ERK1/2 controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell of the cortical collecting duct of the mouse kidney.

The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 pathway as shown in vivo by immunostaining of phosphorylated active ERK1/2 (pERK1/2). The MEK1/2-ERK1/2 pathway controls many different ion transports both in proximal and distal nephron, raising the question of whether this pathway is involved in the basal and/or hormone-dependent transepithelial sodium reabsorption in the principal cell of the cortical collecting duct (CCD), a process mediated by the apical epithelial sodium channel and the basolateral sodium pump (Na,K-ATPase). To answer this question we used ex vivo microdissected CCDs from normal mouse kidney or in vitro cultured mpkCCDcl4 principal cells. Significant basal levels of pERK1/2 were observed ex vivo and in vitro. Aldosterone and vasopressin, known to up-regulate sodium reabsorption in CCDs, did not change ERK1/2 activity either ex vivo or in vitro. Basal and aldosterone- or vasopressin-stimulated sodium transport was down-regulated by the MEK1/2 inhibitor PD98059, in parallel with a decrease in pERK1/2 in vitro. The activity of Na,K-ATPase but not that of epithelial sodium channel was inhibited by MEK1/2 inhibitors in both unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface biotinylation showed that intrinsic activity rather than cell surface expression of Na,K-ATPase was controlled by pERK1/2. PD98059 also significantly inhibited the activity of Na,K-ATPase ex vivo. Our data demonstrate that the ERK1/2 pathway controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell and indicate that basal constitutive activity of the ERK1/2 pathway is a critical component of this control.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and killed them on day 11 to perform [125I]epibatidine binding autoradiograms on serial coronal slices. Results showed significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory cortex, motor cortex, auditory cortex, retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 33% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The percentage of up-regulation correlated positively with the density of serotonin transporters, according to the serotonergic profile of MDMA. The heteromeric nAChR increase in concrete areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term treatment with MDMA.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and killed them on day 11 to perform [125I]epibatidine binding autoradiograms on serial coronal slices. Results showed significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory cortex, motor cortex, auditory cortex, retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 33% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The percentage of up-regulation correlated positively with the density of serotonin transporters, according to the serotonergic profile of MDMA. The heteromeric nAChR increase in concrete areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term treatment with MDMA.

Protracted treatment with MDMA induces heteromeric nicotinic receptor up-regulation in rat brain: an autoradiography study.

Previous studies indicate that 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) can induce heteromeric nicotinic acetylcholine receptor (nAChR, mainly of α4β2 subtype) up-regulation. In this study we treated Sprague-Dawley rats twice-daily for 10 days with either saline or MDMA (7 mg/kg) and killed them on day 11 to perform [125I]epibatidine binding autoradiograms on serial coronal slices. Results showed significant increases in nAChR density in the substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, anterior caudate-putamen, somatosensory cortex, motor cortex, auditory cortex, retrosplenial cortex, laterodorsal thalamus nuclei, amygdala, postsubiculum and pontine nuclei. These increases ranged from 3% (retrosplenial cortex) to 30 and 33% (amygdala and substantia nigra). No increased α4 subunit immunoreactivity was found in up-regulated areas compared with saline-treated rats, suggesting a post-translational mechanism as occurs with nicotine. The percentage of up-regulation correlated positively with the density of serotonin transporters, according to the serotonergic profile of MDMA. The heteromeric nAChR increase in concrete areas could account, at least in part, for the reinforcing, sensitizing and psychiatric disorders observed after long-term treatment with MDMA.

Revisiting sodium and water reabsorption with functional genomics tools.

PURPOSE OF REVIEW: The kidney plays an essential role in maintaining sodium and water balance, thereby controlling the volume and osmolarity of the extracellular body fluids, the blood volume and the blood pressure. The final adjustment of sodium and water reabsorption in the kidney takes place in cells of the distal part of the nephron in which a set of apical and basolateral transporters participate in vectorial sodium and water transport from the tubular lumen to the interstitium and, finally, to the general circulation. According to a current model, the activity and/or cell-surface expression of these transporters is/are under the control of a gene network composed of the hormonally regulated, as well as constitutively expressed, genes. It is proposed that this gene network may include new candidate genes for salt- and water-losing syndromes and for salt-sensitive hypertension. A new generation of functional genomics techniques have recently been applied to the characterization of this gene network. The purpose of this review is to summarize these studies and to discuss the potential of the different techniques for characterization of the renal transcriptome. RECENT FINDINGS: Recently, DNA microarrays and serial analysis of gene expression have been applied to characterize the kidney transcriptome in different in-vivo and in-vitro models. In these studies, a set of new interesting genes potentially involved in the regulation of sodium and water reabsorption by the kidney have been identified and are currently under detailed investigation. SUMMARY: Characterization of the kidney transcriptome is greatly expanding our knowledge of the gene networks involved in multiple kidney functions, including the maintenance of sodium and water homeostasis.

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo.

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

Metabolic activation pattern of distinct hippocampal subregions during spatial learning and memory retrieval.

Activation dynamics of hippocampal subregions during spatial learning and their interplay with neocortical regions is an important dimension in the understanding of hippocampal function. Using the (14C)-2-deoxyglucose autoradiographic method, we have characterized the metabolic changes occurring in hippocampal subregions in mice while learning an eight-arm radial maze task. Autoradiogram densitometry revealed a heterogeneous and evolving pattern of enhanced metabolic activity throughout the hippocampus during the training period and on recall. In the early stages of training, activity was enhanced in the CA1 area from the intermediate portion to the posterior end as well as in the CA3 area within the intermediate portion of the hippocampus. At later stages, CA1 and CA3 activations spread over the entire longitudinal axis, while dentate gyrus (DG) activation occurred from the anterior to the intermediate zone. Activation of the retrosplenial cortex but not the amygdala was also observed during the learning process. On recall, only DG activation was observed in the same anterior part of the hippocampus. These results suggest the existence of a functional segmentation of the hippocampus, each subregion being dynamically but also differentially recruited along the acquisition, consolidation, and retrieval process in parallel with some neocortical sites.

Functional connectivity of reward processing in the brain

Controversial results have been reported concerning the neural mechanisms involved in the processing of rewards and punishments. On the one hand, there is evidence suggesting that monetary gains and losses activate a similar fronto-subcortical network. On the other hand, results of recent studies imply that reward and punishment may engage distinct neural mechanisms. Using functional magnetic resonance imaging (fMRI) we investigated both regional and interregional functional connectivity patterns while participants performed a gambling task featuring unexpectedly high monetary gains and losses. Classical univariate statistical analysis showed that monetary gains and losses activated a similar fronto-striatallimbic network, in which main activation peaks were observed bilaterally in the ventral striatum. Functional connectivity analysis showed similar responses for gain and loss conditions in the insular cortex, the amygdala, and the hippocampus that correlated with the activity observed in the seed region ventral striatum, with the connectivity to the amygdala appearing more pronounced after losses. Larger functional connectivity was found to the medial orbitofrontal cortex for negative outcomes. The fact that different functional patterns were obtained with both analyses suggests that the brain activations observed in the classical univariate approach identifi es the involvement of different functional networks in the current task. These results stress the importance of studying functional connectivity in addition to standard fMRI analysis in reward-related studies.

Structure function relationships of ENaC and its role in sodium handling.

The epithelial sodium channel (ENaC) in the apical membrane of polarized epithelial cells is the rate-limiting step for Na entry into the cell; in series with the basolateral Na pump, it allows the vectorial transepithelial transport of Na ions. ENaC is expressed in different epithelia like the distal nephron or colon, and the airways epithelium. In the lung ENaC controls the composition and the amount of pulmonary fluid, whereas in the distal nephron ENaC under the control of aldosterone and vasopressin, is essential to adapt the amount of Na+ reabsorbed with the daily sodium intake. Activating mutations of ENaC cause severe disturbances of Na+ homeostasis leading to hypertension in human and in mouse models. Functional expression of ENaC in different cell systems allowed the identification of structural domains of the protein that are essential for channel function and/or modulation of channel activity. Site-directed mutations in specific domains of the channel protein lead to channel hyperactivity or channel loss of function. Knowledge about ENaC structure-function relationships opens new opportunities for development of pharmacological tools for controlling ENaC activity, such as channel activators of potential benefit in the treatment of pulmonary edema, or highly potent ENaC blockers with natriuretic effects.

Dépersonnalisation. Données actuelles.

OBJECTIVE: Depersonalization is a fascinating clinical phenomenon referring to a self-consciousness disorder, characterized by emotional detachment from one's own feelings, thoughts, or actions. This article intends to summarize the current literature in this area. METHOD: Using the Medline data base, we reviewed literature addressing the clinical, etiology, nosology, physiopathology, and treatment of depersonalization. CONCLUSIONS: Derealization means that perception of the world and of external reality are altered. These 2 phenomena are often associated. They are not specific to any psychiatric entity and are reported in many different psychiatric syndromes. Many factors, including use of different substances, are involved in their onset. The physiopathology is still little known. However, some conceptual models suggest partial amygdala inhibition combined with activation of other amygdaloid structures. A serotoninergic functioning impairment is indicated in different pharmacologic studies. Different psychotropic drugs, especially serotoninergic antidepressants, have been proposed for pharmacotherapy; however, there are no conclusive randomized studies, and the contribution of psychotherapy in treating these patients is still questioned

Epithelial sodium transport and its control by aldosterone: the story of our internal environment revisited.

Transcription and translation require a high concentration of potassium across the entire tree of life. The conservation of a high intracellular potassium was an absolute requirement for the evolution of life on Earth. This was achieved by the interplay of P- and V-ATPases that can set up electrochemical gradients across the cell membrane, an energetically costly process requiring the synthesis of ATP by F-ATPases. In animals, the control of an extracellular compartment was achieved by the emergence of multicellular organisms able to produce tight epithelial barriers creating a stable extracellular milieu. Finally, the adaptation to a terrestrian environment was achieved by the evolution of distinct regulatory pathways allowing salt and water conservation. In this review we emphasize the critical and dual role of Na(+)-K(+)-ATPase in the control of the ionic composition of the extracellular fluid and the renin-angiotensin-aldosterone system (RAAS) in salt and water conservation in vertebrates. The action of aldosterone on transepithelial sodium transport by activation of the epithelial sodium channel (ENaC) at the apical membrane and that of Na(+)-K(+)-ATPase at the basolateral membrane may have evolved in lungfish before the emergence of tetrapods. Finally, we discuss the implication of RAAS in the origin of the present pandemia of hypertension and its associated cardiovascular diseases.

Estudio del desarrollo y conectividad de las neuronas vasotocinérgicas en el prosencéfalo de pollo

En este Trabajo de Fin de Grado se estudia el origen embrionario de las distintas poblaciones neuronales que forman la amígdala medial extendida. La amígdala es una estructura del cerebro anterior involucrada en otorgar un significado emocional a los estímulos ambientales y en el control de distintos aspectos del comportamiento social (p.ej. comportamientos sexual, maternal, agresivo y afiliativo). Ante dichos estímulos, la amígdala pone en marcha una serie de reacciones de carácter motor, autonómico y endocrino que constituyen la respuesta emocional. Algunos desórdenes de carácter neuropsiquiátrico en humanos están relacionados con una disfunción en el control de las emociones y del comportamiento social, y varios de ellos se asocian a alteraciones en el desarrollo de la amígdala. El objetivo del presente trabajo ha sido investigar el origen de las neuronas de la amígdala medial extendida en embriones de pollo (E15 y E18) mediante ensayos de inmunocitoquímica, técnica utilizada para localizar las células que contienen el neuropéptido vasotocina (AVT) y proteínas reguladoras del desarrollo (la producida a partir del gen Otp) para ayudar en la delimitación de los distintos dominios embrionarios del prosencéfalo y distintas subdivisiones de la amígdala extendida. Los resultados de estos ensayos se combinaron con ensayos de trazado de conexiones para analizar la conectividad de las neuronas vasotocinérgicas de esta estructura. Los resultados obtenidos sugieren que las neuronas AVT-positivas podrían derivar del dominio Supra-Opto-Paraventricular (SPV), y algunas poblaciones alcanzarían su posición definitiva dentro del propio dominio por migración radial, mientras que otras invadirían otros dominios cerebrales por migración tangencial. En conclusión, la investigación proporciona importantes datos que clarifican aspectos relevantes del desarrollo y organización adulta de la amígdala extendida, y ayuda a establecer las bases para una mejor comprensión del control neural de las emociones y el comportamiento social en condiciones normales y patológicas.

Functional connectivity of reward processing in the brain

Controversial results have been reported concerning the neural mechanisms involved in the processing of rewards and punishments. On the one hand, there is evidence suggesting that monetary gains and losses activate a similar fronto-subcortical network. On the other hand, results of recent studies imply that reward and punishment may engage distinct neural mechanisms. Using functional magnetic resonance imaging (fMRI) we investigated both regional and interregional functional connectivity patterns while participants performed a gambling task featuring unexpectedly high monetary gains and losses. Classical univariate statistical analysis showed that monetary gains and losses activated a similar fronto-striatallimbic network, in which main activation peaks were observed bilaterally in the ventral striatum. Functional connectivity analysis showed similar responses for gain and loss conditions in the insular cortex, the amygdala, and the hippocampus that correlated with the activity observed in the seed region ventral striatum, with the connectivity to the amygdala appearing more pronounced after losses. Larger functional connectivity was found to the medial orbitofrontal cortex for negative outcomes. The fact that different functional patterns were obtained with both analyses suggests that the brain activations observed in the classical univariate approach identifi es the involvement of different functional networks in the current task. These results stress the importance of studying functional connectivity in addition to standard fMRI analysis in reward-related studies.

Intracellular membrane trafficking in Osteoclasts The role of direct Rab protein – Rac 1 interactions in the targeting of intracellular vesicles

Osteoclasts are cells responsible for bone resorption. These cells undergo extensive membrane re-organization during their polarization for bone resorption and form four distinct membrane domains, namely the ruffled border, the basolateral membrane, the sealing zone and the functional secretory domain. The endocytic/biosynthetic pathway and transcytotic route(s) are important for the resorption process, since the endocytic/biosynthetic pathway brings the specific vesicles to the ruffled border whereas the transcytotic flow is believed to transport the degraded bone matrix away from the resorption lacuna to the functional secretory domain. In the present study, we found a new transcytotic route from the functional secretory domain to the ruffled border, which may compensate membrane loss from the ruffled border during the resorption process. We also found that lipid rafts are essential for the ruffled border-targeted late endosomal pathways. A small GTP-binding protein, Rab7, has earlier been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts it is involved in the formation of the ruffled border, which displays several features of late endosomal membranes. Here we discovered a new Rab7-interacting protein, Rac1, which is another small GTP-binding protein and binds to the GTP-form of Rab7 in vitro. We demonstrated further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, this colocalization is mainly perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, we suggest that the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments, thus enabling endosomal vesicles to switch tracks from microtubules to microfilaments before their fusion to the ruffled border. We then studied the role of Rab-Rac1 interaction in the slow recycling pathway. We revealed that Rac1 also binds directly to Rab11 and to some other but not all Rab-proteins, suggesting that Rab-Rac1 interaction could be a general regulatory mechanism to direct the intracellular vesicles from microtubule mediated transport to actin filament mediated transport and vice versa. On the basis of our results we thus propose a new hypothesis for these GTPases in the regulation of intracellular membrane flow.

Ultrastructural lesions in the myocardium and kidneys of rabbits in experimental acute Amorimia exotropica poisoning

Abstract: Amorimia exotropica is an important plant associated with sudden death in cattle in Southern Brazil. In order to understand the mechanisms by which A. exotropica causes acute lesions in the heart and kidney of intoxicated animals, an experiment was conducted to determine the histopathology and ultrastructure of myocardial and renal lesions of intoxicated rabbits. After receiving 18g/kg of dried plant, six rabbits died suddenly. At necropsy, the liver was swollen and no other macroscopic lesions were observed. Histologically, centrolobular and midzonal hepatocytes were vacuolated. These vacuoles were strong PAS stained positive, suggesting that they corresponded to glycogen accumulations. In some regions of the ventricular septum and ventricles were found vacuoles of different sizes and the kidneys of two rabbits showed vacuolar degeneration on distal convoluted tubules. Ultrastructurally, the myocardium had cardiomyocytes swelling with separation of myofibrils bundles and rupture and disorganization of the sarcomeres. The mitochondria displayed swelling, disorganization, disruption of the mitochondrial cristae, and electron-dense matrix. Some mitochondria exhibited eccentric projections of their membranes with disruption of both outer and inner membranes. The sarcoplasmic reticulum had no alterations, whereas the T-tubule system was occasionally dilated and ruptured. The kidneys had mitochondrial swelling with disorganization and disruption of the mitochondrial cristae. The vacuoles result from the swelling of the endoplasmatic reticulum and usually were located between two basolateral infoldings and mitochondria, occurring preferentially around the nucleus. The myocytes and T system damages induced by A. exotropica result in acute heart failure and death. Furthermore, this mechanism of cardiotoxicity may be common to all plant containing monofluoroacetate.