of a D2 dopamine receptor antagonist. This suggests that novel object recognition is optimal at intermediate levels of D2 receptor activity.

Predictability of arousal in mouse slow wave sleep by accelerometer data

Arousals can be roughly characterized by punctual intrusions of wakefulness into sleep. In a standard perspective, using human electroencephalography (EEG) data, arousals are associated to slow-wave rhythms and K-complex brain activity. The physiological mechanisms that give rise to arousals during sleep are not yet fully understood. Moreover, subtle body movement patterns, which may characterize arousals both in human and in animals, are usually not detectable by eye perception and are not in general present in sleep studies. In this paper, we focus attention on accelerometer records (AR) to characterize and predict arousal during slow wave sleep (SWS) stage of mice. Furthermore, we recorded the local field potentials (LFP) from the CA1 region in the hippocampus and paired with accelerometer data. The hippocampus signal was also used here to identify the SWS stage. We analyzed the AR dynamics of consecutive arousals using recurrence technique and the determinism (DET) quantifier. Recurrence is a fundamental property of dynamical systems, which can be exploited to characterize time series properties. The DET index evaluates how similar are the evolution of close trajectories: in this sense, it computes how accurate are predictions based on past trajectories. For all analyzed mice in this work, we observed, for the first time, the occurrence of a universal dynamic pattern a few seconds that precedes the arousals during SWS sleep stage based only on the AR signal. The predictability success of an arousal using DET from AR is nearly 90%, while similar analysis using LFP of hippocampus brain region reveal 88% of success. Noteworthy, our findings suggest an unique dynamical behavior pattern preceding an arousal of AR data during sleep. Thus, the employment of this technique applied to AR data may provide useful information about the dynamics of neuronal activities that control sleep-waking switch during SWS sleep period. We argue that the predictability of arousals observed through DET(AR) can be functionally explained by a respiratory-driven modification of neural states. Finally, we believe that the method associating AR data with other physiologic events such as neural rhythms can become an accurate, convenient and non-invasive way of studying the physiology and physiopathology of movement and respiratory processes during sleep.

Behavioral Changes Over Time Following Ayahuasca Exposure in Zebrafish

The combined infusion of Banisteriopsis caapi stem and Psychotria viridis leaves, known as ayahuasca, has been used for centuries by indigenous tribes. The infusion is rich in N, N-dimethyltryptamine (DMT) and monoamine oxidase inhibitors, with properties similar to those of serotonin. Despite substantial progress in the development of new drugs to treat anxiety and depression, current treatments have several limitations. Alternative drugs, such as ayahuasca, may shed light on these disorders. Here, we present time-course behavioral changes induced by ayahuasca in zebrafish, as first step toward establishing an ideal concentration for pre-clinical evaluations. We exposed adult zebrafish to five concentrations of the ayahuasca infusion: 0 (control), 0.1, 0.5, 1, and 3 ml/L (n = 14 each group), and behavior was recorded for 60 min. We evaluated swimming speed, distance traveled, freezing and bottom dwelling every min for 60 min. Swimming speed and distance traveled decreased with an increase in ayahuasca concentration while freezing increased with 1 and 3 ml/L. Bottom dwelling increased with 1 and 3 ml/L, but declined with 0.1 ml/L. Our data suggest that small amounts of ayahuasca do not affect locomotion and reduce anxiety-like behavior in zebrafish, while increased doses of the drug lead to crescent anxiogenic effects. We conclude that the temporal analysis of zebrafish behavior is a sensitive method for the study of ayahuasca-induced functional changes in the vertebrate brain.