Gabriel Maisonnave Arisi

Plasticity‚ synaptic strength‚ and epilepsy: what can we learn from ultrastructural data?

Summary:  Central nervous system synapses have an intrinsic plastic capacity to adapt to new conditions with rapid changes in their structure. Such activity‐dependent refinement occurs during development and learning, and shares features with diseases such as epilepsy. Quantitative ultrastructural studies based on serial sectioning and reconstructions have shown various structural changes associated with synaptic strength involving both dendritic spines and postsynaptic densities (PSDs) during long‐term potentiation (LTP). In this review, we focus on experimental studies that have analyzed at the ultrastructural level the consequences of LTP in rodents, and plastic changes in the hippocampus of experimental models of epilepsy and human tissue obtained during surgeries for intractable temporal lobe epilepsy (TLE). Modifications in spine morphology, increases in the proportion of synapses with perforated PSDs, and formation of multiple spine boutons arising from the same dendrite are the possible sequence of events that accompany hippocampal LTP. Structural remodeling of mossy fiber synapses and formation of aberrant synaptic contacts in the dentate gyrus are common features in experimental models of epilepsy and in human TLE. Combined electrophysiological and ultrastructural studies in kindled rats and chronic epileptic animals have indicated the occurrence of seizure‐ and neuron loss‐induced changes in the hippocampal network. In these experiments, the synaptic contacts on granule cells are similar to those described for LTP. Such changes could be associated with enhancement of synaptic efficiency and may be important in epileptogenesis.

Chelatable zinc modulates excitability and seizure duration in the amygdala rapid kindling model

Zinc is present in high concentration in many structures of the limbic circuitry, however the role of zinc as a neuromodulator in such synapses is still uncertain. In this work, we verified the effects of zinc chelation in an animal model of epileptogenesis induced by amygdala rapid kindling. The basolateral amygdala was electrically stimulated ten times per day for 2 days. A single stimulus was applied on the third day. Stimulated animals received injections of PBS or the zinc chelator diethildythiocarbamate acid (DEDTC) before each stimulus series. Animals were monitored with video-EEG and were perfused 3h after the last stimulus for subsequent neo-Timm and Fluoro-Jade B analysis. Zinc chelation decreased the duration of both behavioral seizures and electrical after-discharges, and also decreased the EEG spikes frequency, without changing the progression of behavioral seizure severity. These results indicate that the zinc ion may have a facilitatory role during kindling progression.

Increased CCL2, CCL3, CCL5, and IL-1β cytokine concentration in piriform cortex, hippocampus, and neocortex after pilocarpine-induced seizures.

BackgroundCytokines and chemokines play an important role in the neuroinflammatory response to an initial precipitating injury such as status epilepticus (SE). These signaling molecules participate in recruitment of immune cells, including brain macrophages (microglia), as well as neuroplastic changes, deterioration of damaged tissue, and epileptogenesis. This study describes the temporal and brain region pattern expression of numerous cytokines, including chemokines, after pilocarpine-induced seizures and discusses them in the larger context of their potential involvement in the changes that precede the development of epilepsy.FindingsAdult rats received pilocarpine to induce SE and 90 min after seizure onset were treated with diazepam to mitigate seizures. Rats were subsequently deeply anesthetized and brain regions (hippocampus, piriform cortex, neocortex, and cerebellum) were freshly dissected at 2, 6, and 24 h or 5 days after seizures. Using methodology identical to our previous studies, simultaneous assay of multiple cytokines (CCL2, CCL3, CCL5, interleukin IL-1β, tumor necrosis factor (TNF-α)), and vascular endothelial growth factor (VEGF) was performed and compared to control rats. These proteins were selected based on existing evidence implicating them in the epileptogenic progression. A robust increase in CCL2 and CCL3 concentrations in the hippocampus, piriform cortex, and neocortex was observed at all time-points. The concentrations peaked with a ~200-fold increase 24 h after seizures and were two orders of magnitude greater than the significant increases observed for CCL5 and IL-1β in the same brain structures. TNF-α levels were altered in the piriform cortex and neocortex (24 h) and in the hippocampus (5 days) after SE.ConclusionsPilocarpine-induced status epilepticus causes a rapid increase of multiple cytokines in limbic and neocortical regions. Understanding the precise spatial and temporal pattern of cytokines and chemokine changes could provide more viable therapeutic targets to reduce, reverse, or prevent the development of epilepsy following a precipitating injury.

Elucidating the Neurotoxicity of the Star Fruit

Caramboxin: Patients suffering from chronic kidney disease are frequently intoxicated after ingesting star fruit. The main symptoms of this intoxication are named in the picture. Bioguided chemical procedures resulted in the discovery of caramboxin, which is a phenylalanine-like molecule that is responsible for intoxication. Functional experiments in vivo and in vitro point towards the glutamatergic ionotropic molecular actions of caramboxin, which explains its convulsant and neurodegenerative properties.

Correlation between shaking behaviors and seizure severity in five animal models of convulsive seizures

Wet dog shakes (WDS) and head shakes (HS) are associated with experimentally induced convulsive seizures. We sought to determine whether these behaviors are correlated or not with major (status epilepticus (SE) or fully kindled animals) or minor (non-SE or partially kindled animals) seizure severity. WDS are directly correlated with SE induced by intracerebral star fruit extract (Averrhoa carambola) injection and with kindled animals in the amygdala fast kindling model. On the other hand, WDS are inversely correlated with SE induced by intracerebral bicuculline and pilocarpine injections. Systemic pilocarpine in animals pretreated with methyl-scopolamine barely induced WDS or HS. The role of shaking behaviors may vary from ictal to anticonvulsant depending on the experimental seizure model, circuitries involved, and stimulus intensity. The physical presence of acrylic helmets may per se inhibit the HS response. Also, methyl-scopolamine, a drug incapable of crossing the blood-brain barrier, can induce HS in animals without acrylic helmets.

Oropouche virus experimental infection in the golden hamster (Mesocrisetus auratus)

Oropouche virus (OROV), of the family Bunyaviridae, is the second most frequent arbovirus causing febrile disease in Brazil. In spite of this, little is known about pathogenesis of OROV infection. This report describes an experimental model of OROV in golden hamster (Mesocricetus auratus). Following subcutaneous inoculation of OROV, over 50% of the animals developed disease characterized by lethargy, ruffled fur, shivering, paralysis, and approximately one third died. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation to collect tissue samples from brain, heart, liver, lung, spleen, muscle and blood for virus titration, histology and OROV immunohistochemistry. OROV was detected in high titers in blood, liver and brain, but not in the other organs. Histopathology revealed meningoencephalitis and hepatitis, with abundant OROV antigen detected in liver and brain. Diffuse galectin-3 immunostaining in brain and liver supports microglial and Kupfer cells activation. This is the first description of an experimental model for OROV infection and should be helpful to study pathogenesis and possibly to test antiviral interventions such as drugs and vaccine candidates.

Morphological Alterations in Newly Born Dentate Gyrus Granule Cells That Emerge after Status Epilepticus Contribute to Make Them Less Excitable

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.

A proliferação de neurônios granulares hipocampais aumenta e os dendritos dos novos neurônios são anormais no modelo experimental de ELT induzida por pilocarpina

Hippocampal doublecortin-positive granular neurons have abnormal dendritic morphology in the temporal lobe epilepsy pilocarpine model To quantify the number and morphology of doublecortin-positive (DCX+) granular neurons in a temporal lobe epilepsy model, Wistar rats were treated with pilocarpine and, 90 min after status epilepticus (SE) onset, with diazepam. Controls received saline instead of pilocarpine. Rats were monitored 8 h per day to record spontaneous recurrent seizures (SRS) and perfused 30 days after SE. A series of sections containing the hippocampus was immunolabeled for DCX. The stereological optical fractionator method was used to count DCX+ neurons in granular cell layer (GCL) and subgranular zone of the dentate gyrus (DG). DCX+ neurons (n = 40) were digitally reconstructed and their dendritic distribution measured with Sholl’s concentric spheres analysis. The first sphere has the radius equal to the GCL thickness followed by spheres with increments of 50 μm in their radii. Neo-Timm staining was performed to evaluate the mossy fiber sprouting. Statistical difference in Student and Mann-Whitney tests at p < 0.05, values are mean ± SEM. Each animal had 8.4 ± 3.7 SRS recorded. The DCX+ counting revealed the presence of 8,987 ± 1,986 neurons in controls DG (n = 7) and 19,337 ± 4,715 neurons in epileptic DG (n = 7). Basal dendrites were observed in 85% of DCX+ neurons in epileptics and in 30% of DCX+ neurons in controls with a length of 63.9 ± 9.7 μm and 19.8 ± 5.3 μm, respectively. Apical dendrites of DCX+ neurons had ~500 μm in both groups, but the epileptics had more nodes (3.9 ± 0.3) and dendrites emerging from GCL (4.1 ± 0.4) than controls (2.47 ± 0.2 nodes and 2.95 ± 0.3 dendrites). The epileptic rats had more dendritic endings (4.55 ± 0.5) than controls (1.7 ± 0.4) in the first 50 μm of the molecular layer (GCL + 50). Epileptic rats had more DCX+ dendritic length in GCL (168 ± 14 μm against 126 ± 11 μm in controls) and GCL + 50 (231 ± 26 μm against 163 ± 17 μm in controls); the contrary occurred in GCL + 100 (94 ± 23 μm against 160 ± 23 μm in controls). Neo-Timm staining revealed a confluent band of silver granules in epileptic DG with a mean thickness of 48 μm (equivalent to GCL + 50) and no band in controls. Hilar basal dendrites were more frequent, long and ramified and the apical dendrites presented intense ramification in the inner molecular layer of epileptic rats. Dendrites were more abundant in regions with mossy fiber sprouting. Formation of synapses between these new dendrites and axons could contribute to the pathological process of hippocampal epileptogenesis.