Suppression of synapsin II inhibits the formation and maintenance of synapses in hippocampal culture.

Numerous synaptic proteins, including several integral membrane proteins, have been assigned roles in synaptic vesicle fusion with or retrieval from the presynaptic plasma membrane. In contrast, the synapsins, neuron-specific phosphoproteins associated with the cytoplasmic surface of synaptic vesicles, appear to play a much broader role, being involved in the regulation of neurotransmitter release and in the organization of the nerve terminal. Here we have administered antisense synapsin II oligonucleotides to dissociated hippocampal neurons, either before the onset of synaptogenesis or 1 week after the onset of synaptogenesis. In both cases, synapsin II was no longer detectable within 24-48 h of treatment. After 5 days of treatment, cultures were analyzed for the presence of synapses by synapsin I and synaptophysin antibody labeling and by electron microscopy. Cultures in which synapsin II was suppressed after axon elongation, but before synapse formation, did not develop synapses. Cultures in which synapsin II was suppressed after the development of synapses lost most of their synapses. Remarkably, with the removal of the antisense oligonucleotides, neurons and their synaptic connections recovered. These studies lead us to conclude that synapsin II is involved in the formation and maintenance of synapses in hippocampal neurons.

Impairment of axonal development and of synaptogenesis in hippocampal neurons of synapsin I-deficient mice.

Synapsin I, the most abundant of all neuronal phosphoproteins, is enriched in synaptic vesicles. It has been hypothesized to regulate synaptogenesis and neurotransmitter release from adult nerve terminals. The evidence for such roles has been highly suggestive but not compelling. To evaluate the possible involvement of synapsin I in synaptogenesis and in the function of adult synapses, we have generated synapsin I-deficient mice by homologous recombination. We report herein that outgrowth of predendritic neurites and of axons was severely retarded in the hippocampal neurons of embryonic synapsin I mutant mice. Furthermore, synapse formation was significantly delayed in these mutant neurons. These results indicate that synapsin I plays a role in regulation of axonogenesis and synaptogenesis.

Hippocampal long-term depression and depotentiation are defective in mice carrying a targeted disruption of the gene encoding the RI beta subunit of cAMP-dependent protein kinase.

The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). We have combined pharmacologic and genetic approaches to demonstrate that PKA activity is required for both homosynaptic LTD and depotentiation and that a specific neuronal isoform of type I regulatory subunit (RI beta) is essential. Mice carrying a null mutation in the gene encoding RI beta were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CA1 synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RI beta mutant mice. Despite a compensatory increase in the related RI alpha protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RI beta. Since the late phase of CA1 LTP also requires PKA but is normal in RI beta mutant mice, our data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits.

Role of the cotransporter KCC2 in cortical excitatory synapse development and febrile seizure susceptibility

Le co-transporteur KCC2 spécifique au potassium et chlore a pour rôle principal de réduire la concentration intracellulaire de chlore, entraînant l’hyperpolarisation des courants GABAergic l’autorisant ainsi à devenir inhibiteur dans le cerveau mature. De plus, il est aussi impliqué dans le développement des synapses excitatrices, nommées aussi les épines dendritiques. Le but de notre projet est d’étudier l’effet des modifications concernant l'expression et la fonction de KCC2 dans le cortex du cerveau en développement dans un contexte de convulsions précoces. Les convulsions fébriles affectent environ 5% des enfants, et ce dès la première année de vie. Les enfants atteints de convulsions fébriles prolongées et atypiques sont plus susceptibles à développer l’épilepsie. De plus, la présence d’une malformation cérébrale prédispose au développement de convulsions fébriles atypiques, et d’épilepsie du lobe temporal. Ceci suggère que ces pathologies néonatales peuvent altérer le développement des circuits neuronaux irréversiblement. Cependant, les mécanismes qui sous-tendent ces effets ne sont pas encore compris. Nous avons pour but de comprendre l'impact des altérations de KCC2 sur la survenue des convulsions et dans la formation des épines dendritiques. Nous avons étudié KCC2 dans un modèle animal de convulsions précédemment validé, qui combine une lésion corticale à P1 (premier jour de vie postnatale), suivie d'une convulsion induite par hyperthermie à P10 (nommés rats LHS). À la suite de ces insultes, 86% des rats mâles LHS développent l’épilepsie à l’âge adulte, au même titre que des troubles d’apprentissage. À P20, ces animaux presentent une augmentation de l'expression de KCC2 associée à une hyperpolarisation du potentiel de réversion de GABA. De plus, nous avons observé des réductions dans la taille des épines dendritiques et l'amplitude des courants post-synaptiques excitateurs miniatures, ainsi qu’un déficit de mémoire spatial, et ce avant le développement des convulsions spontanées. Dans le but de rétablir les déficits observés chez les rats LHS, nous avons alors réalisé un knock-down de KCC2 par shARN spécifique par électroporation in utero. Nos résultats ont montré une diminution de la susceptibilité aux convulsions due à la lésion corticale, ainsi qu'une restauration de la taille des épines. Ainsi, l’augmentation de KCC2 à la suite d'une convulsion précoce, augmente la susceptibilité aux convulsions modifiant la morphologie des épines dendritiques, probable facteur contribuant à l’atrophie de l’hippocampe et l’occurrence des déficits cognitifs. Le deuxième objectif a été d'inspecter l’effet de la surexpression précoce de KCC2 dans le développement des épines dendritiques de l’hippocampe. Nous avons ainsi surexprimé KCC2 aussi bien in vitro dans des cultures organotypiques d’hippocampe, qu' in vivo par électroporation in utero. À l'inverse des résultats publiés dans le cortex, nous avons observé une diminution de la densité d’épines dendritiques et une augmentation de la taille des épines. Afin de confirmer la spécificité du rôle de KCC2 face à la région néocorticale étudiée, nous avons surexprimé KCC2 dans le cortex par électroporation in utero. Cette manipulation a eu pour conséquences d’augmenter la densité et la longueur des épines synaptiques de l’arbre dendritique des cellules glutamatergiques. En conséquent, ces résultats ont démontré pour la première fois, que les modifications de l’expression de KCC2 sont spécifiques à la région affectée. Ceci souligne les obstacles auxquels nous faisons face dans le développement de thérapie adéquat pour l’épilepsie ayant pour but de moduler l’expression de KCC2 de façon spécifique.

Role of the cotransporter KCC2 in cortical excitatory synapse development and febrile seizure susceptibility

Le co-transporteur KCC2 spécifique au potassium et chlore a pour rôle principal de réduire la concentration intracellulaire de chlore, entraînant l’hyperpolarisation des courants GABAergic l’autorisant ainsi à devenir inhibiteur dans le cerveau mature. De plus, il est aussi impliqué dans le développement des synapses excitatrices, nommées aussi les épines dendritiques. Le but de notre projet est d’étudier l’effet des modifications concernant l'expression et la fonction de KCC2 dans le cortex du cerveau en développement dans un contexte de convulsions précoces. Les convulsions fébriles affectent environ 5% des enfants, et ce dès la première année de vie. Les enfants atteints de convulsions fébriles prolongées et atypiques sont plus susceptibles à développer l’épilepsie. De plus, la présence d’une malformation cérébrale prédispose au développement de convulsions fébriles atypiques, et d’épilepsie du lobe temporal. Ceci suggère que ces pathologies néonatales peuvent altérer le développement des circuits neuronaux irréversiblement. Cependant, les mécanismes qui sous-tendent ces effets ne sont pas encore compris. Nous avons pour but de comprendre l'impact des altérations de KCC2 sur la survenue des convulsions et dans la formation des épines dendritiques. Nous avons étudié KCC2 dans un modèle animal de convulsions précédemment validé, qui combine une lésion corticale à P1 (premier jour de vie postnatale), suivie d'une convulsion induite par hyperthermie à P10 (nommés rats LHS). À la suite de ces insultes, 86% des rats mâles LHS développent l’épilepsie à l’âge adulte, au même titre que des troubles d’apprentissage. À P20, ces animaux presentent une augmentation de l'expression de KCC2 associée à une hyperpolarisation du potentiel de réversion de GABA. De plus, nous avons observé des réductions dans la taille des épines dendritiques et l'amplitude des courants post-synaptiques excitateurs miniatures, ainsi qu’un déficit de mémoire spatial, et ce avant le développement des convulsions spontanées. Dans le but de rétablir les déficits observés chez les rats LHS, nous avons alors réalisé un knock-down de KCC2 par shARN spécifique par électroporation in utero. Nos résultats ont montré une diminution de la susceptibilité aux convulsions due à la lésion corticale, ainsi qu'une restauration de la taille des épines. Ainsi, l’augmentation de KCC2 à la suite d'une convulsion précoce, augmente la susceptibilité aux convulsions modifiant la morphologie des épines dendritiques, probable facteur contribuant à l’atrophie de l’hippocampe et l’occurrence des déficits cognitifs. Le deuxième objectif a été d'inspecter l’effet de la surexpression précoce de KCC2 dans le développement des épines dendritiques de l’hippocampe. Nous avons ainsi surexprimé KCC2 aussi bien in vitro dans des cultures organotypiques d’hippocampe, qu' in vivo par électroporation in utero. À l'inverse des résultats publiés dans le cortex, nous avons observé une diminution de la densité d’épines dendritiques et une augmentation de la taille des épines. Afin de confirmer la spécificité du rôle de KCC2 face à la région néocorticale étudiée, nous avons surexprimé KCC2 dans le cortex par électroporation in utero. Cette manipulation a eu pour conséquences d’augmenter la densité et la longueur des épines synaptiques de l’arbre dendritique des cellules glutamatergiques. En conséquent, ces résultats ont démontré pour la première fois, que les modifications de l’expression de KCC2 sont spécifiques à la région affectée. Ceci souligne les obstacles auxquels nous faisons face dans le développement de thérapie adéquat pour l’épilepsie ayant pour but de moduler l’expression de KCC2 de façon spécifique.

Epilepsies Associated With Hippocampal Sclerosis.

Hippocampal sclerosis (HS) is considered the most frequent neuropathological finding in patients with mesial temporal lobe epilepsy (MTLE). Hippocampal specimens of pharmacoresistant MTLE patients that underwent epilepsy surgery for seizure control reveal the characteristic pattern of segmental neuronal cell loss and concomitant astrogliosis. However, classification issues of hippocampal lesion patterns have been a matter of intense debate. International consensus classification has only recently provided significant progress for comparisons of neurosurgical and clinic-pathological series between different centers. The respective four-tiered classification system of the International League Against Epilepsy subdivides HS into three types and includes a term of gliosis only, no-HS. Future studies will be necessary to investigate whether each of these subtypes of HS may be related to different etiological factors or with postoperative memory and seizure outcome. Molecular studies have provided potential deeper insights into the pathogenesis of HS and MTLE on the basis of epilepsy-surgical hippocampal specimens and corresponding animal models. These include channelopathies, activation of NMDA receptors, and other conditions related to Ca(2+) influx into neurons, the imbalance of Ca(2+)-binding proteins, acquired channelopathies that increase neuronal excitability, paraneoplastic and non-paraneoplastic inflammatory events, and epigenetic regulation promoting or facilitating hippocampal epileptogenesis. Genetic predisposition for HS is clearly suggested by the high incidence of family history in patients with HS, and by familial MTLE with HS. So far, it is clear that HS is multifactorial and there is no individual pathogenic factor either necessary or sufficient to generate this intriguing histopathological condition. The obvious variety of pathogenetic combinations underlying HS may explain the multitude of clinical presentations, different responses to clinical and surgical treatment. We believe that the stratification of neuropathological patterns can help to characterize specific clinic-pathological entities and predict the postsurgical seizure control in an improved fashion.

Long-term Postoperative Atrophy Of Contralateral Hippocampus And Cognitive Function In Unilateral Refractory Mtle With Unilateral Hippocampal Sclerosis.

This study aimed to evaluate long-term atrophy in contralateral hippocampal volume after surgery for unilateral MTLE, as well as the cognitive outcome for patients submitted to either selective transsylvian amygdalohippocampectomy (SelAH) or anterior temporal lobe resection (ATL). We performed a longitudinal study of 47 patients with MRI signs of unilateral hippocampal sclerosis (23 patients with right-sided hippocampal sclerosis) who underwent surgical treatment for MTLE. They underwent preoperative/postoperative high-resolution MRI as well as neuropsychological assessment for memory and estimated IQ. To investigate possible changes in the contralateral hippocampus of patients, we included 28 controls who underwent two MRIs at long-term intervals. The volumetry using preoperative MRI showed significant hippocampal atrophy ipsilateral to the side of surgery when compared with controls (p<0.0001) but no differences in contralateral hippocampal volumes. The mean postoperative follow-up was 8.7 years (± 2.5 SD; median=8.0). Our patients were classified as Engel I (80%), Engel II (18.2%), and Engel III (1.8%). We observed a small but significant reduction in the contralateral hippocampus of patients but no volume changes in controls. Most of the patients presented small declines in both estimated IQ and memory, which were more pronounced in patients with left TLE and in those with persistent seizures. Different surgical approaches did not impose differences in seizure control or in cognitive outcome. We observed small declines in cognitive scores with most of these patients, which were worse in patients with left-sided resection and in those who continued to suffer from postoperative seizures. We also demonstrated that manual volumetry can reveal a reduction in volume in the contralateral hippocampus, although this change was mild and could not be detected by visual analysis. These new findings suggest that dynamic processes continue to act after the removal of the hippocampus, and further studies with larger groups may help in understanding the underlying mechanisms.

Longitudinal Analysis Of Hippocampal T2 Relaxometry In Fmtle.

To investigate the degree of T2 relaxometry changes over time in groups of patients with familial mesial temporal lobe epilepsy (FMTLE) and asymptomatic relatives. We conducted both cross-sectional and longitudinal analyses of T2 relaxometry with Aftervoxel, an in-house software for medical image visualization. The cross-sectional study included 35 subjects (26 with FMTLE and 9 asymptomatic relatives) and 40 controls; the longitudinal study was composed of 30 subjects (21 with FMTLE and 9 asymptomatic relatives; the mean time interval of MRIs was 4.4 ± 1.5 years) and 16 controls. To increase the size of our groups of patients and relatives, we combined data acquired in 2 scanners (2T and 3T) and obtained z-scores using their respective controls. General linear model on SPSS21® was used for statistical analysis. In the cross-sectional analysis, elevated T2 relaxometry was identified for subjects with seizures and intermediate values for asymptomatic relatives compared to controls. Subjects with MRI signs of hippocampal sclerosis presented elevated T2 relaxometry in the ipsilateral hippocampus, while patients and asymptomatic relatives with normal MRI presented elevated T2 values in the right hippocampus. The longitudinal analysis revealed a significant increase in T2 relaxometry for the ipsilateral hippocampus exclusively in patients with seizures. The longitudinal increase of T2 signal in patients with seizures suggests the existence of an interaction between ongoing seizures and the underlying pathology, causing progressive damage to the hippocampus. The identification of elevated T2 relaxometry in asymptomatic relatives and in patients with normal MRI suggests that genetic factors may be involved in the development of some mild hippocampal abnormalities in FMTLE.

Hippocampal Dysplasia With Balloon Cells: Case Report And Discussion On Classification.

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Long-term Spinal Ventral Root Reimplantation, But Not Bone Marrow Mononuclear Cell Treatment, Positively Influences Ultrastructural Synapse Recovery And Motor Axonal Regrowth.

We recently proposed a new surgical approach to treat ventral root avulsion, resulting in motoneuron protection. The present work combined such a surgical approach with bone marrow mononuclear cells (MC) therapy. Therefore, MC were added to the site of reimplantation. Female Lewis rats (seven weeks old) were subjected to unilateral ventral root avulsion (VRA) at L4, L5 and L6 levels and divided into the following groups (n = 5 for each group): Avulsion, sealant reimplanted roots and sealant reimplanted roots plus MC. After four weeks and 12 weeks post-surgery, the lumbar intumescences were processed by transmission electron microscopy, to analyze synaptic inputs to the repaired α motoneurons. Also, the ipsi and contralateral sciatic nerves were processed for axon counting and morphometry. The ultrastructural results indicated a significant preservation of inhibitory pre-synaptic boutons in the groups repaired with sealant alone and associated with MC therapy. Moreover, the average number of axons was higher in treated groups when compared to avulsion only. Complementary to the fiber counting, the morphometric analysis of axonal diameter and g ratio demonstrated that root reimplantation improved the motor component recovery. In conclusion, the data herein demonstrate that root reimplantation at the lesion site may be considered a therapeutic approach, following proximal lesions in the interface of central nervous system (CNS) and peripheral nervous system (PNS), and that MC therapy does not further improve the regenerative recovery, up to 12 weeks post lesion.

Effects of Commonly Used Solubilizing Agents on a Model Nerve-Muscle Synapse

Solubility represents a limiting factor when testing new compounds in animal experiments, since solubilizing agents generally have pharmacological effects that can interfere with the studied substance. Vehicles are commonly used for solubilizing certain substances including apolar and polar extracts obtained from medicinal plants. In this study, fifteen vehicles were investigated on mice neuromuscular preparations. A known in vitro neuroblocker myotoxin from Bothrops jararacussu venom, bothropstoxin-I, was used as a pharmacological tool for testing the medicinal potential of apolar and polar extracts (hexane, dichloromethane, ethyl acetate and methanol) obtained from Casearia sylvestris Sw. leaves, which in turn were used for testing their solubility and concomitantly to produce no change on basal response of indirectly stimulated mouse phrenic nerve-diaphragm preparations. Taken together in vitro biological system and extracts solubility, our results showed that dimethyl sulphoxide and polyethylene glycol 400 were the better vehicles, and methanol extract solubilized on PEG 400 was the only one able to act against the paralysis induced by the myotoxin. Thus, this study points out to the relevant role that vehicles exhibit for extracting the potential pharmacological activity of plants in a given test system.

Nuclear calcium signaling evoked by cholinergic stimulation in hippocampal CA1 pyramidal neurons

The cholinergic system is thought to play an important role in hippocampal-dependent learning and memory. However, the mechanism of action of the cholinergic system in these actions in not well understood. Here we examined the effect of muscarinic receptor stimulation in hippocampal CA1 pyramidal neurons using whole-cell recordings in acute brain slices coupled with high-speed imaging of intracellular calcium. Activation of muscarinic acetylcholine receptors by synaptic stimulation of cholinergic afferents or application of muscarinic agonist in CA1 pyramidal neurons evoked a focal rise in free calcium in the apical dendrite that propagated as a wave into the soma and invaded the nucleus. The calcium rise to a single action potential was reduced during muscarinic stimulation. Conversely, the calcium rise during trains of action potentials was enhanced during muscarinic stimulation. The enhancement of free intracellular calcium was most pronounced in the soma and nuclear regions. In many cases, the calcium rise was distinguished by a clear inflection in the rising phase of the calcium transient, indicative of a regenerative response. Both calcium waves and the amplification of action potential-induced calcium transients were blocked the emptying of intracellular calcium stores or by antagonism of inositol 1,4,5-trisphosphate receptors with heparin or caffeine. Ryanodine receptors were not essential for the calcium waves or enhancement of calcium responses. Because rises in nuclear calcium are known to initiate the transcription of novel genes, we suggest that these actions of cholinergic stimulation may underlie its effects on learning and memory.

Overshadowing in conditioned taste aversion or in conditioned emotional response after neonatal ventral hippocampal lesions in rats

The neonatal hippocampus lesion thought to model schizophrenia should show the same modifications in behavioural tests as other models, especially pharmacological models. namely decreased latent inhibition, blocking and overshadowing. The present study is set out to evaluate overshadowing in order to complement our previous studies, which had tested latent inhibition. ""Overshadowing"" refers to the decreased conditioning that occurs when the to-be-conditioned stimulus is combined with another stimulus at the conditioning stage. We used the same two Pavlovian conditioning paradigms as in our previous works, namely conditioned taste aversion (CTA) and conditioned emotional response (CER). A sweet taste overshadowed a salty conditioned stimulus, and a tone overshadowed a flashing light. Totally different stimuli were used to counter possible sensory biases. The protocols were validated with two groups of Sprague Dawley rats. The same two protocols were then applied to a cohort of rats whose ventral hippocampus had been destroyed when they were 7 days old. Only rats with extended ventral hippocampus lesions were included. The overall effect of Pavlovian conditioning was attenuated, significantly so in the conditioned emotional response paradigm, but overshadowing appeared not to be modified in either the conditioned emotional response or the conditioned taste aversion paradigm. (C) 2008 Elsevier B.V. All rights reserved.

Expression of vitamin D receptor mRNA in the hippocampal formation of rats submitted to a model of temporal lobe epilepsy induced by pilocarpine

Vitamin D (VD), is a steroid hormone with multiple functions in the central nervous system (CNS), producing numerous physiological effects mediated by its receptor (VDR). Clinical and experimental studies have shown a link between VD dysfunction and epilepsy. Along these lines, the purpose of our work was to analyze the relative expression of VDR mRNA in the hippocampal formation of rats during the three periods of pilocarpine-induced epilepsy. Male Wistar rats were divided into five groups: (1) control group; rats that received saline 0.9%, i.p. and were killed 7 days after its administration (CTRL, n = 8), (2) SE group; rats that received pilocarpine and were killed 4 h after SE (SE, n = 8), (3) Silent group-7 days; rats that received pilocarpine and were killed 7 days after SE (SIL 7d, n = 8), (4) Silent group-14 days; rats that received pilocarpine and were killed 14 days after SE (SIL 14d, n = 8), (5) Chronic group; rats that received pilocarpine and were killed 60 days after the first spontaneous seizure, (chronic, n = 8). The relative expression of VDR mRNA was determined by real-time PCR. Our results showed an increase of the relative expression of VDR mRNA in the SIL 7 days, SIL 14 days and Chronic groups, respectively (0.060 +/- 0.024; 0.052 +/- 0.035; 0.085 +/- 0.055) when compared with the CTRL and SE groups (0.019 +/- 0.017; 0.019 +/- 0.025). These data suggest the VDR as a possible candidate participating in the epileptogenesis process of the pilocarpine model of epilepsy. (C) 2008 Elsevier Inc. All rights reserved.