Search

Background Zika virus (ZIKV) infection during pregnancy may cause major congenital defects, including microcephaly, ocular, articular and muscle abnormalities, which are collectively defined as Congenital Zika Syndrome. Here, we performed an in-depth characterization of the effects of congenital ZIKV infection (CZI) in immunocompetent mice. Methods Pregnant dams were inoculated with ZIKV on embryonic day 5.5 in the presence or absence of a sub-neutralizing dose of a pan-flavivirus monoclonal antibody (4G2) to evaluate the potential role of antibody-dependent enhancement phenomenon (ADE) during short and long outcomes of CZI. Findings ZIKV infection induced maternal immune activation (MIA), which was associated with occurrence of foetal abnormalities and death. Therapeutic administration of AH-D antiviral peptide during the early stages of pregnancy prevented ZIKV replication and death of offspring. In the post-natal period, CZI was associated with a decrease in whole brain volume, ophthalmologic abnormalities, changes in testicular morphology, and disruption in bone microarchitecture. Some alterations were enhanced in the presence of 4G2 antibody. Interpretation Our results reveal that early maternal ZIKV infection causes several birth defects in immunocompetent mice, which can be potentiated by ADE phenomenon and are associated with MIA. Additionally, antiviral treatment with AH-D peptide may be beneficial during early maternal ZIKV infection. Fund This work was supported by the Brazilian National Science Council (CNPq, Brazil), Minas Gerais Foundation for Science (FAPEMIG), Funding Authority for Studies and Projects (FINEP), Coordination of Superior Level Staff Improvement (CAPES), National Research Foundation of Singapore and Centre for Precision Biology at Nanyang Technological University.

Although the functional role for newborn neurons in neural circuits is still matter of investigation, there is no doubt that neurogenesis modulates learning and memory in rodents. In general, boosting neurogenesis before learning, using genetic-target tools or drugs, improves hippocampus-dependent memories. However, inhibiting neurogenesis may yield contradictory results depending on the type of memory evaluated. Here we tested the hypothesis that inhibiting constitutive neurogenesis would compromise social recognition memory (SRM). Male Swiss mice were submitted to three distinct procedures to inhibit neurogenesis: (1) intra-cerebral infusion of Cystosine-β-D-Arabinofuranoside (AraC); (2) intra-peritoneal injection of temozolomide (TMZ) and (3) cranial gamma irradiation. All three methods decreased cell proliferation and neurogenesis in the dentate gyrus of the dorsal (dDG) and ventral hippocampus (vDG), and the olfactory bulb (OB). However, the percentage inhibition diverged between methods and brain regions. Ara-C, TMZ and gamma irradiation impaired SRM, though only gamma irradiation did not cause side effects on weight gain, locomotor activity and anxiety. Finally, we examined the contribution of cell proliferation in vDG, dDG and OB to SRM. The percent of inhibition in the dDG correlates with SRM, independently of the method utilized. This correlation was observed for granular cell layer of OB and vDG, only when the inhibition was induced by gamma irradiation. Animal's performance was restrained by the inhibition of dDG cell proliferation, suggesting that cell proliferation in the dDG has a greater contribution to SRM. Altogether, our results demonstrate that SRM, similarly to other hippocampus-dependent memories, has its formation impaired by reducing constitutive neurogenesis.

The idea of the epileptic brain being highly excitable and facilitated to synchronic activity has guided pharmacological treatment since the early twentieth century. Although tackling epilepsy's seizure-prone feature, by tonically modifying overall circuit excitability and/or connectivity, the last 50 years of drug development has not seen a substantial improvement in seizure suppression of refractory epilepsies. This review presents a new conceptual framework for epilepsy in which the temporal dynamics of the disease plays a more critical role in both its understanding and therapeutic strategies. The repetitive epileptiform pattern (characteristic during ictal activity) and other well-defined electrographic signatures (i.e., present during the interictal period) are discussed in terms of the sequential activation of the circuit motifs. Lessons learned from the physiological activation of neural circuitry are used to further corroborate the argument and explore the transition from proper function to a state of instability. Furthermore, the review explores how interfering in the temporally dependent abnormal connectivity between circuits may work as a therapeutic approach. We also review the use of probing stimulation to access network connectivity and evaluate its power to determine transitional states of the dynamical system as it moves towards regions of instability, especially when conventional electrographic monitoring is proven inefficient. Unorthodox cases, with little or no scalp electrographic correlate, in which ictogenic circuitry and/or seizure spread is temporally restricted to neurovegetative, cognitive, and motivational areas are shown as possible explanations for sudden death in epilepsy (SUDEP) and other psychiatric comorbidities. In short, this review presents a paradigm shift in the way that we address the disease and is aimed to encourage debate rather than narrow the rationale epilepsy is currently engaged in. This article is part of the Special Issue “NEWroscience 2018".

The brain oscillations may play a critical role in synchronizing neuronal assemblies in order to establish appropriate sensory-motor integration. In fact, studies have demonstrated phase-amplitude coupling of distinct oscillatory rhythms during cognitive processes. Here we investigated whether olfacto-hippocampal coupling occurs when mice are detecting familiar odors located in a spatially restricted area of a new context. The spatial olfactory task (SOT) was designed to expose mice to a new environment in which only one quadrant (target) contains odors provided by its own home-cage bedding. As predicted, mice showed a significant higher exploration preference to the target quadrant; which was impaired by olfactory epithelium lesion (ZnSO4). Furthermore, mice were able to discriminate odors from a different cage and avoided the quadrant with predator odor 2,4,5-trimethylthiazoline (TMT), reinforcing the specificity of the SOT. The local field potential (LFP) analysis of non-lesioned mice revealed higher gamma activity (35-100Hz) in the main olfactory bulb (MOB) and a significant theta phase/gamma amplitude coupling between MOB and dorsal hippocampus, only during exploration of home-cage odors (i.e. in the target quadrant). Our results suggest that exploration of familiar odors in a new context involves dynamic coupling between the olfactory bulb and dorsal hippocampus.

The unpredictability of spontaneous and recurrent seizures significantly impairs the quality of life of patients with epilepsy. Probing neural network excitability with deep brain electrical stimulation (DBS) has shown promising results predicting pathological shifts in brain states. This work presents a proof-of-principal that active electroencephalographic (EEG) probing, as a seizure predictive tool, is enhanced by pairing DBS and the electrographic seizure itself. The ictogenic model used consisted of inducing seizures by continuous intravenous infusion of pentylenetetrazol (PTZ — 2.5 mg/ml/min) while a probing DBS was delivered to the thalamus (TH) or amygdaloid complex to detect changes prior to seizure onset. Cortical electrophysiological recordings were performed before, during, and after PTZ infusion. Thalamic DBS probing, but not amygdaloid, was able to predict seizure onset without any observable proconvulsant effects. However, previously pairing amygdaloid DBS and epileptic polyspike discharges (day-1) elicited distinct preictal cortically recorded evoked response (CRER) (day-2) when compared with control groups that received the same amount of electrical pulses at different moments of the ictogenic progress at day-1. In conclusion, our results have demonstrated that the pairing strategy potentiated the detection of an altered brain state prior to the seizure onset. The EEG probing enhancement method opens many possibilities for both diagnosis and treatment of epilepsy.

Purpose To combine the technique of respiratory gating and compressed sensing (CS) with the objective of accelerating mouse abdominal magnetic resonance imaging (MRI). Materials and methods To obtain the maximum acceleration, phase-encoding data from a phantom and mouse were obtained on a 4.7 Tesla scanner using the respiratory gating technique. The fully sampled data (FSD) were used to construct reference images and to provide samples to simulate retrospective undersampled data (UD) acquisition using respiratory gating. The UD and 95% of the UD on acceleration 2–5 rates were acquired and used for image reconstruction by CS. Quantitative assessment of reconstructed images was performed by structural similarity index (SSIM), peak signal-to-noise ratio (PSNR) and root mean square error (RMSE). Results The proposed method can accelerate phantom and mouse abdominal MRI acquisition between 2 and 4 rates by reducing the amount of FSD. For phantom UD acquisition, the mean time was reduced in 45.9% and for the acquisition of 95% of UD in 67.8%. For mouse abdominal image UD acquisition, the mean time was reduced in 44.6% and for the acquisition of 95% of UD in 62.5%. The metrics results show that the reconstructed image from UD and 95% of UD by using CS maintains an optimal agreement with their reference images (similarity above 0.88 for phantom and 0.93 for mouse). Conclusion This study presents a novel approach to accelerate mouse abdominal MRI combining respiratory gating technique and CS without the use of expensive hardware and capable of achieving up to 4 acceleration rate without image degradation.

Given the known effects of undernutrition over protein synthesis, we promoted neonatal undernutrition to evaluate its effect over the neuroplasticity induced by the pilocarpine model of epilepsy and also over spontaneous seizure expression. A well-nourished group (WN), fed ad libitum rat chow diet, and an undernourished group (UN), fed 60% of the amount of diet consumed by a WN group, were submitted to status epilepticus (SE) through pilocarpine injection at 45 days of age. Thereafter, animals were behaviorally monitored for 6h daily to quantify seizures. On the 120th day, electroencephalography (EEG) was recorded and rats were sacrificed to measure proteins and glutamate release from hippocampus. Neo-Timm staining was used to detect mossy fiber sprouting. The results indicate no statistical difference in the latency for the first spontaneous recurrent seizure (SRS), in the number of daily SRS, or in EEG epileptiform activity duration between groups. However, PILO promoted more K(+)-stimulated glutamate release in the hippocampus slices from WN animals when compared to the UN group. It was also found a lower degree of mossy fibers sprouting in UN group. Data from this work, thus, indicate that the decreased neuroplasticity as currently measured does not directly impact on the manifestation of spontaneous seizures.

The role of physical exercise as a neuroprotective agent against ischemic injury has been extensively discussed. Nevertheless, the mechanisms underlying the effects of physical exercise on cerebral ischemia remain poorly understood. Here, we investigate the hypothesis that physical exercise increases ischemic tolerance by decreasing the induction of cellular apoptosis and glutamate release. Rats (n = 50) were submitted to a swimming exercise protocol for 8 weeks. Hippocampal slices were then submitted to oxygen and glucose deprivation. Cellular viability, pro-apoptotic markers (Caspase 8, Caspase 9, Caspase 3, and apoptosis-inducing factor), and glutamate release were analyzed. The percentage of cell death, the amount of glutamate release, and the expression of the apoptotic markers were all decreased in the exercise group when compared to the sedentary group after oxygen and glucose deprivation. Our results suggest that physical exercise protects hippocampal slices from the effects of oxygen and glucose deprivation, probably by a mechanism involving both the decrease of glutamatergic excitotoxicity and apoptosis induction. Exercise reduces excitotoxicity and apoptosis. The mechanisms underlying neuroprotective effects of regular exercise are still not fully understood. Here, we showed that swim training promotes neuroprotection decreasing pro-apoptotic markers as caspases 8, 9, 3, and apoptose-Inducing Factor (AIF) as well as glutamate release from hippocampal slices. Our data reinforce the idea that exercise affords a neuroprotective effect.

Background Epilepsy is a common neurological disease affecting over 40 million people worldwide. The foremost important challenge of epileptologists has been to control and predict the recurrent and spontaneous seizures of epileptic patients. The application of low frequency electrical stimulation (LFS) in deep brain structures has shown promising results in seizure control. However, the use of LFS as a probing strategy for seizure prediction, thus contributing to a closed loop solution, is still poorly explored. Objective To improve seizure prediction by producing gradually increasing phase-locked pre-ictal electrographical responses, due to the short-term plastic changes in epileptogenic neural networks, thus behaving as a “programmed” surrogate marker. Methods Urethane anesthetized rats were divided into 3 groups: the PTZ-noES group was injected with pentylenetetrazole (PTZ 4 mg/ml/min flow rate) i.v. without electrical stimulation (ES); the ES-noPTZ group received ES (0.5 Hz, 0.1 ms pulse width and 0.6 mA) to the amygdaloid complex and the PTZ + ES group received simultaneously i.v. PTZ infusion and ES. After each condition, electrographical parameters and c-Fos expression of regions of interest were evaluated. Results Although the PTZ + ES group had no evident change in the sustained electrographic seizure onset, duration and/or frequency spectrum; c-Fos labeling showed a different expression pattern when compared to the PTZ-noES and ES-noPTZ. Also, PTZ + ES formed a gradually increasing evoked potential; confirming the strong coupling of reverberant neural networks induced by ES – phase locked to stimuli. Conclusion ES induces a detectable temporal rearrangement of pre-ictal activity, which has suggestive applicability to seizure prediction.

Maternal immune activation (MIA) during pregnancy in rodents increases the risk of the offspring to develop schizophrenia-related behaviors, suggesting a relationship between the immune system and the brain development. Here we tested the hypothesis that MIA induced by the viral mimetic polyinosinic-polycytidylic acid (poly I:C) in early or late gestation of mice leads to behavioral and neuroanatomical disorders in the adulthood. On gestational days (GDs) 9 or 17 pregnant dams were treated with poly I:C or saline via intravenous route and the offspring behaviors were measured during adulthood. Considering the progressive structural neuroanatomical alterations in the brain of individuals with schizophrenia, we used magnetic resonance imaging (MRI) to perform brain morphometric analysis of the offspring aged one year. MIA on GD9 or GD17 led to increased basal locomotor activity, enhanced motor responses to ketamine, a psychotomimetic drug, and reduced time spent in the center of the arena, suggesting an increased anxiety-like behavior. In addition, MIA on GD17 reduced glucose preference in the offspring. None of the treatments altered the relative volume of the lateral ventricles. However, a decrease in brain volume, especially for posterior structures, was observed for one-year-old animals treated with poly I:C compared with control groups. Thus, activation of the maternal immune system at different GDs lead to neuroanatomical and behavioral alterations possibly related to the positive and negative symptoms of schizophrenia. These results provide insights on neuroimmunonological and neurodevelopmental aspects of certain psychopathologies, such as schizophrenia.

Epilepsy is a neurological disease related to the occurrence of pathological oscillatory activity, but the basic physiological mechanisms of seizure remain to be understood. Our working hypothesis is that specific sensory processing circuits may present abnormally enhanced predisposition for coordinated firing in the dysfunctional brain. Such facilitated entrainment could share a similar mechanistic process as those expediting the propagation of epileptiform activity throughout the brain. To test this hypothesis, we employed the Wistar audiogenic rat (WAR) reflex animal model, which is characterized by having seizures triggered reliably by sound. Sound stimulation was modulated in amplitude to produce an auditory steady-state-evoked response (ASSR; −53.71 Hz) that covers bottom-up and top-down processing in a time scale compatible with the dynamics of the epileptic condition. Data from inferior colliculus (IC) c-Fos immunohistochemistry and electrographic recordings were gathered for both the control Wistar group and WARs. Under 85-dB SLP auditory stimulation, compared to controls, the WARs presented higher number of Fos-positive cells (at IC and auditory temporal lobe) and a significant increase in ASSR-normalized energy. Similarly, the 110-dB SLP sound stimulation also statistically increased ASSR-normalized energy during ictal and post-ictal periods. However, at the transition from the physiological to pathological state (pre-ictal period), the WAR ASSR analysis demonstrated a decline in normalized energy and a significant increase in circular variance values compared to that of controls. These results indicate an enhanced coordinated firing state for WARs, except immediately before seizure onset (suggesting pre-ictal neuronal desynchronization with external sensory drive). These results suggest a competing myriad of interferences among different networks that after seizure onset converge to a massive oscillatory circuit.

Accumulating evidence from different animal models has contributed to the understanding of the bidirectional comorbidity associations between the epileptic condition and behavioral abnormalities. A strain of animals inbred to enhance seizure predisposition to high-intensity sound stimulation, the Wistar audiogenic rat (WAR), underwent several behavioral tests: forced swim test (FST), open-field test (OFT), sucrose preference test (SPT), elevated plus maze (EPM), social preference (SP), marble burying test (MBT), inhibitory avoidance (IAT), and two-way active avoidance (TWAA). The choice of tests aimed to investigate the correlation between underlying circuits believed to be participating in both WAR's innate susceptibility to sound-triggered seizures and the neurobiological substrates associated with test performance. Comparing WAR with its Wistar counterpart (i.e., resistant to audiogenic seizures) showed that WARs present behavioral despair traits (e.g., increased FST immobility) but no evidence of anhedonic behavior (e.g., increased sucrose consumption in SPT) or social impairment (e.g., no difference regarding juvenile exploration in SP). In addition, tests suggested that WARs are unable to properly evaluate degrees of aversiveness (e.g., performance on OFT, EPM, MBT, IAT, and TWAA). The particularities of the WAR model opens new venues to further untangle the neurobiology underlying the co-morbidity of behavioral disorders and epilepsy. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Inputting information to the brain through direct electrical microstimulation must consider how underlying neural networks encode information. One unexplored possibility is that a single electrode delivering temporally coded stimuli, mimicking an asynchronous serial communication port to the brain, can trigger the emergence of different brain states. This work used a discriminative fear-conditioning paradigm in rodents in which 2 temporally coded microstimulation patterns were targeted at the amygdaloid complex. Each stimulus was a binary-coded "word" made up of 10 ms bins, with 1's representing a single pulse stimulus: A-1001111001 and B-1110000111. During 3 consecutive retention tests (i.e., day-word: 1-B; 2-A, and 3-B), only binary-coded words previously paired with a foot-electroshock elicited proper aversive behavior. To determine the neural substrates recruited by the different stimulation patterns, c-Fos expression was evaluated 90 min after the last retention test. Animals conditioned to word-B, after stimulation with word-B, demonstrated increased hypothalamic c-Fos staining. Animals conditioned to word-A, however, showed increased prefrontal c-Fos labeling. In addition, prefrontal-cortex and hypothalamic c-Fos staining for, respectively, word-B- and word-A-conditioned animals, was not different than that of an unpaired control group. Our results suggest that, depending on the valence acquired from previous learning, temporally coded microstimulation activates distinct neural networks and associated behavior.

Epilepsia é uma afecção neurológica que atinge em média de 1% da população mundial, sendo que 25% são resistentes ao tratamento medicamentoso. Um dos fatores mais incapacitantes dessa síndrome é a imprevisibilidade de ocorrência das crises epilépticas (CE). Há um grande interesse no desenvolvimento de dispositivos capazes de predizer a ocorrência de CE. O objetivo deste trabalho foi utilizar a estimulação elétrica cerebral profunda (ES) como forma de sondagem dos estados da rede neural em um modelo de crise epiléptica por infusão intravenosa de pentilenotetrazol (PTZ). Eletrodos bipolares foram cirurgicamente implantados na região do núcleo anterior do tálamo ou do complexo amigdaloide de ratos Wistar, assim como micro parafusos na região do cortex parietal para aquisição de EEG. Os animais foram divididos em três grupos: PTZ-noES (infusão de PTZ sem ES), ES-noPTZ (ES concomitante a infusão de salina i.v.) e PTZ+ES (simultaneamente infusão de PTZ e aplicação de ES). A estimulação talâmica (0.5 Hz, 0.1 ms largura de pulso e 0,8 mA) em animais acordados concomitante à infusão de PTZ (grupo PTZ+ES) resultou em um aumento significativo da atividade cortical, 250ms após o ES, doze segundos antes da ocorrência da crise epiléptica, composto principalmente de frequências de 12 a 30Hz (banda beta). Quando ES (0.5 Hz, 0.1 ms largura de pulso e 0,6mA) foi aplicado no complexo amigdaloide de animais anestesiados juntamente com infusão de PTZ, um aumento significativo da energia do EEG peri estímulo (250ms após ES) foi detectado oitenta segundos antes da ocorrência da crise eletrográfica. Houve também, para ao grupo PTZ+ES, um aumento significativo da expressão de c-Fos em regiões como tálamo, hipotálamo e amigdala contralateral ao eletrodo, evidenciado pela marcação imuno-histoquímica de c-Fos. Em contrapartida, ES (0.5 Hz, 0.1 ms largura de pulso e 0,8 mA) em complexo amigdaloide de animais acordados somente resultou em um aumento de atividade cortical precedente à crise epilética (doze segundos) se o ES foi temporalmente pareado com os disparos epileptiformes 24 horas antes. Em nenhum dos protocolos a ES apresentou característica pró-convulsivas, sem qualquer alteração na latência ou duração das crises (quando se compara os grupos PTZ+ES e PTZ-noES) ou qualquer disparo epileptiforme para o grupo ES-noPTZ. A estimulação elétrica apresenta-se como uma estratégia eficiente de sondagem do estado neural e, futuramente, pode ser usada como uma técnica de previsão de crise epiléptica. Epilepsy is a common neurological disease affecting 1% of world population, whose 25% has no seizure control by pharmacotherapy. The seizure unpredictability is one of the most disable features of this syndrome. Therefore, the designing of devices able to forecast a seizure occurrence is a paramount goal. The aim of this work was to probe the neural system state by deep brain electrical stimulation (DBS) in a pentylenetetrazole (PTZ) seizure model. Bipolar electrodes were surgically implanted in wistar rats anterior thalamus (ANT) or amygaloid complex (AMY) for electrical stimulation and two micro screws on parietal cortex for EEG acquisition. The animals were further divided in three groups: PTZ-noES (i.v. PTZ infusion without DBS), ES-noPTZ (DBS and saline infusion) and PTZ+ES (i.v. PTZ infusion and DBS). ANT DBS on awake animals (0.5 Hz, 0.1ms pulse width, 0,8mA) simultaneously with PTZ infusion showed an increase of cortical activity (250ms after stimuli) twelve seconds prior seizure, mainly in beta band (12-30Hz). DBS (0.5 Hz, 0.1ms pulse width, 0,6mA) applied on AMY of anesthetized animals, concomitantly with PTZ infusion, produced a detectable cortical evoked response 80s prior seizure onset and increase thalamus, hypothalamus, and amygdala (contralateral to electrode) cell activity by c-Fos immunohistochemistry labeling. Nevertheless, AMY DBS on awaked animals only increase cortical EEG activity prior seizure onset (12s) if the electrical stimuli were temporal paired with epileptic discharges 24 hours earlier. DBS used in this study demonstrated no pro-convulsive effect due to any statistical alteration on seizure latency and duration (PTZ + ES compared with PTZ-noES) or any epileptic discharge presented by ES-noPTZ group. The DBS presents as a reliable strategy to probe the neural system state and a possible tool to seizure prediction device.

Electrical stimulation applied to the basolateral amygdala in the pentylenetetrazole animal model of seizures may result in either a proconvulsant or an anticonvulsant effect depending on the interpulse intervals used: periodic or nonperiodic, respectively. We tested the effect of this electrical stimulation temporal coding on the spontaneous and recurrent behavioral seizures produced in the chronic phase of the pilocarpine animal model of temporal lobe epilepsy, an experimental protocol that better mimics the human condition. After 45 days of the pilocarpine-induced status epilepticus, male Wistar rats were submitted to a surgical procedure for the implantation of a bipolar electrical stimulation electrode in the right basolateral amygdala and were allowed to recover for seven days. The animals were then placed in a glass box, and their behaviors were recorded daily on DVD for 6 h for 4 consecutive days (control period). Spontaneous recurrent behavioral seizures when showed in animals were further recorded for an extra 4-day period (treatment period), under periodic or nonperiodic electrical stimulation. The number, duration, and severity of seizures (according to the modified Racine's scale) during treatment were compared with those during the control period. The nonperiodically stimulated group displayed a significantly reduced total number and duration of seizures. There was no difference between control and treatment periods for the periodically stimulated group. Results corroborate previous findings from our group showing that nonperiodic electrical stimulation has a robust anticonvulsant property. In addition, results from the pilocarpine animal model further strengthen nonperiodic electrical stimulation as a valid therapeutic approach in current medical practice. Our working hypothesis is that temporally unstructured electrical stimulation may wield its effect by desynchronizing neural networks involved in the ictogenic process.

Selective blockade of anandamide hydrolysis, through the inhibition of the FAAH enzyme, has anticonvulsant effects, which are mediated by CB1 receptors. Anandamide, however, also activates TRPV1 channels, generally with an opposite outcome on neuronal modulation. Thus, we suggested that the dual FAAH and TRPV1 blockade with N-arachidonoyl-serotonin (AA-5-HT) would be efficacious in inhibiting pentylenetetrazole (PTZ)-induced seizures in mice. We also investigated the contribution of CB1 activation and TRPV1 blockade to the overt effect of AA-5-HT. In the first experiment, injection of AA-5-HT (0.3-3.0 mg/kg) delayed the onset and reduced the duration of PTZ (60 mg)-induced seizures in mice. These effects were reversed by pre-treatment with the CB1 antagonist, AM251 (1.0-3.0 mg/kg). Finally, we observed that administration of the selective TRPV1 antagonist, SB366791 (0.1-1 mg/kg), did not entirely mimic AA-5-HT effects. In conclusion, AA-5-HT alleviates seizures in mice, an effect inhibited by CB1 antagonism, but not completely mimicked by TRPV1 blockage, indicating that the overall effect of AA-5-HT seems to depend mainly on CB1 receptors. This may represent a new strategy for the development of drugs against seizures, epilepsies and related syndromes.

Epilepsy is a severely debilitating brain disease, often associated with premature death, which has an urgent need for alternative methods of treatment. In fact, roughly 25% of patients with epilepsy do not have seizures satisfactorily controlled by pharmacological treatment, and 30% of these patients with treatment-refractory seizures are not even eligible for ablative surgery. Epilepsy is most readily identifiable by its seizures and/or paroxysmal events, mostly viewed as spontaneously recurrent and unpredictable, which are caused by stereotyped changes in neurological function associated with hyperexcitability and hypersynchronicity of the underlying neural networks. Treatment has strongly been based on the fixed goal of depressing neuronal activity, working under the veiled assumption that hyperexcitability would lead to synchronous neuronal activity and, therefore, to seizure. Over the last 20–30 years, the emergence of electrical (ES) of deep brain structures, a practicable option for treating patients with otherwise untreatable seizures, has broadened our understanding of anticonvulsant mechanisms that conceptually differ from those of pharmacological treatment. Conversely, the research on ES therapy applied to epilepsy is contributing significantly to untwine the phenomena of excitation from that of synchronization as potential target mechanisms for abolishing seizures and predicting paroxysmal events. This paper is, thus, an addendum to other reviews on the subject of ES therapy in epilepsy which focuses on the desynchronization effect ES has on epileptogenic neural networks rather than its effect on overall brain excitability. This article is part of a Special Issue entitled “NEWroscience 2013”.

Summary Cannabinoids and drugs that increase endocannabinoid levels inhibit neuronal excitability and restrain epileptic seizures through CB1 receptor activation. Nevertheless, the results have not been entirely consistent, since pro-convulsant effects have also been reported. The present study aimed to further investigate the effects of cannabinoid-related compounds on seizures induced by pentylenetetrazole (PTZ) in rats. Video-EEG recordings were used to determine both electrographic and behavioral thresholds to ictal activity. The animals received injections of WIN-55,212-2 (0.3–3 mg/kg, non-selective) or ACEA (1–4 mg/kg, CB1-selective), two synthetic cannabinoids, or URB-597 (0.3–3 mg/kg), an anandamide-hydrolysis inhibitor (FAAH enzyme inhibitor), followed by PTZ. Both WIN-55,212-2 (1 mg/kg) and ACEA (1–4 mg/kg) reduced the threshold for myoclonic seizures and enhanced epileptiform EEG activity, typical pro-convulsive effects. On the contrary, URB-597 (1 mg/kg) had an anti-convulsive effect, as it increased the threshold for the occurrence of minimal seizures and reduced EEG epileptiform activity. None of the drugs tested altered the tonic–clonic maximal seizure threshold. These data suggest that the effects of CB1 signaling upon seizure activity may depend on how this receptor is activated. Contrary to direct agonists, drugs that increase anandamide levels seem to promote an optimal tonus and represent a promising strategy for treating myoclonic seizures.

Although neuroimmune interactions associated with the development of pain sensitization in models of neuropathic pain have been widely studied, there are some aspects that require further investigation. Thus, we aimed to evaluate whether the local intraneural or perineural injections of dexamethasone, an efficacious anti-inflammatory and immunosuppressant drug, delays the development of both thermal hyperalgesia and mechanical allodynia in an experimental model of neuropathic pain in rats. Hargreaves and electronic von Frey tests were applied. The chronic constriction injury (CCI) of right sciatic nerve was performed. Single intraneural dexamethasone administration at the moment of constriction delayed the development of sensitization for thermal hyperalgesia and mechanical allodynia. However, perineural administration of dexamethasone, at the highest dose, did not delay experimental pain development. These results show that inflammation/immune response at the site of nerve lesion is an essential trigger for the pathological changes that lead to both hyperalgesia and allodynia. In conclusion, this approach opens new opportunities to study cellular and molecular neuroimmune interactions associated with the development of pain derived from peripheral neuropathies.

Our working hypothesis is that constant inter-pulse interval (IPI) electrical stimulation (ES) would resonate with endogenous epileptogenic reverberating circuits, favoring seizure, while random inter-interval ES protocol would promote desynchronization of such neural networks, interfering with the abnormal recruitment of neural structures. Male Wistar rats were stereotaxically implanted with a monopolar ES carbon-fiber electrode (minimizing fMRI artifact) in the amygdala. A 7T fMRI scanner was used to evaluate brain activity during ES, fixed four pulses per second ratio, using either a periodic IPI (ES-P) or random IPI (non-periodic ES-NP) stimulation paradigm. Appropriate imaging protocols were used to compare baseline BOLD (blood oxygen level dependent) MRI with scans during ES. A second series of experiments, both without stimuli and under the same ES paradigms, were evaluated during continuous infusion of pentylenetetrazole (PTZ, 4mg/ml/min) through an i.v. catheter. Our results show that temporal lobe activation during ES-P or ES-NP did not present any statistical differences during ES. However, during PTZ infusion, PTZ-P facilitated recruitment of the temporal lobe ipsilateral to ES while PTZ-NP showed significantly less activation ipsilateral to ES and, in turn, less inter-hemispheric differences. Altogether, our results support the hypothesis of reverberating circuits being synchronized by ES-P and desynchronized by ES-NP. Time-coded low frequency stimulation may be an interesting alternative treatment for patients with refractory epilepsy.

Our working hypothesis is that constant interpulse interval (IPI) electrical stimulation would resonate with endogenous epileptogenic reverberating circuits, inducing seizures, whereas a random interinterval electrical stimulation protocol would promote desynchronization of such neural networks, producing an anticonvulsant effect. Male Wistar rats were stereotaxically implanted with a bipolar electrical stimulation electrode in the amygdala. Pentylenetetrazole (10 mg/ml/min) was continuously infused through an intravenous catheter to induce seizures while four different patterns of temporally coded electrical stimulation were applied: periodic stimulation (PS), pseudo-randomized IPI stimulation (LH), restrictively randomized IPI stimulation (IH), and bursts of 20-ms IPIs (burst). PS decreased the pentylenetetrazole threshold to forelimb clonus, whereas IH increased the threshold to forelimb clonus and to generalized tonic–clonic seizures. We hypothesize that PS facilitates forelimb clonus by reverberating with epileptogenic circuits in the limbic system, whereas IH delays forelimb clonus and generalized tonic–clonic seizures by desynchronizing the epileptic neural networks in the forebrain–midbrain–hindbrain circuits.

In the PTZ animal model of epilepsy, electrical stimulation applied to the amygdaloid complex may result in either pro-convulsive or anticonvulsant effect, depending on the temporal pattern used (i.e. periodic-PS and non-periodic-NPS electrical stimulation). Our hypothesis is that the anatomical target is a determinant factor for the differential effect of temporally-coded patterns on seizure outcome. The threshold dose of PTZ to elicit forelimb clonus and generalized tonic–clonic seizure behavior was measured. The effect of amygdaloid complex PS on forelimb clonus threshold showed a pro-convulsive effect while NPS was anticonvulsant. NPS also significantly increased generalized tonic–clonic threshold; while PS, although at lower threshold levels, did not present statistical significance. Thalamus stimulation did not affect forelimb clonus threshold and showed similar anticonvulsant profiles for both PS and NPS on generalized tonic–clonic threshold. In summary, the anatomical target is a determinant factor on whether temporally-coded ES differentially modulates seizure outcome.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A epilepsia é um distúrbio neurológico que afeta milhões de pessoas em todo o mundo, enquanto a epilepsia do lobo temporal (ELT) responde por 40% de todos os casos de epilepsia, sendo a forma mais comum em adultos. A família de receptores de potencial transiente (TRP) é um grupo de canais catiônicos envolvidos em diferentes processos fisiológicos, como a homeostase iônica. O acúmulo de cálcio nos neurônios do hipocampo tem sido associado à etiologia da epilepsia, o que motiva o estudo da família TRP, cujos canais são altamente permeáveis a esse íon. O objetivo deste estudo foi avaliar os efeitos dos bloqueadores TRPA1 e TRPV1 nas crises epilépticas, alterações histológicas e bioquímicas, além da liberação de glutamato e do conteúdo de cálcio citosólico em modelo de crise epiléptica induzida por pilocarpina ou pentilenotetrazol ou em preparações sinaptossomais de córtex. Para isso, animais C57Bl/6 (8-12 semanas de idade) receberam injeção intra-hipocampal de pilocarpina (40 μg), 30 minutos após a injeção intraperitoneal de bloqueador de TRPA1, TRPV1 ou veículo. Os bloqueadores do receptor TRPA1, AP18 (1, 3 e 10 mg/Kg) e do receptor TRPV1, SB366791 (0,1; 0,3 e 1 mg/Kg) foram utilizados nos experimentos in vivo. As concentrações de 0,1; 1; 10 e 30 μM foram usadas nas preparações de sinaptossomas para ambos os bloqueadores. Vinte e quatro horas após o status epilepticus (SE) induzido pela pilocarpina, os animais foram eutanasiados para a coleta dos tecidos cerebrais. Metade de seus cérebros foram removidos e processados para posteriores análises histológicas para avaliação de morte e viabilidade neuronal, marcação microglial e astrocitária. A outra metade dos cérebros dos animais foi removida para posterior análise de BDNF e dos níveis de citocinas, além da ativação da proteína quinase B (Akt) e da quinase regulada por sinal extracelular (Erk). As crises epilépticas induzidas pela pilocarpina foram avaliadas de acordo com a escala de Racine. Foi revelada forte associação entre AP18 e o desenvolvimento de SE, bem como o bloqueador e crises tônicoclônicas. O AP18 (3 mg/Kg) aumentou as crises classificadas como 3, 4 e 5. Por outro lado, o AP18 (10 mg/Kg) aumentou a latência mediana para os animais desenvolverem SE em relação ao grupo pilocarpina. A pilocarpina aumentou a marcação de neurônios em processo de degeneração evidenciada pela marcação por Fluoro-Jade C (FJC), sendo esta exacerbada pela administração de AP18 (1 e 3 mg/kg). De forma surpreendente, a droga induziu uma resposta dicotômica na microgliose induzida pela pilocarpina, pois exacerbou este parâmetro no giro denteado (GD) (3 mg/Kg) e reduziu em CA1 (1 e 3 mg/Kg). Observamos uma redução na reatividade astrocitária com AP18 (1 e 10 mg/Kg) induzida por pilocarpina, enquanto a dose de 3 mg/Kg de AP18 aumentou essa marcação no GD. Além disso, o AP18 reduziu os níveis de BDNF, TNF-α e CX3CL1 e aumentou os níveis de IL-10 comparado ao grupo pilocarpina. Em relação ao receptor TRPV1, foi observada forte associação entre SB366791 e o desenvolvimento do SE. A frequência de SE e convulsões tônico-clônicas no grupo SB366791 (0,1 e 0,3 mg/Kg) foi maior do que no grupo pilocarpina. A crise comportamental foi acompanhada por um aumento da morte neuronal. AP18 e SB366791 não alteraram nenhuma das análises com preparações de sinaptossomas corticais. Finalmente, nas crises induzidas por pentilenotetrazol, AP18 e SB366791 não alteraram a latência para o desenvolvimento de SE. O presente estudo mostrou que a administração sistêmica dos bloqueadores de TRPA1 e TRPV1, AP18 ou SB366791, respectivamente, aumentou a gravidade das crises epilépticas e danos neuronais decorrentes do SE induzido pela injeção intra-hipocampal de pilocarpina. Nossos resultados sugerem que AP18 e SB366791 apresentam efeitos pró-convulsivantes no modelo de pilocarpina, mas não de pentilenotetrazol. Estudos complementares nos ajudarão a caracterizar os mecanismos subjacentes desses canais na ELT. Epilepsy is a neurological disorder that affects millions of people around the world, while temporal lobe epilepsy (ELT) accounts for 40% of all epilepsy cases, being the most common form in adults. The family of transient potential receptors (TRP) are a group of cation channels involved in different physiological processes, such as ion homeostasis. Calcium accumulation in the neurons of the hippocampus has been associated with the etiology of epilepsy, which motivates the study of the TRP family, whose channels are highly permeable to this ion. The aim of this study was to evaluate the effects of TRPA1 and TRPV1 blockers on seizures, histological, and biochemical changes, besides glutamate release and cytosolic calcium content in model of pilocarpine or pentylenetetrazole -induced seizures or in cortex synaptosomal preparations. For this, C57Bl/6 animals (8-12 weeks of age) received intrahippocampal pilocarpine injection (40 μg), 30 minutes after intraperitoneal injection of TRPA1, TRPV1 blocker or vehicle. Blockers of the TRPA1, AP18 receptor (1, 3 and 10 mg/Kg) and TRPV1 receptor, SB366791 (0.1; 0.3 and 1 mg/Kg) were used in the in vivo experiments. Concentrations of 0.1; 1; 10 and 30 μM were used in the preparation of synaptosomes for both blockers. Twenty-four hours after pilocarpine-induced status epilepticus (SE), animals were euthanized to collect brain tissues. Half of their brains were removed and processed, for further histological techniques to evaluate neuronal death and viability, microglia and astrocyte staining. The other half of the animal’s brains were removed for further analysis of BDNF and cytokines levels, besides the protein kinase B (Akt) and extracellular-signal-regulated kinase (Erk) activation. The seizures induced by pilocarpine were evaluated according to Racine’s scale of seizures. Strong association between AP18 and the development of SE was revealed, as well as the blocker and tonic-clonic seizures. AP18 (3 mg/Kg) increased the seizures classified as 3, 4 and 5. On the other hand, AP18 (10 mg/Kg) increased the median latency for the animals to develop the SE as compared with pilocarpine group. Pilocarpine increased the labeling of neurons in the degeneration process evidenced by Fluoro-Jade C (FJC) staining, which was exacerbated by the administration of AP18 (1 and 3 mg/kg). Surprisingly, the drug induced a dichotomous response in microgliosis induced by pilocarpine, as it exacerbated in dentate gyrus (DG) with AP18 (3 mg/kg) and decreased in CA1 with doses of 1 and 3 mg/kg of AP18. We observed a reduction in astrocyte reactivity with AP18 (1 and 10 mg/kg) induced by pilocarpine, while the dose of 3 mg/kg of AP18 increased this marking in DG. In addition, AP18 reduced the levels of BDNF, TNF-α and CX3CL1, and enhanced IL-10 levels compared to the pilocarpine group. In relation to TRPV1, strong association was observed between SB366791 and the development of the SE. The frequency of SE and tonic-clonic seizures in the SB366791 group (0.1 and 0.3 mg/Kg) was higher than the pilocarpine group. The behavioral seizure was accompanied by an enhancement of neuronal death. AP18 and SB366791 did not change any of the analysis with cortical synaptosomes preparations. Finally, in the pentylenetetrazole-induced seizures AP18 and SB366791 did not change the latency for SE development. The present study showed that the systemic administration of the TRPA1 and TRPV1 blockers, AP18 or SB366791, respectively, increased the severity of epileptic seizures and neuronal damage resulting from the SE induced by the intra-hippocampal injection of pilocarpine. Our results suggested that AP18 and SB366791 show pro-convulsive effects in the pilocarpine model, but not in pentylenetetrazole- induced seizures. Further studies will allow the characterization of the underlying mechanisms of these channels in TLE.

Este trabalho envolve a extração de características e reconhecimento de padrões que indiquem estados ictogênicos em registros eletrográficos. A parte preliminar do trabalho avalia o desempenho de um método de decomposição de modos de sinais, a Transformada de Wavelet Empírica (EWT), em um problema de classificação de uma base de dados conhecida em epilepsia. São diferenciados registros de três classes; crise, inter-ictais e de pacientes normais, com exatidão de até 94.5%, valor compatível com outros métodos na literatura, demonstrando a viabilidade do método. Em seguida, hipóteses são propostas a respeito da ictogênese em ratos da linhagem WAR, um modelo animal de Crises Audiogênicas. Estas são avaliadas em registros do Colículo Inferior, obtidos em protocolos de experimentos baseados na sondagem acústica, onde tons de 10kHz modulados em amplitude são apresentados, evocando respostas auditivas em regime permanente. Estas podem ser consideradas assinaturas eletrográficas do processamento auditivo ao longo do tempo de cada animal, realçando atributos relativos à magnitude e sincronismo da resposta com o estímulo. O primeiro protocolo utiliza sons com intensidade 85dB, insuficiente para desencadear Crises Audiogênicas nos animais, modulados em amplitude em 53.71Hz (3 trials) e 92.77Hz (3 trial), em trials intercalados. É abordada a hipótese de que o processamento auditivo de WARs é diferenciado de animais fisiológicos, sendo necessária a aplicação de estímulos auditivos para ressaltar a diferença. A maior amplitude da resposta confirmou o processamento auditivo anormal de WARs, indicando a hiperexcitabilidade do Colículo Inferior destes animais. Porém, a atividade basal também é diferenciada entre os grupos, com animais ictogênicos apresentando maior energia nas bandas de alta frequência (Gamma), além de episódios semelhantes a fusos de alta amplitude. A segunda hipótese proposta, que circuitos neurais de WARs possuem tendência a sincronizar mais facilmente com estímulos externos, foi confirmada pelo maior índice de acoplamento de fase (PLV) entre estímulo e resposta destes animais. Com os atributos de energia e PLV utilizados nas abordagens das duas primeiras hipóteses, foram testados classificadores para identificação de registros provenientes de animais WAR. A amplitude da resposta evocada possui o maior poder preditivo, sobretudo quando a moduladora de 53.71Hz é utilizada (96% de exatidão), em comparação com a de 92.77Hz (80% de exatidão). O uso de mais atributos (como energia de outras bandas e PLV) possibilita a melhora no desempenho no segundo caso, aumentando de 80% para 87%. Isto indica a possibilidade de usar atributos de um protocolo de sondagem como biomarcadores, com aplicações em ferramentas diagnósticas. Em seguida, a dinâmica dos mesmos atributos ao longo de crises é analisada, de modo a confirmar ou refutar a hipótese que o processamento auditivo de WARs é prejudicado durante e após uma crise audiogênica. As características são extraídas de registros do segundo protocolo, onde uma estimulação sonora de maior intensidade (110dB) induz crises tônico-clônicas em animais WAR. Foi mostrado que, apesar de alterações transitórias durante a crise, a energia da resposta evocada não é alterada significativamente entre os períodos pré-ictal, ictal e pós-ictal. Isto contrasta com as energias em outras bandas de frequência, que aumentam consideravelmente durante a crise, devido ao recrutamento pelo foco ictal, mas diminuem no pós-ictal. O sincronismo da resposta com o estímulo, quantificado pelo índice PLV, confirma parte desta conclusão. Antes da crise, o PLV está abaixo dos valores normais encontrados em WARs, possivelmente devido a interações de circuitos responsáveis pela resposta com grupos do foco ictal. Assim, a resposta ainda estaria presente, mas a capacidade de acompanhar o estímulo seria prejudicada. Durante a crise há somente alterações transitórias do PLV, sobretudo próximo ao final. No período pós-ictal, como grande parte das oscilações endógenas do sistema está em período refratário, mas este ainda é responsivo a estímulos externos, os índices de PLV atingem o valor máximo. Assim, a dinâmica na resposta evocada indica que circuitos que fazem parte do processamento auditivo, apesar de estarem envolvidos no processo que leva a uma CA, não fazem parte efetivamente de circuitos da via ictogênica, pois continuam responsivos durante e após a crise audiogênica. This work deals with the extraction of features related to excitability and synchronism in electrographic signals, for evaluating hypotheses about auditory processing in WAR animals, an animal model for audiogenic seizures. These hypotheses are evaluated in Local Field Potential (LFP) signals from the Inferior Colliculus, obtained through experiment protocols based on the acoustic probing of the brain. Amplitude Modulated tones are presented to evoke Auditory Steady State Responses (ASSR). These can be considered as electrographic signatures of the auditory processing over time of each animal, highlighting features related to response magnitude and synchronism. The first protocol employs tones with intensity of 85 dB, enough to evoke the ASSR, but without triggering audiogenic seizures, modulated in amplitude at 53.71 Hz or 92.77 Hz, in interleaved trials. The first hypothesis states that the auditory processing in WARs is distinct from physiologic animals. The application of auditory stimuli would be needed to elicit responses which would make this difference visible. The larger amplitude response in WARs confirmed the hypothesis, indicating the hyperexcitability of the Inferior Colliculus of these animals. However, basal activity is also different between the groups, with ictogenic animals having greater energy in high frequency bands (gamma and beta), besides the occurrence of episodes resembling High Amplitude Spindles. The second hypothesis proposed that neural circuits in WARs have a tendency to synchronize more easily with external stimuli. This was confirmed by the higher phase coupling index (PLV) between stimulus and response of these animals. A diagnostic tool is then proposed, on which these features extracted from the probing protocol are used as inputs to classifiers that identify animals prone to audiogenic seizures. The ASSR was found to have the greatest predictive power, achieving accuracy of 96% with a modulating frequency of 53 Hz. The use of other features such as energies of endogenous EEG rhythms enables the improvement of performance, when a modulating frequency of 92 Hz is used. This indicates the possibility of using features from a probing protocol as diagnostic biomarkers. The second protocol involves the presentation of high intensity (110dB) AM tones in order to elicit audiogenic seizures in WARs only. The dynamic of the features presented earlier is analyzed in the pre-ictal, ictal and post-ictal periods, with the purpose of evaluating the hypothesis that the auditory processing in WARs is impaired during and after a seizure. It was shown that, although transitory changes happen during the seizure, the energy of the evoked response is not significantly changed across these three periods. This contrasts with the energies in other frequency bands, which significantly increase during the seizure due to recruitment by the ictal focus, followed by a refractory period with decreased activity after the seizure. The synchrony of the response with the stimulus, measured by the PLV index, confirms part of this conclusion. Before the seizure, the PLV is below the normal range found in WARs, possibly due to interactions of the response circuits with ictogenic ones. Thus, despite the magnitude of the response not being changed before the seizure, the ability to follow the stimulus is slightly impaired. During the seizure, there are only transient changes of the PLV, especially near its end. In the post-ictal period, most of the endogenous oscillations are in refractory period, but the system is still responsive to external sound stimuli, as evidenced by the persistence and synchrony of the ASSR. Thus, the dynamics of the ASSR suggest that the circuitry of the auditory xi pathway, despite being involved in the processes leading to audiogenic seizures, may not effectively be part of the ictogenic focus.