Electrical stimulation alleviates depressive-like behaviors of rats: investigation of brain targets and potential mechanisms.

Deep brain stimulation (DBS) is a promising therapy for patients with refractory depression. However, key questions remain with regard to which brain target(s) should be used for stimulation, and which mechanisms underlie the therapeutic effects. Here, we investigated the effect of DBS, with low- and high-frequency stimulation (LFS, HFS), in different brain regions (ventromedial prefrontal cortex, vmPFC; cingulate cortex, Cg; nucleus accumbens (NAc) core or shell; lateral habenula, LHb; and ventral tegmental area) on a variety of depressive-like behaviors using rat models. In the naive animal study, we found that HFS of the Cg, vmPFC, NAc core and LHb reduced anxiety levels and increased motivation for food. In the chronic unpredictable stress model, there was a robust depressive-like behavioral phenotype. Moreover, vmPFC HFS, in a comparison of all stimulated targets, produced the most profound antidepressant effects with enhanced hedonia, reduced anxiety and decreased forced-swim immobility. In the following set of electrophysiological and histochemical experiments designed to unravel some of the underlying mechanisms, we found that vmPFC HFS evoked a specific modulation of the serotonergic neurons in the dorsal raphe nucleus (DRN), which have long been linked to mood. Finally, using a neuronal mapping approach by means of c-Fos expression, we found that vmPFC HFS modulated a brain circuit linked to the DRN and known to be involved in affect. In conclusion, HFS of the vmPFC produced the most potent antidepressant effects in naive rats and rats subjected to stress by mechanisms also including the DRN.

Intracranial self stimulation upregulates the expression of synaptic plasticity related genes and Arc protein expression in rat hippocampus.

Post-training lateral hypothalamus (LH) intracranial self stimulation (ICSS) has a reliable enhancing effect on explicit memory formation evaluated in hippocampus-dependent tasks such as the Morris water maze. In this study, the effects of ICSS on gene expression in the hippocampus are examined 4.5 h post treatment by using oligonucleotide microarray and real-time PCR, and by measuring Arc protein levels in the different layers of hippocampal subfields through immunofluorescence. The microarray data analysis resulted in 65 significantly regulated genes in rat ICSS hippocampi compared to sham, including cAMP-mediated signaling as one of the most significantly enriched Database for Annotation, Visualization and Integrated Discovery (DAVID) functional categories. In particular, expression of CREB-dependent synaptic plasticity related genes (c-Fos, Arc, Bdnf, Ptgs-2 and Crem and Icer) was regulated in a time-dependent manner following treatment administration. Immunofluorescence results showed that ICSS treatment induced a significant increase in Arc protein expression in CA1 and DG hippocampal subfields. This empirical evidence supports our hypothesis that the effect of ICSS on improved or restored memory functions might be mediated by increased hippocampal expression of activity-dependent synaptic plasticity related genes, including Arc protein expression, as neural mechanisms related to memory consolidation.

Intracranial self-stimulation induces expression of learning and memory-related genes in rat amygdala.

Intracranial self-stimulation (ICSS) in the lateral hypothalamus improves memory when administered immediately after a training session. In our laboratory, ICSS has been shown as a very reliable way to increase two-way active avoidance (TWAA) conditioning, an amygdala-dependent task. The aim of this work was to study, in the rat amygdala, anatomical and molecular aspects of ICSS, using the same parameters facilitating TWAA. First, we examined the activation of ipsilateral and contralateral lateral (LA) and basolateral (BLA) amygdala, the main amygdalar regions involved in the TWAA, by the immunohistochemical determination of c-Fos protein expression. Second, we tested the effects of the ICSS treatment on the expression of 14 genes related to learning and memory processes using real-time polymerase chain reaction. Results showed a bilateral increase in c-Fos protein expression in LA and BLA nuclei after ICSS treatment. We also found that Fos, brain-derived nerve growth factor (BDNF), Arc, inducible cAMP early repressor (ICER), COX-2, Dnajb1, FKpb5 and Ret genes were upregulated in the amygdala 90 min and 4.5 h post ICSS. From this set of genes, BDNF, Arc and ICER are functionally associated with the cAMP-responsive element-mediated gene transcription molecular pathway that plays a pivotal role in memory, whereas Dnajb1 and Ret are associated with protein folding required for plasticity or neuroprotection. Our results suggest that ICSS induces expression of genes related with synaptic plasticity and protein folding functions in the rat amygdaloid area, which may be involved in the molecular mechanisms by which ICSS may improve or restore memory functions related to this brain structure.

The influence of engineered Fe(2)O(3) nanoparticles and soluble (FeCl(3)) iron on the developmental toxicity caused by CO(2)-induced seawater acidification.

An embryo development assay using a common test organism, the edible mussel (Mytilus galloprovincialis), exposed to both Fe(2)O(3) nanoparticles and soluble FeCl(3) at 3 acidic pHs, has provided evidence for the following: (1) CO(2) enriched seawater adjusted to pH projections for carbon capture leakage scenarios (CCS) significantly impaired embryo development; (2) under natural pH conditions, no significant effect was detected following exposure of embryos to Fe, no matter if in nano- or soluble form; (3) at pH of natural seawater nano-Fe particles aggregate into large, polydisperse and porous particles, with no biological impact detected; (4) at pH 6 and 7, such aggregates may moderate the damage associated with CO(2) enrichment as indicated by an increased prevalence of normal D-shell larvae when nano-Fe was present in the seawater at pH 7, while soluble iron benefited embryo development at pH 6, and (5) the observed effects of iron on pH-induced development toxicity were concentration dependent.

Intracranial self-stimulation to the lateral hypothalamus, a memory improving treatment, results in hippocampal changes in gene expression.

Intracranial self-stimulation (ICSS) within the medial forebrain bundle of the lateral hypothalamus (LH) facilitates consolidation of implicit and explicit memories for a variety of learning paradigms in rats. However, the neural and molecular mechanisms involved in memory facilitation by ICSS are not known. Here, we investigated the influence of ICSS treatment on hippocampal gene expression in order to identify potential signaling pathways and cellular processes involved in ICSS-mediated cognitive improvements. Immunohistochemistry studies demonstrated that ICSS caused a rapid induction of c-Fos expression in hippocampal cornu ammonis (CA) 3 and dentatus gyrus areas. Moreover, using microarray or quantitative real-time polymerase chain reaction (PCR) analysis, we showed that ICSS modulates the expression of 62 hippocampal genes shortly after training. Most of the proteins encoded by these genes, such as calmodulin-dependent-phosphodiesterase 1 A (Pde1a), are part of signal transduction machineries or are related to anti-apoptosis, as heat shock 70 kDa protein 1A (Hspa1a). Importantly, 10 of the regulated genes have been previously related with learning and memory or neural plasticity, including the cocaine and amphetamine-regulated transcript (Cart), adenylate cyclase activating polypeptide 1 (Adcyap1), serum/glucocorticoid regulated kinase (Sgk), Ret proto-oncogene (Ret), and Fos. The fact that the Fos gene was differentially expressed in our microarray experiments validated our findings from our immunohistochemical studies mentioned above. In addition, using quantitative real-time PCR, we confirmed the observed expression changes for several of the genes identified by our microarray analyses. Our results suggest that ICSS may facilitate learning and memory by regulation of multiple signaling pathways in the hippocampus that may promote neuroplasticity.

Unidentified extracellular prokaryotes within the byssal threads of the deep-sea vent mussel Bathymodiolus azoricus.

Bacterial symbiosis and/or parasitism is widespread in hydrothermal bivalves, and is typically developed in gills, with a lower incidence in mantle and digestive glands, while it has never been described in byssus. Using ultrastructural examination, we provide evidence for the existence of a potentially new group of filamentous prokaryotic organism in Bathymodiolus azoricus byssus, with putative parasitic influence. Additionally, a cystic, undefined organism was found with an unclear physiological role within the spongy net of the byssus plaque. Our results indicate that in spite of its antibacterial protective sheath, byssus gives access to prokaryotic organisms becoming prone to failure through damaged collagen fibres.

Effect of culture conditions on the obtention of boar epididymal epithelial cell monolayers.

The goal of this study was to investigate the effect of the collagenase digestion time, the initial density of fragments and the culture temperature on the obtention of a boar epididymal epithelial cell culture, which is a useful methodology for the study of epididymal functions. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was only obtained when an adequate enzymatic digestion of the connective tissue surrounding the epididymal tubule was performed. For the correct digestion of caput and corpus fragments two collagenase digestions of 2 and 1h, respectively, were enough. Cauda fragments, however, needed two collagenase digestions of 3h each. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained regardless of the initial density tested (15, 30, 60 and 90fragments/well). However, cultures originated from 15 and 30fragments/well showed higher cell concentration during the first 2 weeks of culture than cultures originated from 60 and 90fragments/well. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained at both 32 and 37 degrees Celsius, but at 32 degrees Celsius cells grew very slowly and confluence was not reached until a week later than it was with cells growing at 37 degrees Celsius. In conclusion, we have observed that the time of digestion with collagenase is an important factor for the successful establishment of boar epididymal cell monolayers, and that the initial density of fragments and the culture temperature should be taken into account.

Lectin histochemistry of the boar bulbourethral glands.

The present study describes, for the first time, the glycosidic content of boar bulbourethral glands using lectin histochemistry. Fourteen horseradish peroxidase- or digoxigenin-labelled lectins with different carbohydrate specificities were used in samples obtained from 3 healthy Landrace boars. The results obtained indicate that endpiece and duct cells synthesize and secrete mainly O-glycoproteins with alpha- and beta-D-N-acetylgalactosamine, beta-D-galactose-beta(1-->3)-D-N-acetylgalactosamine, D-N-acetylglucosamine and neuraminic acid residues. Glycoproteins secreted by bulbourethral glands have a role in the protection and lubrication of the urethra. In addition, they may be also involved in the regulation of the sperm metabolic activity and in the maintenance of the structural integrity of acrosomal and plasma membranes.

Evaluation of boar sperm maturation after co-incubation with caput, corpus and cauda epididymal cultures [corrected].

Boar sperm from the proximal caput epididymis were co-incubated with 1, 4, 7, 10 and 14-day old caput, corpus and cauda epididymal cultures for 24, 48 and 72 h. Boar kidney epithelial cells (LLC-PK1) and ECM alone were used as negative controls. Sperm motility, morphology and membrane integrity were studied to evaluate boar sperm maturation in vitro. Our results showed that epithelial cell monolayers (10, 14-day old) create a suitable microenvironment for the survival of proximal caput sperm and the maintenance of sperm motility over a 72 h period. Moreover, corpus epididymal tubule fragments in culture (1, 4-day old) are capable of promoting the migration of the cytoplasmic droplet along the sperm tail after 24h of co-incubation.

Semen quality of postpubertal boars during increasing and decreasing natural photoperiods.

The present study analyses the effects of increasing and decreasing photoperiods on the semen quality of 20 selected postpubertal Landrace boars. The boars were exposed, throughout 75 days, to increasing and decreasing photoperiods of natural light, a constant temperature of 21 +/- 1 degrees C and 60-70% of humidity, fed with a nutritious diet and, submitted to a rhythm of semen collection of twice a week. During the last 2 weeks of each treatment, semen samples were analysed and the parameters measured were: ejaculate volume and pH, sperm concentration, sperm production and the number of semen doses per ejaculate, sperm vitality, sperm motility, osmotic resistance of spermatozoa and sperm morphology. The comparative analysis between increasing and decreasing photoperiods indicated that the semen quality of boars exposed to a decreasing photoperiod was reduced as a consequence of decreases in sperm concentration, sperm production and the number of semen doses. There was no difference between increasing and decreasing photoperiods in terms of sperm vitality and sperm motility, nor in the osmotic resistance of spermatozoa to isotonic and hypotonic media. The analysis of sperm morphology showed significantly lower frequencies of mature and immature spermatozoa with a distal cytoplasmic droplet, and significantly higher frequencies of immature spermatozoa with a proximal droplet in boars exposed to the decreasing photoperiod. These results indicate that the sperm quality of the selected boars decreased during decreasing photoperiods, in comparison with increasing photoperiods, mainly due to impaired testicular function.

Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene.

To investigate to what extent myeloablation, graft size, and ex vivo manipulation influence the engraftment and long-term survival of transduced murine hematopoietic cells, groups of C57BL/6J (CD45.2) mice receiving total body irradiation (TBI) (1-9 Gy) or no irradiation were transplanted with either transduced bone marrow (BM) cells, at two cell doses, or with fresh BM cells from B6/SJL (CD45.1) congenic mice. Short (40 days) and long-term (5 months) engraftment and transgene expression were measured by FACS analysis. No donor cells were detected in the hematopoietic tissues of non-myeloablated mice, whereas in the irradiated animals, levels of engraftment correlated well with the dose of TBI administered. Similar percentages of transgene-expressing cells were found in the grafted hematopoietic cells of all groups of mice, regardless of the dose of TBI administered or the level of engraftment achieved. This suggests that the engrafted animals could become tolerant to the transgene product (enhanced green fluorescent protein, EGFP). Our results indicate that TBI facilitates the engraftment of manipulated hematopoietic cells in a dose-dependent manner, that mice engrafted with EGFP(+) hematopoietic cells probably acquire tolerance to EGFP, and that increasing the graft size and reducing the ex vivo manipulation required for retroviral gene transfer of hematopoietic cells also enhances their engrafting potential.

The cycle of the seminiferous epithelium in Landrace boars.

The present study describes the morphological features of the eight stages of the seminiferous epithelium in Landrace boars according to the tubular morphology method, as well as their relative frequency, length, and duration. In Landrace boars the pre-meiotic stages occupied the 31.9 +/- 19.9% of the spermatogenic cycle and had a total length of 1788.8 +/- 1153.0 microm and a duration of 2.78 days; they were mainly characterised by the presence of leptotene and pachytene spermatocytes and round spermatids. Meiotic stages, with a relative frequency of 16.4 +/- 6.8%, a length of 787.1 +/- 603.1 microm and a duration of 1.41 days, contained spermatocytes in advanced meiosis I and/or in meiosis II and elongating spermatids grouped in bundles. Post-meiotic stages occupied the 50.6 +/- 20.4% of the spermatogenic cycle and had a length of 2096.8 +/- 1175.0 microm and a duration of 4.37 days; the most important event of these stages was the spermiation, which included the complete remodelling of sperm head and tail and the releasing of spermatozoa into the lumen, as well as the formation of residual bodies. From data obtained we concluded that both germ cell associations of the stages maintain constant among Landrace boars, and that the relative frequency, length and duration of the stages were directly dependent of the cytological transformations on the seminiferous tubules.

Drifting towards a diffuse control model of exploratory motor learning: A comparison of global and within-trial performance measures.

Accurate measurement is crucial for understanding the processes that underlie exploratory patterns in motor learning. Accordingly, measures of learning should be sensitive to the changes that take place during skill acquisition. Most studies, however, use trial-based global measures that assess performance but do not actually measure gradual changes taking place within trials. The present study attempted to remedy this shortcoming by analysing a visual adaptation task, and comparing traditional global measures of learning with new, within-trial measures. Movement time was the only global measure sensitive to changes in the movement trajectory during learning. Three new measures were expected to reveal changes to the movement trajectory that are associated with learning: (i) the length of runs, (ii) change of trajectory angle in relation to the target, and (iii) drift (change in distance from the target). All three measures were sensitive to learning and indicated a gradual straightening of the movement trajectories over trials. Furthermore, three different methods to partition trajectories into segments were examined. The new within-trial measures, irrespective of partitioning method, prove promising for the development of a diffuse control model of exploratory learning.

Avoidance responses to aluminium in the freshwater bivalve Anodonta cygnea.

This study examined the effect of aluminium (Al) on the filtering behaviour (shell opening or gape) of the freshwater bivalve Anodonta cygnea L in neutral fresh water. Parallel measurements of Al concentration in the soft tissues were made to examine the relationship between changes in behaviour and accumulation of Al. The number of lysosomal granules in the gill, kidney and digestive gland were counted, as lysosomes are known to be involved in the excretion and detoxification of trace metals. The bivalves were exposed to two environmentally relevant concentrations of added Al i.e. 250 and 500 microg l(-1) (9.25 and 18.5 microM l(-1)) at neutral pH for 15 days and shell movement monitored continuously. Aluminium affected the mussels' filtering activity, producing an avoidance reaction whose magnitude was concentration-dependent; 250 microg l(-1) added Al produced no detectable change, while 500 microg l(-1) Al reduced mean duration of shell opening by 50%. This effect was irreversible over a 15 day recovery period. Tissue levels of Al after 15 days exposure were an order of magnitude higher in animals exposed to 250 microg l(-1) added Al than in those exposed to 500 microg l(-1). This was consistent with the inhibition of filtering activity due to valve closure at the higher concentration, which may have prevented uptake of Al. In addition, probable different chemical speciation of Al in the water column (soluble for 250 and colloidal for 500 microg l(-1)) may lead to marked differences in tissue uptake. The kidney and digestive gland were the main sites of accumulation of Al and concentrations remained significantly elevated 15 days after transfer of animals to clean water. It is suggested that mucus plays a role in the exclusion of Al as elevated concentrations were measured in the pseudofaeces of animals during and after exposure. Lysosomal granules may be involved in the intracellular handling and detoxification of Al as numbers increased significantly in all organs during exposure and continued to increase after the animals were transferred to clean water. The present study provides evidence for the bioavailability and toxicity of Al to mussels at neutral pH and at concentrations which are known to enter neutral freshwaters when mobilised by natural or anthropogenic acidity. The changes in behaviour and uptake of Al in the mussel observed in this investigation are, therefore, likely to be reflected in the natural environment and the degree to which Al affects the 'fitness' of the mussel populations and the transfer of Al through the food chain merit investigation.

A kinematic analysis of hand selection in a reaching task.

A group of left- and right-handers was tested on a task requiring them to reach out and pick up an object with either the left or the right hand. We varied the eccentricity of the target object (a small glass) and the required accuracy level, by filling the glass with liquid. We recorded (a) frequency of left or right hand use, (b) hand preference using a handedness questionnaire, and (c) the trajectories of the reaches using a movement registration system. It was found that the stronger the hand preference, the further in contralateral space the shift occurred between left and right hand use. Not only did the transition point corresponding to the shift between the two hands correlate with the point where their deceleration times were equal, but these locations closely coincided. These findings suggest that people are highly skilled perceivers of their own action capabilities, and that they are able to select the action mode that is most suited to perform a given task. We argue that laterality should be understood in terms of asymmetries in action modes.

Efecto del fraccionamiento de la dosis sobre la DL50/30 por irradiación corporal total en el ratón / The dose fractionation effect on LD50/30 after total body irradiation in mice

Introducción: El efecto del fraccionamiento de la dosis sobre la eficacia biológica de las radiaciones es conocido. Sin embargo, su importancia sobre las células hemopoyéticas siempre ha sido considerada como poco relevante, en base a su escasa capacidad de reparación de daño subletal. Propósito: Evaluar la variación de la DL50/30 en un modelo murino, en función del intervalo entre dos fracciones de irradiación corporal total. Material y métodos: Hemos utilizado ratones hembra, de la cepa B6D2F1, de 8 semanas de edad y un peso medio de 20 g, que fueron mantenidas en ambiente estéril hasta el final de la experiencia, y sometidas a una irradiación corporal total mediante fotones gamma de Co-60, con una dosis total de 10 Gy en dos fracciones de 5 Gy, separadas por intervalos de tiempo crecientes, comprendidos entre 0 y 50 minutos. La curva de mortalidad se obtuvo con el porcentaje de animales fallecidos de cada grupo para cada intervalo, durante los 40 días siguientes al tratamiento. Resultados: A medida que el intervalo entre las dos fracciones aumenta, la mortalidad decrece: pasa del 78,89 por ciento para un intervalo entre fracciones de 0 minutos al 19,04 por ciento para un intervalo de 50 minutos. Conclusiones: Las células hemopoyéticas responsables de la mortalidad de los animales, tienen una capacidad de reparación del daño subletal, que se manifiesta cuando el intervalo entre fracciones aumenta (AU)

Granulocyte colony-stimulating factor administered as a single intraperitoneal injection modifies the lethal dose95/30 in irradiated B6D2F1 mice.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that stimulates the proliferation of progenitor myeloid cells. We have previously demonstrated that recombinant human G-CSF (rhG-CSF) significantly improves survival of lethally irradiated B6D2F1 mice when administered as a single intraperitoneal dose of 1 mg/kg 2 hours after a lethal dose (LD)95/30 irradiation. In our model, rhG-CSF is also able to modify the LD95/30 in irradiated animals and 1.1 has been found to be the dose modification factor (the ratio of LD95/30 for mice treated with rhG-CSF to that for control animals).

Effects of orotic acid on ischaemic/reperfused myocardial function and glycogen content in isolated working rat hearts.

The present study was carried out to investigate the effect of orotic acid on ischaemic/reperfused myocardial function and cardiac glycogen content in isolated working rat hearts. In a preliminary series of studies, hearts isolated from male Wistar rats (300-350 g) were perfused with oxygenated Krebs-Henseleit buffer containing cumulative concentrations of orotic acid from 0.01 to 10.00 mg l-1. In the concentration range of 0.01-0.10 mg l-1, orotic acid significantly improved left ventricular function. Therefore, in the second series of studies, rats were treated intravenously with 0.01 mg kg-1 orotic acid for 4 days. Hearts were then isolated and subjected to 30 min of global no-flow ischaemia followed by 10 min of reperfusion. Orotic acid treatment significantly improved post-ischaemic myocardial function and increased pre-ischaemic and post-ischaemic glycogen content of the heart. We conclude that orotic acid improves ischaemic/reperfused cardiac performance and this effect may be based on the elevation of myocardial glycogen content.