Vulnerability to climate change of a microendemic lizard species from the central Andes.

Given the rapid loss of biodiversity as consequence of climate change, greater knowledge of ecophysiological and natural history traits are crucial to determine which environmental factors induce stress and drive the decline of threatened species. Liolaemus montanezi (Liolaemidae), a xeric-adapted lizard occurring only in a small geographic range in west-central Argentina, constitutes an excellent model for studies on the threats of climate change on such microendemic species. We describe field data on activity patterns, use of microhabitat, behavioral thermoregulation, and physiology to produce species distribution models (SDMs) based on climate and ecophysiological data. Liolaemus montanezi inhabits a thermally harsh environment which remarkably impacts their activity and thermoregulation. The species shows a daily bimodal pattern of activity and mostly occupies shaded microenvironments. Although the individuals thermoregulate at body temperatures below their thermal preference they avoid high-temperature microenvironments probably to avoid overheating. The population currently persists because of the important role of the habitat physiognomy and not because of niche tracking, seemingly prevented by major rivers that form boundaries of their geographic range. We found evidence of habitat opportunities in the current range and adjacent areas that will likely remain suitable to the year 2070, reinforcing the relevance of the river floodplain for the species' avoidance of extinction.

Low microbial biomass within the reproductive tract of mid-lactation dairy cows: A study approach.

The microbiome from the reproductive tract is being investigated for its putative effect on fertility, embryo development, and health status of the human or animal host postpartum. Besides the presence of a vaginal microbiome, recent studies have claimed the existence and putative role of the uterine microbiome. Yet, the extremely low bacterial numbers and high eukaryotic/prokaryotic DNA ratio make this a highly challenging environment to study with next-generation sequencing (NGS) techniques. Here, we describe the methodological challenges that are typically encountered when performing an accurate analysis of low microbial biomass samples, illustrated by data of our own observational study. In terms of the research question, we compared the microbial composition throughout different parts of the reproductive tract of clinically healthy, mid-lactation Holstein-Friesian cows. Samples were collected from 5 dairy cows immediately after killing. Swabs were taken from the vagina, and from 4 pre-established locations of the uterine endometrium. In addition to the conventional DNA extraction blank controls, sterile swabs rubbed over disinfected disposable gloves and the disinfected surface of the uterus (tunica serosa) before incision were taken as sampling controls. The DNA extraction, DNA quantification, quantitative PCR of the 16S rRNA genes, and 16S rRNA gene sequencing were performed. In terms of NGS data analysis, we performed prevalence-based filtering of putative contaminant operational taxonomic units (OTU) using the decontam R package. Although the bacterial composition differed between the vagina and uterus, no differences in bacterial community structure (α and ß diversity) were found among the different locations in the uterus. At phylum level, uterine samples had a greater relative abundance of Proteobacteria, and a lesser relative abundance of Firmicutes than vaginal samples. The number of shared OTU between vagina and uterus was limited, suggesting the existence of bacterial transmission routes other than the transcervical one to the uterus. The mid-lactation bovine genital tract is a low microbial biomass environment, which makes it difficult to distinguish between its constitutive versus contaminant microbiome. The integration of key controls is therefore strictly necessary to decrease the effect of accidentally introduced contaminant sequences and improve the reliability of results in samples with low microbial biomass.

Estrategias de Comunicación para el control de criaderos de Aedes aegypti resultantes del brote epidémico de Dengue de 2017-2018 en Asunción y Central, Paraguay / Communication Strategies for controlling Aedes aegypti breading sites as a result of Dengue epidemic 2017-2018 in Asunción and Central, Paraguay

Introducción: Los criaderos del mosquito vector Aedes aegypti que impactaron en el brote epidémico de Dengue de 2017-2018, en Asunción y Departamento Central, Paraguay, son los mismos apuntados desde el 2011, pero la relación de la población con los criaderos se ha modificado en el transcurso de esos años. Objetivo: Describir evidencias de la relación población-criaderos y apuntar estrategias de comunicación para el control del vector Aedes aegypti. Metodología: Estudio descriptivo-analítico que tomó dos aportes, siendo el primero los registros de criaderos en base de datos en el período de 2 de enero a 5 de abril de 2018, compilados a partir de Rastrillaje realizado por el Servicio Nacional de Erradicación del Paludismo (SENEPA) durante el brote epidémico, y el segundo las técnicas cualitativas empleadas durante la Misión Internacional de OPS a Paraguay entre 2 a 13 de abril de 2018 para apoyo técnico al brote de Dengue. Resultados: Se denotó que para el control más efectivo de los criaderos de Aedes aegypti la atención debe estar centrada en estrategias de comunicación que sean segmentadas por audiencia. De un lado porque las conductas para el control vectorial están asimiladas por la población en grados diferenciados y por otro porque cada criadero impacta una audiencia específica. Conclusión: La información obtenida con este estudio descriptivo-analítico permite impulsar acciones y estrategias de comunicación segmentada por audiencia que pueden realizarse por intermedio de la Estrategia de Gestión Integrada (EGI-Dengue/Vectores) de Paraguay. Palabras Claves: Comunicación; Aedes aegypti; Criaderos; Paraguay

Introduction: The Aedes aegypti breeding sites that affected the 2017-2018 Dengue outbreak, in Asunción and Central Department, Paraguay, are the same as those reported since 2011. However, the relationship between people and breeding sites has been modified in the course of those years. Objective: Describing evidence of the populationbreeding site relationship and the communication strategies for Aedes aegypti prevention and control. Methodology: A descriptive-analytical study considering data about breeding sites from January 2 to April 5, 2018, compiled by SENEPA, and the results of the qualitative technics used by PAHO International Mission in Paraguay from April 2 to 13, 2018, aiming technical support for the Dengue outbreak. Results: For a more effective control of Aedes aegypti breeding sites, attention should be given to communication strategies segmented by audience. On one hand, individuals in different degrees have already assimilated conducts, and on the other because each breeding sites affects a specific audience. Conclusion: The information obtained with this descriptive-analytical study can be used as a baseline for actions and communication strategies that are segmented by audience. They could be implemented through the Integrated Management Strategy (EGIDengue/ Vectors) of Paraguay. Keywords: Communication; Aedes aegypti; breeding site; Paraguay

Criaderos de Aedes aegypti en la ciudad de Asunción, Paraguay durante los años 2011-2014 / Aedes aegypti breeding site in the city of Asunción, Paraguay in the 2011-2014 periods

INTRODUCCIÓN: Las poblaciones propensas a sufrir la aparición del dengue y otras enfermedades transmitidas por el Aedes aegypti en aquellas que no cuentan con un adecuado sistema de eliminación de residuos (desechos sólidos) y el suministro de agua potable es deficiente, lo cual contribuye a la aparición de los criaderos del mosquito vector. OBJETIVO: Describir los tipos de criaderos de Aedes aegypti en la ciudad de Asunción en el periodo 2011-2014. METODOLOGÍA: Estudio observacional descriptivo sobre los criaderos registrados en la base de datos del Servicio Nacional de Erradicación del Paludismo del levantamiento larval de Aedes aegypti en Asunción en el periodo 2011-2014. Resultados: Se denotó el predominio de criaderos inservibles, representando entre el 58% y el 72% del total de criaderos positivos encontrados, seguidos de los criaderos útiles (26% y el 40%), y por último los criaderos naturales (≤2%). Los criaderos útiles, inservibles y naturales exhiben una homogeneidad en los diferentes años analizados, representada por floreros, bebedero con agua para los criaderos considerados de utilidad para el ciudadano; neumáticos usados, cubetas descartables y desechos sólidos para los criaderos inservibles y axilas de hojas en lo que respecta a criaderos naturales. CONCLUSIÓN: La información obtenida podrá optimizar las decisiones y acciones del control vectorial del Aedes aegypti en lo referente a la focalización de los criaderos y su posterior disposición final por parte de la comuna o entidades responsables.

INTRODUCTION: The populations prone to dengue and other diseases transmitted by Aedes aegypti are those that do not have an adequate waste disposal system (solid waste) and the drinking water supply is deficient, which contributes to the emergence of mosquito vector breeding containers. OBJECTIVE: To describe the types of Aedes aegypti breeding site in the city of Asunción in the 2011-2014period. METHODOLOGY: Observational descriptive study on breeding siteregistered in the database of the National Malaria Eradication Service of the larval survey of Aedes aegypti in Asunción in the 2011- 2014period. Results: There was a predominance of unusable breeding sites, representing between 58% and 72% of the total number of positive breeding, followed by the useful breeding site (26% and 40%), and finally the natural breeding sites (≤2%) . The useful, unusable and natural breeding places exhibit a homogeneity in the different years analyzed, represented by vases, drinker with water for the breeding site considered useful for people; used tires, disposable buckets and unserviceable solid waste for useless hatcheries and leaf armpits for natural breeding site. CONLCUSION: The information obtained will allow optimizing decisions and actions onAedes aegypti vector control in relation to the targeting of breeding sites and their subsequent final disposal by the commune or responsible entities.

In vitro colonisation of the distal colon by Akkermansia muciniphila is largely mucin and pH dependent.

Host mucin is the main constituent of the mucus layer that covers the gut epithelium of the host, and an important source of glycans for the bacteria colonising the intestine. Akkermansia muciniphila is a mucin-degrading bacterium, abundant in the human gut, that is able to produce acetate and propionate during this degradation process. A. muciniphila has been correlated with human health in previous studies, but a mechanistic explanation is lacking. In this study, the main site of colonisation was characterised alongside additional conditions, such as differences in colon pH, prebiotic supplementation and variable mucin supply. To overcome the limitations of in vivo studies concerning variations in mucin availability and difficult access to proximal regions of the colon, a dynamic in vitro gut model (SHIME) was used. In this model, A. muciniphila was found to colonise the distal colon compartment more abundantly than the proximal colon ((±8 log copies/ml compared to ±4 log copies/ml) and the preference for the distal compartment was found to be pH-dependent. The addition of mucin caused a specific increase of A. muciniphila (±4.5 log increase over two days), far exceeding the response of other bacteria present, together with an increase in propionate. These findings suggest that colonisation and mucin degradation by A. muciniphila is dependent on pH and the concentration of mucin. Our results revealed the preference of A. muciniphila for the distal colon environment due to its higher pH and uncovered the quick and stable response of A. muciniphila to mucin supplementation.

An endoplasmic reticulum trafficking signal regulates surface expression of ß4 subunit of a voltage- and Ca²âº-activated K⁺ channel.

Voltage-dependent and calcium-activated K⁺ (MaxiK, BK) channels are widely expressed in many tissues and organs where they play various physiological roles. Here we report discovery of a functional trafficking signal in MaxiK channel accessory ß4 subunit that could regulate activity of MaxiK α subunit (hSlo) on the plasma membrane. We demonstrate that ß4 is mostly retained within the cell and removal or mutation of ß4 trafficking signal significantly enhances its surface expression in HEK293T expression system. In hippocampal slices and cultured neurons we also observed significant ß4 expressions within the neurons. Finally, we show that unlike SV1 and ß1 subunits, ß4 shows no dominant-negative effect on MaxiK channel α subunit. Taken together, we propose ß4 subunit of MaxiK channel is mostly retained within the cells without interfering with other subunits. Removal of ß4 retention signal increases its surface expression that may lead to reduction of the MaxiK channel activity and neuronal excitability.

A new species of Silvinichthys (Siluriformes, Trichomycteridae) lacking pelvic fins from mid-elevation localities of the southern Andes, with comments on the genus.

Silvinichthys huachi new species, is described from a stream along the lower slope of the Andean Cordillera in the Provincia de San Juan, Argentina. It shares the distinctive modifications characteristic of Silvinichthys, but is distinguished from the four previously described congeners by the combination of a lack of the pelvic fin and the pelvic girdle, details of pigmentation and various meristic and morphometric features. Silvinichthys huachi is apparently endemic to the type locality situated within an arid region of western central Argentina in the Andino Cuyana Province. Major gaps in the range of species of Silvinichthys may indicate that the origin of the genus predates the uplift events that subdivided drainages along the eastern slopes of the Andean Cordillera in west central Argentina. Silvinichthys huachi is hypothesized to be the sister species of Silvinichthys bortayro.

P38 MAPK contributes to resistance and invasiveness of HER2- overexpressing breast cancer.

Intrinsic or acquired resistance to the HER2-targeted therapy trastuzumab is a clinical concern in the treatment of patients with HER2-over-expressing metastatic breast cancers. We demonstrate here that multiple models of intrinsic and acquired resistance exhibit increased phosphorylation of p38 MAPK. Kinase inhibition of p38 rescued trastuzumab sensitivity in cells with acquired resistance. In addition, knockdown of p38 increased sensitivity to trastuzumab in an intrinsically resistant cell line. We previously reported that expression of growth differentiation factor 15 (GDF15) is increased in trastuzumab-resistant HER2-overexpressing breast cancer cells. In this study, we found that exogenous GDF15 or stable overexpression of GDF15 stimulated p38 phosphorylation in HER2-positive cells, suggesting a possible mechanism by which p38 is activated in resistant cells.GDF15 stable clones showed significantly increased invasiveness, which was rescued by p38 kinase inhibition, suggesting that p38 plays a role in the pro-invasive phenotype conferred by GDF15. Importantly, immunohistochemical analysis of a breast tumor tissue array indicated a significant (p=0.0053) correlation between HER2 and phosphorylated p38 specifically in GDF15-positive tissues. Our results suggest that p38 signaling drives trastuzumab resistance and invasiveness in HER2-overexpressing breast cancer. Upstream growth factor signals that have previously been implicated in trastuzumab resistance, such as GDF15, may contribute to the increased phosphorylation of p38 found in resistant cells.

MOLECULAR INSIGHTS ON THE FUNCTIONAL MICROBIAL COMMUNITY FROM ORGANIC AND MINERAL GROWING MEDIA AND ITS INTERACTION WITH AGROBACTERIUM RHIZOGENES.

Despite numerous preventative measures, the hairy roots syndrome is an increasing problem in greenhouse horticulture. A recent survey of 177 tomato, cucumber and eggplant growers in Flanders (Belgium) revealed an increase of this disease in the last two years, with about 26% of all the tomato crops showing the syndrome. In this study, we compared the physicochemical and microbial community characteristics of inorganic and organic growing media in relation to the presence of the causative agent of the hairy roots, the plant pathogen Agrobacterium rhizogenes. We aimed to identify how the microbial and environmental interactions influenced the development and spread of this disease in a soilless cultivation system. Multivariate statistical analysis performed to assess the characteristics of each growing media revealed key variables impacting the hosted microbial community. Thus, humidity, pH, potassium and conductivity were drivers of the differences among microbial community composition. High throughput sequencing analysis of the bacterial family abundance of the communities present in organic media indicated potential competitive interactions with A. rhizogenes. Based on our hypothesis that growing media hosted a particular microbiota with potential for modulating hairy roots, we determined how the environment in organic media is reshaped to avoid establishment of A. rhizogenes. Our methodology provides a comprehensive insight into the complex bacterial interactions in horticultural media, which may be potentially applied for the development of effective control strategies and decrease in economic losses.

Morphological and electrophysiological properties of pyramidal-like neurons in the stratum oriens of Cornu ammonis 1 and Cornu ammonis 2 area of Proechimys.

Proechimys (Rodentia: Echimyidae) is a neotropical rodent of the Amazon region that has been successfully colonized in the laboratory and used for experimental medicine. Preliminary studies indicated that Proechimys (casiragua) rodents express an atypical resistance to developing a chronic epileptic condition in common models of temporal lobe epilepsy. Moreover, previous investigation of our laboratory described a remarkably different Proechimy's cytoarchitecture organization of the hippocampal CA2 subfield. In the present study, we investigated the intrinsic neuronal properties and morphological characteristics of the Proechimys's hippocampal pyramidal neurons of the CA1 and CA2 areas. A comparative approach was performed using neurons recorded in Wistar rats. A striking finding in Proechimys rodents was the presence of large pyramidal-like neurons throughout the stratum oriens from CA2 to CA1 area. In order to confirm such distinctive feature of the Proechimys's hippocampus, we performed Nissl staining and immunohistochemistry for neurofilament protein SM311. CA2 pyramidal neurons in the stratum pyramidale of Proechimys exhibited a significantly higher input resistance and lower time constant when compared to corresponding cell groups in the same area of the Wistar rat's. This newly identified population of pyramidal-shaped neurons in stratum oriens of Proechimys exhibited distinct electrophysiological and morphological properties. This included larger capacitance, lower input resistance, larger rheobase, long latency to first action potential and slower firing frequency. In addition, the apical dendrites of these neurons were oriented in parallel to apical dendrites of regular pyramidal neurons in stratum pyramidale. Moreover, these neurons were immunoreactive to SM311 as the majority of the neurons of the pyramidal layer. The functional role of these hippocampal neurons of the rodent Proechimys deserves further investigation.

Effects of sampling location and time, and host animal on assessment of bacterial diversity and fermentation parameters in the bovine rumen.

AIMS: To investigate, using culture-independent methods, whether the ruminal bacterial structure, population and fermentation parameters differed between sampling locations and time. METHODS AND RESULTS: The detectable bacteria and fermentation parameters in the digesta from five locations in the rumen of three cows at three time points were analysed. The PCR-denaturing gradient gel electrophoresis (PCR-DGGE) profiles were similar among digesta samples from five locations (95.4%) and three time points (93.4%) within cows; however, a lower similarity was observed for samples collected from different host animals (85.5%). Rumen pH and concentration of volatile fatty acids (VFA) were affected by time points of sampling relative to feeding. CONCLUSIONS: The detectable bacterial structure in the rumen is highly conserved among different locations and over time, while the quantity of individual bacterial species may change diurnally in response to the feeding. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supplies the fundamental understanding of the microbial ecology in the rumen, which is essential for manipulation of ruminal microflora and subsequent improvement in animal production.

Convulsions induced by methylmalonic acid are associated with glutamic acid decarboxylase inhibition in rats: a role for GABA in the seizures presented by methylmalonic acidemic patients?

Methylmalonic acid (MMA) is an endogenous convulsing compound that accumulates in methylmalonic acidemia, an inborn error of the metabolism characterized by severe neurological dysfunction, including seizures. The mechanisms by which MMA causes seizures involves the activation of the N-methyl-D-aspartate (NMDA) receptors, but whether GABAergic mechanisms are involved in the convulsions induced by MMA is not known. Therefore, in the current study we investigated the involvement of GABAergic mechanisms in the convulsions induced by MMA. Adult rats were injected (i.c.v.) with muscimol (46 pmol/1 microl), baclofen (0.03, 0.1 and 0.3 micromol/1 microl), MK-801 (6 nmol/1 microl), pyridoxine (2 micromol/4 microl) or physiological saline (0.15 micromol/1 microl). After 30 min, MMA (0.3, 0.1 and 3 micromol/1 microl) or NaCl (6 micromol/1 microl, i.c.v.) was injected. The animals were immediately transferred to an open field and observed for the appearance of convulsions. After behavioral evaluation, glutamic acid decarboxylase (GAD) activity was determined in cerebral cortex homogenates by measuring the 14CO2 released from l-[14C]-glutamic acid. Convulsions were confirmed by electroencephalographic recording in a subset of animals. MMA caused the appearance of clonic convulsions in a dose-dependent manner and decreased GAD activity in the cerebral cortex ex vivo. GAD activity negatively correlated with duration of MMA-induced convulsions (r=-0.873, P<0.01), in an individual basis. Muscimol, baclofen, MK-801 and pyridoxine prevented MMA-induced convulsions, but only MK-801 and pyridoxine prevented MMA-induced GAD inhibition. These data suggest GABAergic mechanisms are involved in the convulsive action of MMA, and that GAD inhibition by MMA depends on the activation of NMDA receptors. While in this study we present novel data about the role of the GABAergic system in MMA-induced convulsions, the central role of NMDA receptors in the neurochemical actions of MMA is further reinforced since they seem to trigger GABAergic failure.

Electrophysiological and morphological heterogeneity of slow firing neurons in medial septal/diagonal band complex as revealed by cluster analysis.

Slow firing septal neurons modulate hippocampal and neocortical functions. Electrophysiologically, it is unclear whether slow firing neurons belong to a homogeneous neuronal population. To address this issue, whole-cell patch recordings and neuronal reconstructions were performed on rat brain slices containing the medial septum/diagonal band complex (MS/DB). Slow firing neurons were identified by their low firing rate at threshold (<5 Hz) and lack of time-dependent inward rectification (Ih). Unsupervised cluster analysis was used to investigate whether slow firing neurons could be further classified into different subtypes. The parameters used for the cluster analysis included latency for first spike, slow after-hyperpolarizing potential, maximal frequency and action potential (AP) decay slope. Neurons were grouped into three major subtypes. The majority of neurons (55%) were grouped as cluster I. Cluster II (17% of neurons) exhibited longer latency for generation of the first action potential (246.5+/-20.1 ms). Cluster III (28% of neurons) exhibited higher maximal firing frequency (25.3+/-1.7 Hz) when compared with cluster I (12.3+/-0.9 Hz) and cluster II (11.8+/-1.1 Hz) neurons. Additionally, cluster III neurons exhibited faster action potentials at suprathreshold. Interestingly, cluster II neurons were frequently located in the medial septum whereas neurons in cluster I and III appeared scattered throughout all MS/DB regions. Sholl's analysis revealed a more complex dendritic arborization in cluster III neurons. Cluster I and II neurons exhibited characteristics of "true" slow firing neurons whereas cluster III neurons exhibited higher frequency firing patterns. Several neurons were labeled with a cholinergic marker, Cy3-conjugated 192 IgG (p75NTR), and cholinergic neurons were found to be distributed among the three clusters. Our findings indicate that slow firing medial septal neurons are heterogeneous and that soma location is an important determinant of their electrophysiological properties. Thus, slow firing neurons from different septal regions have distinct functional properties, most likely related to their diverse connectivity.

Mobile cauda equina schwannomas.

Although very rare, cases of cauda equina tumours migrating several levels have been known to occur. We report two cases of cauda equina schwannomas which migrated from its primary site, one caudally and the other one rostrally as confirmed by magnetic resonance imaging perioperatively. The first patient was a 27-year-old man and the second a 41-year-old man; both presented with back pain. This report aims to remind clinicians that certain tumours attached to the roots in the lumbar spinal canal may migrate.

KCNMB1 regulates surface expression of a voltage and Ca2+-activated K+ channel via endocytic trafficking signals.

Voltage-dependent and calcium-activated K(+) (MaxiK, BK) channels are ubiquitously expressed and have various physiological roles including regulation of neurotransmitter release and smooth muscle tone. Coexpression of the pore-forming alpha (hSlo) subunit of MaxiK channels with a regulatory beta1 subunit (KCNMB1) produces noninactivating currents that are distinguished by high voltage/Ca(2+) sensitivities and altered pharmacology [McManus OB, Helms LM, Pallanck L, Ganetzky B, Swanson R, Leonard RJ (1995) Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron 14:645-650; Wallner M, Meera P, Ottolia M, Kaczorowski G, Latorre R, Garcia ML, Stefani E, Toro L (1995) Characterization of and modulation by a beta-subunit of a human maxi K(Ca) channel cloned from myometrium. Receptors Channels 3:185-199]. We now show that beta1 can regulate hSlo traffic as well, resulting in decreased hSlo surface expression. beta1 subunit expressed alone is able to reach the plasma membrane; in addition, it exhibits a distinct intracellular punctated pattern that colocalizes with an endosomal marker. Coexpressing beta1 subunit with hSlo, switches hSlo's rather diffuse intracellular expression to a punctate cytoplasmic localization that overlaps beta1 expression. Furthermore, coexpressed beta1 subunit reduces steady-state hSlo surface expression. Site-directed mutagenesis underscores a role of a putative endocytic signal at the beta1 C-terminus in the control of hSlo surface expression. We propose that aside from its well-established role as regulator of hSlo electrical activity, beta1 can regulate hSlo expression levels by means of an endocytic mechanism. This highlights a new beta1 subunit feature that regulates hSlo channels by a trafficking mechanism.

Septal GABAergic neurons are selectively vulnerable to pilocarpine-induced status epilepticus and chronic spontaneous seizures.

The septal region of the basal forebrain plays a critical role modulating hippocampal excitability and functional states. Septal circuits may also play a role in controlling abnormal hippocampal hyperexcitability in epilepsy. Both lateral and medial septal neurons are targets of hippocampal axons. Since the hippocampus is an important epileptogenic area in temporal lobe epilepsy, we hypothesize that excessive excitatory output will promote sustained neurodegeneration of septal region neurons. Pilocarpine-induced status epilepticus (SE) was chosen as a model to generate chronic epileptic animals. To determine whether septal neuronal populations are affected by hippocampal seizures, immunohistochemical assays were performed in brain sections obtained from age-matched control, latent period (7 days post-SE) and chronically epileptic (more than one month post-SE survival) rats. An anti-NeuN (neuronal nuclei) antibody was used to study total neuronal numbers. Anti-ChAT (choline acetyltransferase), anti-GAD (glutamic acid decarboxylase) isoenzymes (65 and 67), and anti-glutamate antibodies were used to reveal cholinergic, GABAergic and glutamatergic neurons, respectively. Our results revealed a significant atrophy of medial and lateral septal areas in all chronically epileptic rats. Overall neuronal density in the septum (medial and lateral septum), assessed by NeuN immunoreactivity, was significantly reduced by approximately 40% in chronically epileptic rats. The lessening of neuronal numbers in both regions was mainly due to the loss of GABAergic neurons (80-97% reduction in medial and lateral septum). In contrast, populations of cholinergic and glutamatergic neurons were spared. Overall, these data indicate that septal GABAergic neurons are selectively vulnerable to hippocampal hyperexcitability, and suggest that the processing of information in septohippocampal networks may be altered in chronic epilepsy.

Succinate increases neuronal post-synaptic excitatory potentials in vitro and induces convulsive behavior through N-methyl-d-aspartate-mediated mechanisms.

Succinate is a dicarboxylic acid that accumulates due to succinate dehydrogenase inhibition by malonate and methylmalonate exposure. These neurotoxins cause increased excitability and excitotoxic damage, which can be prevented by administering high amounts of succinate. In the present study we investigated whether succinate alters hippocampal field excitatory post-synaptic potentials. Bath application of succinate at intermediate concentrations (0.3-1 mM) increased the slope of field excitatory post-synaptic potentials in hippocampal slices, and at high concentrations (above 1 mM) did not alter or decrease field excitatory post-synaptic potentials slope. Succinate-induced enhancement of field excitatory post-synaptic potentials slope was abolished by the addition of d-2-amino-5-phosphonovaleric acid (50 microM) to the perfusate, supporting the involvement of N-methyl-d-aspartate receptors in the excitatory effect of this organic acid. Accordingly, succinate (0.8-7.5 micromol) i.c.v. administration caused dose-dependent convulsive behavior in mice. The i.c.v. co-administration of MK-801 (7 nmol) fully prevented succinate-induced convulsions, further suggesting the involvement of N-methyl-d-aspartate receptors in the convulsant action of succinate. Our data indicate that accumulation of moderate amounts of succinate may contribute to the excitotoxicity induced by succinate dehydrogenase inhibitors, through the activation of N-methyl-d-aspartate receptors.

Deficit in hippocampal long-term potentiation in monosodium glutamate-treated rats.

Rats subjected to monosodium glutamate (MSG) administration during the neonatal period present chronic neuroendocrine dysfunction associated with marked cognitive deficits. Long-term potentiation (LTP) in the hippocampus provides a model suited for the study of mammalian brain plasticity and memory formation. In the present work, we used the LTP protocol to investigate the synaptic plasticity in the hippocampal CA1 area of adult rats subjected to MSG treatment during the first 10 days of life. Synaptic transmission in CA1 area was analyzed using extracellular field recordings in response to Schaffer's collateral fiber stimulation in hippocampal slices. Animals injected with MSG exhibited a dramatic decrement of LTP field excitatory postsynaptic potentials (fEPSPs) compared to control group. Analysis of percent enhancement of fEPSP slope at 2 min after high frequency stimulation (HFS) increased by 189.3 +/- 33.2% in slices from control rats and 129.45 +/- 18.5% (p < 0.01) in slices from MSG-treated rats. Additionally, MSG-treated animals failed to maintain or consolidate LTP as revealed by a significant reduction in fEPSP slope enhancement over time after HFS. The mean fEPSP slope, 60 min after HFS, was 154.28 +/- 21% of the average baseline slope in control slices versus only 124.4 +/- 15% in MSG-treated rats (p < 0.01). At 90 min after HFS, slices from controls reached a potentiation of 44.5 +/- 2.9%, whereas the MSG group displayed an overall response enhancement of 17.65 +/- 2.7% of basal levels (p < 0.01). These findings indicate that MSG-treated rats display a chronic impairment of CA1 synaptic plasticity.

Alterations of the neocortical GABAergic system in the pilocarpine model of temporal lobe epilepsy: neuronal damage and immunocytochemical changes in chronic epileptic rats.

A wealth of previous studies reported pathological alterations in extrahippocampal regions in mesial temporal lobe epilepsy. Previous experimental findings have also demonstrated that the entorhinal cortex and the neocortex are damaged in different animal models of acute limbic seizures. The present study was aimed at verifying possible alterations in neocortical areas, and, in particular, structural changes of GABAergic interneurons in the sensorimotor cortex, in pilocarpine-induced chronic epilepsy in the rat. Series of sections were Nissl stained and processed for immunocytochemistry using antibodies that recognize nonphosphorylated neurofilament (SMI311), glial fibrillary acidic protein (GFAP), the calcium-binding protein parvalbumin (PV) which is expressed by a subset of cortical GABAergic neurons, the GABA transporter (GAT1), and isoform 65 of glutamic acid decarboxylase (GAD65), the GABA synthetic enzyme. Epileptic rats showed decreased cortical thickness, and diffuse gliosis was observed with GFAP antibody. Neurofilament alterations were also detected in sections processed using SMI311 antiserum. In addition, a diffuse decrease of PV, GAD65, and GAT1 immunoreactivity was observed in the sensorimotor cortex. Altered expression of PV, GAD65, and GAT1 pointed out specific neocortical disturbances in GABAergic inhibition, which could play a crucial role in seizure generation and expression. Thus, the present findings indicate that damage of GABAergic interneurons could be strictly associated with neocortical hyperexcitability in temporal lobe epilepsy.