Dystrophin immunoreactivity in normal and Duchenne human fetal neurons in culture.

Dystrophin, the protein product defective in Duchenne muscular dystrophy (DMD), is present in all types of muscle and in the brain. The function of the protein is unknown and its role in the brain is unclear, although 30% of DMD patients show nonprogressive mental retardation. We have therefore studied the localisation of dystrophin in cultures of normal and DMD human fetal neurons using antibodies raised to different regions of the protein. Dystrophin immunoreactivity was demonstrated in the soma and axon hillock of normal neurons and appeared to be associated with the inner part of the cell membrane, although some intracellular staining was also observed. Positive dystrophin staining was present only in cells with fully developed neuronal features, although not all the neurons were positive. Glial cells were always negative for the antigen. Immunostaining with antibodies to the brain spectrins indicate that the dystrophin antibodies did not crossreact with these proteins. The possibility of cross-reactivity with other proteins is discussed. Studies of cells cultured from a DMD fetus also showed specific dystrophin immunostaining in neurons, although the muscle was generally negative for dystrophin. However, the localisation of dystrophin immunostaining and that of the brain spectrins and neurofilaments appeared abnormal, as did the overall morphology of the cells. This suggests that dystrophin may play a role during brain development and dystrophin deficiency results in abnormal neuronal features. This would be consistent with the nonprogressive nature of the mental retardation observed in DMD patients.

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons

Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The calibre of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. In this study we quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (Dmean) in neurites from 0.12 ± 0.05 µm2/s to 0.61 ± 0.03 µm2/s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9 ± 1.9 to 15 ± 4.9 µm/s, whereas a velocity increase from 9 ± 1.3 to 14 ± 3 µm/s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.

Vegetable Oils Deacidification by Solvent Extraction: Liquid-Liquid Equilibrium Data for Systems Containing Sunflower Seed Oil at 298.2 K

Liquid-liquid equilibrium experimental data for refined sunflower seed oil, artificially acidified with commercial oleic acid or commercial linoleic acid and a solvent (ethanol + water), were determined at 298.2 K. This set of experimental data and the experimental data from Cuevas et al.,(1) which were obtained from (283.2 to 333.2) K, for degummed sunflower seed oil-containing systems were correlated using NRTL and UNIQUAC models with temperature-dependent binary parameters. The deviation between experimental and calculated compositions presented average values of (1.13 and 1.41) % for NRTL and UNIQUAC equations, respectively, indicating that the models were able to correctly describe the behavior of compounds under different temperature and solvent hydration.

Gelatin-based films containing hydrophobic plasticizers and saponin from Yucca schidigera as the surfactant

The aim of this study was to evaluate the incorporation of hydrophobic plasticizers (acetyltributyl citrate - ATB, tributyl citrate - TB and acetyltriethyl citrate - ATC) in a matrix of gelatin, using the saponin extracted from Yucca schidigera (yucca) as emulsifier, in the production of biodegradable emulsified films using the casting technique. High levels of hydrophobic plasticizers were incorporated, reaching up to 75% of plasticizer in relation to the protein (w/w) for ATB and TB, and up to 60% for ATC. The minimum values of water vapor permeability were 0.08, 0.07 and 0.06 g mm m(-2) h(-1) kPa(-1) for ATB, TB and ATC respectively, with no significant differences (p > 0.05). The water solubility of the films ranged from 21% to 59.5%. Although the WVP decreased, both scanning electron microscopy and laser scanning confocal microscopy indicated that the incorporation of the hydrophobic plasticizers did not occur homogeneously in the film matrix. (C) 2010 Elsevier Ltd. All rights reserved.

Dielectric properties and physical features of phosphate glasses containing iron oxide

Microwave techniques were applied to the study of dielectric properties of phosphate glasses on the basis of contributions from permanent and induced dipolar polarization of local structural units interacting with the electrical component of the electromagnetic radiation. The dielectric constant of the selected glass system (100-x)(50P(2)O(5)center dot 25Li(2)O center dot 25Na(2)O)center dot xFe(2)O(3), where 0 <= x <= 21 is in mol%, was measured using a microwave setup assembled to measure the phase shift of the standing wave pattern produced by the insertion of the sample. It is shown that the Fe2+ ions contribute effectively to the dielectric constant, as expected from the interactions of the dipoles of the local charge compensation pairs with the microwave radiation. However, there is the possibility of occurrence of some ions Fe3+, in general, at low iron content, which reinforces the glass structure and, therefore, decreases the dielectric constant. There is a gradual conversion from Fe3+ to Fe2+ as the iron ions increases. This is possibly the reason of the anomaly in the dielectric constant values observed in the results. These assumptions can be checked by results of electronic paramagnetic resonance (EPR) and optical absorption (OA). The dielectric constant of the glasses studied in this work was found to increase with the temperature in the range of 25-330 degrees C. (C) 2007 Elsevier B.V. All rights reserved.

Downregulation of VAPB expression in motor neurons derived from induced pluripotent stem cells of ALS8 patients

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope, since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but, in contrast to over-expression systems, cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8, in agreement with the observed reduction of VAPB in sporadic ALS.

Postnatal fluoxetine treatment affects the development of serotonergic neurons in rats

The goal of the present study was to investigate morphological changes in the serotonergic neurons/terminals in the dorsal (DR) and median (MnR) raphe nuclei and on the hippocampal dentate gyrus (DG) in neonatal rats treated from the 1st to the 21st postnatal day with fluoxetine (10 mg/kg sc, daily) or drug vehicle (0 9% saline 1 ml/kg). The results show that postnatal chronic treatment with fluoxetine promoted. (1) a smaller body weight increase during the pre-weaning period; (2) smaller number of 5-HT neurons in the DR, (3) smaller 5-HT neuronal cell bodies (area, perimeter and diameter) in the DR and the MnR and (4) diminished serotonergic terminals in the DG. These data suggest that the development of the serotonergic system was impaired and that early exposure to fluoxetine damaged the morphology of 5-HT neurons in young adult rats While these findings are consistent with other work, more studies are needed to better clarify the effects of postnatal chronic treatment with fluoxetine on the serotonergic system and, consequently, on the functions modulated by serotonin (C) 2010 Elsevier Ireland Ltd All rights reserved

Chemical identity and connections of medial preoptic area neurons expressing melanin-concentrating hormone during lactation

Lactation is an energy-demanding process characterized by massive food and water consumption, cessation of the reproductive cycle and induction of maternal behavior. During lactation, melanin-concentrating hormone (MCH) mRNA and peptide expression are increased in the medial preoptic area (MPO) and in the anterior paraventricular nucleus of the hypothalamus. Here we show that MCH neurons in the MPO coexpress the GABA synthesizing enzyme GAD-67 mRNA. We also show that MCH neurons in the MPO of female rats are innervated by neuropeptides that control energy homeostasis including agouti-related protein (AgRP), alpha-melanocyte stimulating hormone (alpha MSH) and cocaine- and amphetamine-regulated transcript (CART). Most of these inputs originate from the arcuate nucleus neurons. Additionally, using injections of retrograde tracers we found that CART neurons in the ventral premammillary nucleus also innervate the MPO. We then assessed the projections of the female MPO using injections of anterograde tracers. The MPO densely innervates hypothalamic nuclei related to reproductive control including the anteroventral periventricular nucleus, the ventrolateral subdivision of the ventromedial nucleus (VMHvl) and the ventral premammillary nucleus (PMV). We found that the density of MCH-ir fibers is increased in the VMHvl and PMV during lactation. Our findings suggest that the expression of MCH in the MPO may be induced by changing levels of neuropeptides involved in metabolic control. These MCH/GABA neurons may, in turn, participate in the suppression of cyclic reproductive function and/or sexual behavior during lactation through projections to reproductive control sites. (C) 2009 Elsevier B.V. All rights reserved.

Exercise changes the size of cardiac neurons and protects them from age-related neurodegeneration

Aging leads to changes in cardiac structure and function. Evidence suggests that the practice of regular exercise may prevent disturbances in the cardiovascular system during aging. We studied the effects of aging on the morphology and morphometry of cardiac neurons in Wistar rats and investigated whether a lifelong moderate exercise program could exert a protective effect toward some deleterious effects of aging. Aging caused a significant decline (28%) in the number of NADH-diaphorase-stained cardiac Animals submitted to a daily session of 60 min, 5 day/week, at 1.1 km/h of running in treadmill over the entire life span exhibited a reversion of the observed decline in the number of cardiac neurons. However, most interesting was that the introduction of this lifelong exercise protocol dramatically altered the sizes of cardiac neurons. There was a notable increase in the percentage of small neurons in the rats of the exercise group compared to the sedentary animals. This is the first time that a protective effect of lifelong regular aerobic exercise has been demonstrated on the deleterious effects of aging in cardiac neurons. (C) 2009 Elsevier GmbH. All rights reserved.

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments In many cases, suitable treatment is problematic as the therapeutic target remains unknown Basic fibroblast growth factor (bFGF, FGF 2) is involved in neuronal maintenance and wound repair following nervous system lesions It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances Peripheral cranial somatic motor neurons, i e hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF 2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life Moreover, the modulatory effects of astroglial FGF 2 and the Ca+2 binding protein S100 beta have been postulated in paracrine mechanisms after neuronal lesions In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72 h or 11 days Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, ghat fibrillary acidic protein (GFAP, as a marker of astrocytes), S100 beta and FGF-2 The number of Nissl positive neurons of axotomized XII nucleus did not differ from controls The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72 h and 11 days after the surgery The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus, however, the nerve transection increased the number of FGF-2 ghat profiles by 72 h and 11 days Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF 2 immunoreactivity in axotomized XII neurons by 72 h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of ghat nuclei by 72h and 11 days after the two lesions S100 beta decreased in astrocytes of 11-day transected XII nucleus The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology (C) 2009 Elsevier GmbH All rights reserved

Atrophy and Neuron Loss: Effects of a Protein-Deficient Diet on Sympathetic Neurons

Protein deficiency is one of the biggest public health problems in the world, accounting for about 30-40% of hospital admissions in developing countries. Nutritional deficiencies lead to alterations in the peripheral nervous system and in the digestive system. Most studies have focused on the effects of protein-deficient diets on the enteric neurons, but not on sympathetic ganglia, which supply extrinsic sympathetic input to the digestive system. Hence, in this study, we investigated whether a protein-restricted diet would affect the quantitative structure of rat coeliac ganglion neurons. Five male Wistar rats (undernourished group) were given a pre- and postnatal hypoproteinic diet receiving 5% casein, whereas the nourished group (n = 5) was fed with 20% casein (normoproteinic diet). Blood tests were carried out on the animals, e.g., glucose, leptin, and triglyceride plasma concentrations. The main structural findings in this study were that a protein-deficient diet (5% casein) caused coeliac ganglion (78%) and coeliac ganglion neurons (24%) to atrophy and led to neuron loss (63%). Therefore, the fall in the total number of coeliac ganglion neurons in protein-restricted rats contrasts strongly with no neuron losses previously described for the enteric neurons of animals subjected to similar protein-restriction diets. Discrepancies between our figures and the data for enteric neurons (using very similar protein-restriction protocols) may be attributable to the counting method used. In light of this, further systematic investigations comparing 2-D and 3-D quantitative methods are warranted to provide even more advanced data on the effects that a protein-deficient diet may exert on sympathetic neurons. (C) 2009 Wiley-Liss, Inc.

Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are modulated by photic and non-photic stimuli. In rodents, direct photic stimuli reach the SCN mainly through the retinohypothalamic tract (RHT), whereas indirect photic stimuli are mainly conveyed by the geniculohypothalamic tract (GHT). In rodents, retinal cells form a pathway that reaches the intergeniculate leaflet (IGL) where they establish synapses with neurons that express neuropeptide Y (NPY), hence forming the GHT projecting to the SCN. In contrast to the RHT, which has been well described in primates, data regarding the presence or absence of the IGL and GHT in primates are contradictory. Some studies have suggested that an area of the pregeniculate nucleus (PGN) of primates might be homologous to the IGL of rodents, but additional anatomical and functional studies on primate species are necessary to confirm this hypothesis. Therefore, this study investigated the main histochemical characteristics of the PGN and the possible existence of the GHT in the SCN of the primate Cebus, comparing the distribution of NPY immunoreactivity, serotonin (5-HT) immunoreactivity and retinal terminal fibers in these two structures. The results show that a collection of cell bodies containing NPY and serotonergic immunoreactivity and retinal innervations are present within a zone that might be homologous to the IGL of rodents. The SCN also receives dense retinal innervations and we observed an atypical distribution of NPY- and 5-HT-immunoreactive fibers without regionalization in the ventral part of the nucleus as described for other species. These data may reflect morphological differences in the structures involved in the regulation of circadian rhythms among species and support the hypothesis that the GHT is present in some higher primates (diurnal animals). (C) 2009 Elsevier B.V. All rights reserved.