Synaptic Vesicle Protein 2A Expression in Glutamatergic Terminals Is Associated with the Response to Levetiracetam Treatment.

Synaptic vesicle protein 2A (SV2A), the target of the antiepileptic drug levetiracetam (LEV), is expressed ubiquitously in all synaptic terminals. Its levels decrease in patients and animal models of epilepsy. Thus, changes in SV2A expression could be a critical factor in the response to LEV. Epilepsy is characterized by an imbalance between excitation and inhibition, hence SV2A levels in particular terminals could also influence the LEV response. SV2A expression was analyzed in the epileptic hippocampus of rats which responded or not to LEV, to clarify if changes in SV2A alone or together with glutamatergic or GABAergic markers may predict LEV resistance. Wistar rats were administered saline (control) or pilocarpine to induce epilepsy. These groups were subdivided into untreated or LEV-treated groups. All epileptic rats were video-monitored to assess their number of seizures. Epileptic rats with an important seizure reduction (>50%) were classified as responders. SV2A, vesicular γ-aminobutyric acid transporter and vesicular glutamate transporter (VGLUT) expression were assessed by immunostaining. SV2A expression was not modified during epilepsy. However, responders showed ≈55% SV2A-VGLUT co-expression in comparison with the non-responder group (≈40%). Thus, SV2A expression in glutamatergic terminals may be important for the response to LEV treatment.

Levetiracetam Reduced the Basal Excitability of the Dentate Gyrus without Restoring Impaired Synaptic Plasticity in Rats with Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE), the most common type of focal epilepsy, affects learning and memory; these effects are thought to emerge from changes in synaptic plasticity. Levetiracetam (LEV) is a widely used antiepileptic drug that is also associated with the reversal of cognitive dysfunction. The long-lasting effect of LEV treatment and its participation in synaptic plasticity have not been explored in early chronic epilepsy. Therefore, through the measurement of evoked field potentials, this study aimed to comprehensively identify the alterations in the excitability and the short-term (depression/facilitation) and long-term synaptic plasticity (long-term potentiation, LTP) of the dentate gyrus of the hippocampus in a lithium-pilocarpine rat model of TLE, as well as their possible restoration by LEV (1 week; 300 mg/kg/day). TLE increased the population spike (PS) amplitude (input/output curve); interestingly, LEV treatment partially reduced this hyperexcitability. Furthermore, TLE augmented synaptic depression, suppressed paired-pulse facilitation, and reduced PS-LTP; however, LEV did not alleviate such alterations. Conversely, the excitatory postsynaptic potential (EPSP)-LTP of TLE rats was comparable to that of control rats and was decreased by LEV. LEV caused a long-lasting attenuation of basal hyperexcitability but did not restore impaired synaptic plasticity in the early chronic phase of TLE.

Analysis of Differential Expression of Synaptic Vesicle Protein 2A in the Adult Rat Brain.

Synaptic vesicle protein 2A (SV2A), which plays an important role in the pathophysiology of epilepsy, is a unique vesicular protein recognized as a pharmacological target of anticonvulsant drugs. Furthermore, SV2A is a potential synaptic density marker, as it is ubiquitously expressed throughout the brain in all nerve terminals independently of their neurotransmitter content. Due to the growing interest in this protein, we thoroughly analyzed SV2A levels, expression patterns and colocalization in both excitatory and inhibitory synapses among different brain structures in healthy rats. In addition, we discuss the main semiquantitative methodologies used to study SV2A because these techniques might represent powerful tools for evaluating synaptic changes associated with brain disorders. Our results showed that the SV2A expression levels differed among the analyzed structures, and a positive correlation between the SV2A mRNA copy number and protein level was observed by Western blot. In addition, immunohistochemistry demonstrated slight but consistent asymmetrical SV2A levels in different laminated structures, and SV2A expression was increased by up to 40% in some specific layers compared to that in others. Finally, triple immunofluorescence revealed strong SV2A colocalization with GABAergic terminals, mainly around the principal cells, suggesting that SV2A primarily participates in this inhibitory system in different rat brain structures. Although the SV2A protein is considered a good candidate marker of synaptic density, our data show that changes in its expression in pathological processes must be viewed as not only increased or decreased synapse numbers but also in light of the type of neurotransmission being affected.

Differential expression of synaptic vesicle protein 2A after status epilepticus and during epilepsy in a lithium-pilocarpine model.

Synaptic vesicle protein 2A (SV2A) has become an attractive target of investigation because of its role in the pathophysiology of epilepsy; SV2A is expressed ubiquitously throughout the brain in all nerve terminals independently of their neurotransmitter content and plays an important but poorly defined role in neurotransmission. Previous studies have shown that modifications in the SV2A protein expression could be a direct consequence of disease severity. Furthermore, these SV2A modifications may depend on specific changes in the nerve tissue following the induction of epilepsy and might be present in both excitatory and inhibitory terminals. Thus, we evaluated SV2A protein expression throughout the hippocampi of lithium-pilocarpine rats after status epilepticus (SE) and during early and late epilepsy. In addition, we determined the γ-aminobutyric acid (GABA)ergic or glutamatergic nature associated with SV2A modifications. Wistar rats were treated with lithium-pilocarpine to induce SE and subsequently were shown to present spontaneous recurrent seizures (SRS). Later, we conducted an exhaustive semi-quantitative analysis of SV2A optical density (OD) throughout the hippocampus by immunohistochemistry. Levels of the SV2A protein were substantially increased in layers formed by principal neurons after SE, mainly because of GABAergic activity. No changes were observed in the early stage of epilepsy. In the late stage of epilepsy, there were minor changes in SV2A OD compared with the robust modifications of SE; however, SV2A protein expression generally showed an increment reaching significant differences in two dendritic layers and hilus, without clear modifications of GABAergic or glutamatergic systems. Our results suggest that the SV2A variations may depend on several factors, such as neuronal activity, and might appear in both excitatory and inhibitory systems depending on the epilepsy stage.

Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations.

Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression.

El pirofosfato de tiamina reduce el daño celular inducido por hipoxia en el cerebro de ratas neonatas / Thiamine pyrophosphate reduces hypoxia-induced cellular damage in neonatal rat brain

La encefalopatía por hipoxia es causa de discapacidad y requiere de nuevas estrategias terapéuticas. El pirofosfato de tiamina (PPT) es un cofactor esencial de enzimas fundamentales en el metabolismo de la glucosa, cuya disminución puede conducir a la falla en la síntesis de ATP y a la muerte celular. El objetivo de este estudio fue determinar si la administración de PPT, puede reducir el daño celular en un modelo de hipoxia neonatal en ratas. Animales de 11 días de edad fueron tratados con PPT (130 mg/kg) en dosis única o solución salina, una hora antes del protocolo de hipoxia o al término de ésta. Los cerebros fueron colectados para la evaluación del daño celular. Además, se tomaron muestras sanguíneas para evaluar los indicadores gasométricos de presión de dióxido de carbono (PaCO2) y de oxígeno (PaO2) en sangre arterial y pH. Los resultados muestran que la administración de PPT previa a la inducción de hipoxia, reduce el daño celular y restablece los indicadores gasométricos. Estos datos indican que el uso de PPT reduce el daño inducido por la hipoxia en animales neonatos.

Hypoxic encephalopathy is a leading cause of disability and requires new therapeutic strategies. Thiamine pyrophosphate (TPP) is an essential cofactor of fundamental enzymes involved in glucose metabolism. TPP reduction may lead to ATP synthesis failure and cell death. The objective of this study was to determine if TPP administration can reduce cellular damage in a model of neonatal hypoxia in rats. Eleven day old animals were treated with TPP (130 mg/kg) as a single dose or with saline solution one hour before the hypoxia protocol or after ending the protocol. The brains were collected to evaluate cellular damage. Blood samples were also collected to evaluate arterial oxygen tension (PaO2), carbon dioxide tension (PaCO2) and acidity (pH). The results showed that TPP administration previous to hypoxia induction reduces cellular damage and reestablishes arterial blood gases. These data indicate that TPP use reduces the damage induced by hypoxia in neonatal animals.

The age-dependent change in olfactory periglomerular neuronal populations is not affected by interrupting subventricular neuroblast migration in adult rats.

The olfactory bulb (OB) is rich in the number and variety of neurotransmitter and neuropeptide containing cells, in particular in the glomerular layer. Several reports suggest that numbers of some periglomerular phenotypes could change depending on age. However, it is unclear whether the different classes of periglomerular interneurons are modified or are maintained stable throughout life. Thus, our first objective was to obtain the absolute number of cells belonging to the different periglomerular phenotypes at adulthood. On the other hand, the olfactory bulb is continously supplied with newly generated periglomerular neurons produced by stem cells located in the subventricular zone (SVZ) and rostral migratory stream. Previously, we demonstrated that the implantation of a physical barrier completely prevents SVZ neuroblast migration towards the OB. Then, another objective of this study was to evaluate whether stopping the continuous supply of SVZ neuroblasts modified the different periglomerular populations throughout time. In summary, we estimated the total number of TH-IR, CalB-IR, CalR-IR and GAD-IR cells in the OB glomerular layer at several time points in control and barrier implanted adult rats. In addition, we estimated the volume of glomerular, granular and complete OB. Our main finding was that the number of the four main periglomerular populations is age-dependent, even after impairment of subventricular neuroblast migration. Furthermore, we established that these changes do not correlate with changes in the volume of glomerular layer.

The rostral migratory stream is a neurogenic niche that predominantly engenders periglomerular cells: in vivo evidence in the adult rat brain.

In vitro studies support the existence of adult neural stem cells in the rostral migratory stream (RMS). The evidence supporting this possibility in vivo is scarce. We then explore this issue by taking advantage of a rat model in which a physical barrier implanted in the brain interrupted the migration of neuroblasts derived from the SVZ along the RMS at the level of its vertical limb. The presence of local stem cells and neurogenesis were then established by estimating the number of nuclei labeled with bromo-deoxyuridine (BrdU), the number of doublecortin-positive neuroblasts and the existence of cells displaying co-localization of BrdU and Sox-2 immunoreactivity along the RMS, at different time points following barrier implantation. Estimations of the number of the granular and periglomerular neurons integrated into the corresponding layers of the olfactory bulb of implanted rats established that stem cells in the RMS give rise predominantly to periglomerular neurons. Our results then support the notion that the RMS is indeed a region in which neurogenesis is taking place in the adult brain. They also support that the relative location of the neurogenic niche might imprint, at least in some degree, the identity and lineage of the neuroblasts arising from them.