Dense Vesicles Research Articles

Possible Signaling Pathways Mediating Neuronal Calcium Sensor-1-Dependent Spatial Learning and Memory in Mice.

Intracellular Ca2+ signaling regulates diverse functions of the nervous system. Many of these neuronal functions, including learning and memory, are regulated by neuronal calcium sensor-1 (NCS-1). However, the pathways by which NCS-1 regulates these functions remain poorly understood. Consistent with the findings of previous reports, we revealed that NCS-1 deficient (Ncs1-/-) mice exhibit impaired spatial learning and memory function in the Morris water maze test, although there was little change in their exercise activity, as determined via treadmill-analysis. Expression of brain-derived neurotrophic factor (BDNF; a key regulator of memory function) and dopamine was significantly reduced in the Ncs1-/- mouse brain, without changes in the levels of glial cell-line derived neurotrophic factor or nerve growth factor. Although there were no gross structural abnormalities in the hippocampi of Ncs1-/- mice, electron microscopy analysis revealed that the density of large dense core vesicles in CA1 presynaptic neurons, which release BDNF and dopamine, was decreased. Phosphorylation of Ca2+/calmodulin-dependent protein kinase II-α (CaMKII-α, which is known to trigger long-term potentiation and increase BDNF levels, was significantly reduced in the Ncs1-/- mouse brain. Furthermore, high voltage electric potential stimulation, which increases the levels of BDNF and promotes spatial learning, significantly increased the levels of NCS-1 concomitant with phosphorylated CaMKII-α in the hippocampus; suggesting a close relationship between NCS-1 and CaMKII-α. Our findings indicate that NCS-1 may regulate spatial learning and memory function at least in part through activation of CaMKII-α signaling, which may directly or indirectly increase BDNF production.

Distinguishing styles of explosive eruptions at Erebus, Redoubt and Taupo volcanoes using multivariate analysis of ash morphometrics

The style and dynamics of volcanic eruptions control the level and type of hazards posed for local populations and can have a temporary long-range impact on climate if eruptions are extremely energetic. The purpose of this study is to provide a statistical approach to ash morphometrics in order to provide a means by which to evaluate diverse eruption styles and mechanisms of fragmentation. The methodology presented can be applied to tephra deposits worldwide and may aid volcanic hazard mitigation by better defining a volcano's history of explosive behavior.Ash-sized grains were collected from tephra deposits on Mount Erebus, Antarctica (<10ka, phonolitic unit SC4), Mount Redoubt, Alaska (2009, andesitic events 2–4 & 9–18), and the Taupo volcano, New Zealand (1.8ka, rhyolitic unit 3D). Coarse ash from each deposit was carefully hand-sieved to ~1mm diameter and display diverse morphologies that vary from grains that are moderately vesicular and more rectangular (blocky) to highly vesiculated (spongy) grains that vary from angular to sub-rounded. A total of 264 grains were imaged by scanning electron microscopy. Morphometric properties were determined using image processing software and then evaluated by several statistical methods.Discriminant analysis of all parameters was found to be the best at differentiating the tephra deposits and allowing for interpretation of eruptive styles in conjunction with field observations. A linear array of data forming a positive slope in factor space, which explains >99% of the total data variance, is interpreted to represent a continuum between fragmentations involving water-magma interaction (“wet”) to grains that were formed predominately by magmatic (“dry”) fragmentation mechanisms. The Taupo Hatepe ash, which was deposited from a phreatoplinian eruption column, has the highest factor values in the array, which signifies, in part, more rectangular/blocky morphologies with smooth grain edges. Factor values for the 2009 Redoubt eruption (events 2–4) are nearly as high as Hatepe ash and based on this we suggest that it was produced, in part, by phreatomagmatic fragmentation. This is supported by field observations that document melting and eruption through glacial ice during the early phases of the 2009 activity. Redoubt ash grains from later stages of the same eruption (events 9–18) show a significant shift to lower values in factor space (more irregular/vesiculated grains) and are interpreted to be a consequence of ‘dryer’ conditions. Coarse ash data from Mount Erebus are completely separated from Taupo and Redoubt grains in factor space due primarily to the difference in mean gray value, which is a proxy for vesicle density and size. The vesicle characteristics (larger and deeper) are consistent with documented strombolian-style activity and the scatter in grain shape data support fragmentation by a mixture of wet and dry processes as has previously been proposed based on deposit characteristics and resemblance to tephra produced by current activity.

EFFECT OF AMYLOID FIBRILS ON ELECTROKINETIC PROPERTIES OF LIPID VESICLES

The influence of the lysozyme and serum albumin in their native and amyloid forms on the electrokinetic behavior of the negatively charged uni- and multilamellar liposomes from the zwitterionic lipid phosphatidylcholine and anionic lipid cardiolipin has been investigated using the microelectrophoresis technique. The zeta - potential, the surface electrostatic potential and surface charge density of the lipid vesicles have been determined upon varying the lipid-to-protein molar ratio. The complex dependencies of the electrophoretic mobility on the protein concentration and reversal of the surface charge observed for the multilamellar vesicles have been explained by the multilayer protein adsorption on the liposomal surface. It has been found that the native and fibrillar proteins differ in their ability to modify the charge state of the model membranes.

From nucleus pulposus mesenchymal stem cells towards neural differentiation: an interesting prospect

Regenerative medicine arouses great interest for the treatment of many neurological diseases. Since nucleus pulposus of the invertebral discs is a postembryonic vestige of the notochord, it has been hypothesized that mesenchymal stem cells (MSCs) isolated from nucleus pulposus (NP-MSCs) can more easily differentiate into neurons. In this study, MSCs from nucleus pulposus were successfully isolated and characterized. Then, neural differentiation was induced by using a medium consisted of DMEM/F12 supplemented with B27 and the growth factors FGF and EGF for 10 days. Immunocytochemistry, molecular studies, SEM and TEM microscopy analyses were performed. NP-MSCs exhibited the typical features of MSCs, revealing spindle-shape morphology, specific immunophenotype attributable to MSCs and the ability to differentiate in osteogenic and chondrogenic lineages. After neurodifferentiation induction, compared to NP-MSCs in only DMEM/F12, proliferation rate decreased and cells changed morphology acquiring an increased number of the so-called neural-like extensions. Neural progenitor marker NESTIN and mature neuronal marker ENOLASE-2 were up-regulated, while GFAP was not detected. Moreover, cells after differentiation were small rounded and fusiform, with tendency to organize in clumps; they had elongated extrusions containing oriented cytoskeletal elements, classifiable as microtubules and intermediate filaments, as visualized by SEM and TEM microscopy. Dense vesicles similar to lipid droplet were also observed. NP-MSCs in differentiation medium were able to form neurospheres. In conclusion, even if more analysis have to be done and the way to treat neurodegenerative disease with regenerative medicine is still long, NP-MSCs represent a promising resource.

Rapid and facile quantitation of polyplex endocytic trafficking

Design of safe and effective synthetic nucleic acid delivery vectors such as polycation/DNA or polycation/siRNA complexes (polyplexes) will be facilitated by quantitative understanding of the mechanisms by which such materials escort cargo from the cell surface to the nucleus. In particular, the mechanisms of cellular internalization by various endocytosis pathways and subsequent endocytic vesicle trafficking have been shown to strongly affect nucleic acid delivery efficiency. Fluorescence microscopy and subcellular fractionation methods are commonly employed to follow intracellular trafficking of biomolecules and nanoparticulate delivery systems such as polyplexes. However, it is difficult to obtain quantitative data from microscopy and subcellular fractionation is experimentally difficult and low throughput. We have developed a method for quantifying the transport of polyplexes through important endocytic vesicles. The method is based on polymerization of 3,3′-diaminobenzidine by endocytosed horseradish peroxidase, causing an increase in the vesicle density, resistance to being solubilized by detergent and quenching of fluorophores within the vesicles, which makes them easy to separate and quantify. Using this method in HeLa cells, we have observed polyethylenimine/siRNA polyplexes initially appearing in early endosomes and rapidly moving to other compartments within 30min post-transfection. At the same time, we observed the kinetics of accumulation of the polyplexes in lysosomes at a similar rate. The results from the new method are consistent with similar measurements by confocal fluorescence microscopy and subcellular fractionation of endocytic vesicles on a Percoll gradient. The relative ease of this new method will aid investigation of gene delivery mechanisms by providing the means to rapidly quantify endocytic trafficking of polyplexes and other vectors.

Targeting Autophagy As a Therapeutic Strategy in Acute Myeloid Leukemia

Introduction: Autophagy is a process whereby cells digest their own organelles in conditions of stress, such as low nutrient concentration, hypoxia or exposure to chemotherapy. It is well established that acute myeloid leukemia (AML) cells presiding within a hypoxic microenvironment are markedly less sensitive to cytarabine chemotherapy than normoxic cells. We hypothesized that AML cells survive and mediate chemoresistance by invoking autophagy mediators, specifically light chain 3B protein (LC3B). LC3B is a key regulator in the fusion of autophagosomes and lysosomes, giving rise to autophagolysosomes.Methods: Human AML cells (HEL, HL60) were grown under normoxic (21% O2) and hypoxic (1% O2) conditions for 24-72 hours. Cells were treated with cytarabine chemotherapy and/or the autophagy inhibitors, bafilomycin A1 (Baf) and chloroquine (CQ). Viability was determined using trypan blue cell counting and tetrazolium dye MTT. Apoptosis and cell death were measured by flow cytometry for annexin-FITC and propidium iodide. Autophagy was assessed by flow cytometry using Cyto-ID Green Dye (Enzo Life Sciences), fluorescent microscropy for acridine orange dye accumulation, and western blot analysis. HEL cells were also transfected with an anti-LC3B siRNA and non-target sequences or anti-GAPDH siRNA as negative and positive controls, respectively. Real-time PCR with LC3B-specific probes, along with Western blotting with a rabbit anti-LC3B primary antibody, were performed to assess for changes in LC3B mRNA and protein levels.Results: Autophagy in human AML cell lines was significantly increased following 24-72 hours of low oxygenation (1% O2) as compared with normoxia and was a predominantly late response to prolonged hypoxia (> 48 hours). We determined that AML cells exposed to chronic hypoxia were able to overcome an initial growth restriction with a corresponding increase in LC3B levels by Western blotting. Treatment of AML with ARA-C under hypoxia resulted in further dose-dependent increases in autophagic vesicles in AML cells consistent with enhanced autophagy induction. In contrast, exposure of hypoxic HEL cells to the autophagy inhibitors (Baf, CQ), led to arrest in autophagic flux (via higher levels of the LC3BII isoform expression as compared with LC3B I) and decreased autophagic vesicle density (as measured by fluorescent microscopy for acridine orange). Combination treatment with autophagy inhibitors (Baf, CQ) and ARA-C chemotherapy significantly enhanced apoptosis and cell death of AML cells under hypoxia as compared with single agent therapy.To further confirm these effects were LC3B dependent, we used anti-LC3B versus nonspecific siRNA to knockdown protein expression in HEL cells. HEL cells with anti-LC3B siRNA exhibited markedly decreased cell viability under prolonged hypoxia as compared to control siRNA transfected cells. Cells also demonstrated enhanced cell death following ARA-C treatment under hypoxia with the most pronounced effects ranging from 48h up to 72h from the start of treatment (Figure 1).Conclusions: Our experiments demonstrate that the autophagy pathway is upregulated in human AML cells under hypoxic conditions and likely confers a rescue pathway under such conditions to promote leukemia survival. Using pharmacological inhibitors of autophagy and anti-LC3B siRNA technology, we show that targeting the autophagy pathway can render hypoxic AML cells more susceptible to cell death and enhance sensitivity to cytarabine chemotherapy. These preclinical results show promise for further exploring autophagy as a therapeutic target in acute myeloid leukemia. [Display omitted] DisclosuresScott:Immunogen: Research Funding. Wang:Immunogen: Research Funding; Incyte: Speakers Bureau.

Influence of arbuscular mycorrhizal fungi on the vegetative development of citrus rootstocks1

ABSTRACT The use of arbuscular mycorrhizal fungi (AMF) in the production of rootstocks is an alternative to accelerate plant growth. However, their response depends on the symbionts species and environment. This study aimed at evaluating the influence of AMF species [Scutelospora heterogama, Gigaspora margarita, Glomus etunicatum, Acaulospora sp. and a control (non-inoculated)] on the vegetative development of citrus rootstocks {citrange 'Fepagro C37 Reck' [P. trifoliata (L.) Raf. x C. sinensis (L.) Osbeck.] and 'Kumquat' [Fortunella hindsii (L.) Swingle]}. The experimental design consisted of split-plot randomized blocks, with 10 plants per plot and 3 replications. Height, stem diameter, number of leaves, leaf area and fresh and dry root and shoot mass were evaluated. The colonization of AMF in the roots was also assessed, determining the percentage of colonization and density of hyphae, arbuscules and vesicles. The rootstocks showed no difference for the plant growth parameters, in the absence of AMF. The AMF species colonized the rootstocks roots, but were only effective in accelerating the citrange 'Fepagro C37 Reck' growth, especially when inoculated with Scutelospora heterogama, Gigaspora margarita and Glomus etunicatum. The influence of AMF on vegetative development depends on the citrus rootstock species.

Sphingosine 1-phosphate lyase ablation disrupts presynaptic architecture and function via an ubiquitin- proteasome mediated mechanism.

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.

Perinatal exposure to lead (Pb) induces ultrastructural and molecular alterations in synapses of rat offspring.

Lead (Pb), environmentally abundant heavy-metal pollutant, is a strong toxicant for the developing central nervous system. Pb intoxication in children, even at low doses, is found to affect learning and memorizing, with devastating effects on cognitive function and intellectual development. However, the precise mechanism by which Pb impairs synaptic plasticity is not fully elucidated. The purpose of this study was to investigate the effect of pre- and neonatal exposure to low dose of Pb (with Pb concentrations in whole blood below 10μg/dL) on the synaptic structure and the pre- and postsynaptic proteins expression in the developing rat brain. Furthermore, the level of brain-derived neurotrophic factor (BDNF) was analyzed. Pregnant female Wistar rats received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring, while the control animals received drinking water. During the feeding of pups, mothers from the Pb-group were continuously receiving PbAc. Pups of both groups were weaned at postnatal day 21 and then until postnatal day 28 received only drinking water. 28-day old pups were sacrificed and the ultrastructural changes as well as expression of presynaptic (VAMP1/2, synaptophysin, synaptotagmin-1, SNAP25, syntaxin-1) and postsynaptic (PSD-95) proteins were analyzed in: forebrain cortex, cerebellum and hippocampus. Our data revealed that pre- and neonatal exposure to low dose of Pb promotes pathological changes in synapses, including nerve endings swelling, blurred and thickened synaptic cleft structure as well as enhanced density of synaptic vesicles in the presynaptic area. Moreover, synaptic mitochondria were elongated, swollen or shrunken in Pb-treated animals. These structural abnormalities were accompanied by decrease in the level of key synaptic proteins: synaptotagmin-1 in cerebellum, SNAP25 in hippocampus and syntaxin-1 in cerebellum and hippocampus. In turn, increased level of synaptophysin was noticed in the cerebellum, while the expression of postsynaptic PSD-95 was significantly decreased in forebrain cortex and cerebellum, and raised in hippocampus. Additionally, we observed the lower level of BDNF in all brain structures in comparison to control animals. In conclusion, perinatal exposure to low doses of Pb caused pathological changes in nerve endings associated with the alterations in the level of key synaptic proteins. All these changes can lead to synaptic dysfunction, expressed by the impairment of the secretory mechanism and thereby to the abnormalities in neurotransmission as well as to the neuronal dysfunction.

Epigenetic Regulation of SNAP25 Prevents Progressive Glutamate Excitotoxicty in Hypoxic CA3 Neurons

Exposure to global hypoxia and ischemia has been reported to cause neurodegeneration in the hippocampus with CA3 neurons. This neuronal damage is progressive during the initial phase of exposure but maintains a plateau on prolonged exposure. The present study on Sprague Dawley rats aimed at understanding the underlying molecular and epigenetic mechanisms that lead to hypoxic adaptation of CA3 neurons on prolonged exposure to a global hypoxia. Our results show stagnancy in neurodegeneration in CA3 region beyond 14days of chronic exposure to hypobaria simulating an altitude of 25,000ft. Despite increased synaptosomal glutamate and higher expression of NR1 subunit of NMDA receptors, we observed decrease in post-synaptic density and accumulation of synaptic vesicles at the pre-synaptic terminals. Molecular investigations involving western blot and real-time PCR showed duration-dependent decrease in the expression of SNAP-25 resulting in reduced vesicular docking and synaptic remodeling. ChIP assays for epigenetic factors showed decreased expression of H3K9Ac and H3K14Ac resulting in SNAP-25 promoter silencing during prolonged hypoxia. Administration of sodium butyrate, a non-specific HDAC inhibitor, during 21days hypoxic exposure prevented SNAP-25 downregulation but increased CA3 neurodegeneration. This epigenetic regulation of SNAP-25 promoter was independent of increased DNMT3b expression and promoter methylation. Our findings provide a novel insight into epigenetic factors-mediated synaptic remodeling to prevent excitotoxic neurodegeneration on prolonged exposure to global hypobaric hypoxia.

Gender difference in valproic acid-induced neuroprotective effects on APP/PS1 double transgenic mice modeling Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive impairment with gender difference in specific cognitive ability domains, pathology, and risk of AD. Since valproic acid (VPA) is a widely used mood stabilizer and an antiepileptic drug, which exhibits multiple neuroprotective activities on AD, this study intended to investigate the gender difference in the effect of VPA on APP/PS1 double transgenic mice modeling AD. Behavioral experiments showed that VPA reduced the autonomous behaviors, improved learning and memory, and exhibited gender differences in AD mice compared with the control mice. The decrease in senile plaque, amyloid β (Aβ) 40, and Aβ42 caused by VPA in the male AD mice was more notable than that in the female AD mice. Meanwhile, VPA protected brain cells from dying notably in the male AD mice but only slightly in the female AD mice, and VPA treatment thickened the postsynaptic density and markedly increased the number and density of presynaptic vesicles in both male and female AD mice. However, the effects of rescuing early synaptic structural and functional deficits by VPA were more obvious in the male mice. Overall, these results supported the hypothesis that gender difference significantly influences AD and indicated that VPA may be a promising remedy for AD if basic biological differences and gender specificity were prudently taken into account.

Conductive Hearing Loss Has Long-Lasting Structural and Molecular Effects on Presynaptic and Postsynaptic Structures of Auditory Nerve Synapses in the Cochlear Nucleus.

Sound deprivation by conductive hearing loss increases hearing thresholds, but little is known about the response of the auditory brainstem during and after conductive hearing loss. Here, we show in young adult rats that 10 d of monaural conductive hearing loss (i.e., earplugging) leads to hearing deficits that persist after sound levels are restored. Hearing thresholds in response to clicks and frequencies higher than 8 kHz remain increased after a 10 d recovery period. Neural output from the cochlear nucleus measured at 10 dB above threshold is reduced and followed by an overcompensation at the level of the lateral lemniscus. We assessed whether structural and molecular substrates at auditory nerve (endbulb of Held) synapses in the cochlear nucleus could explain these long-lasting changes in hearing processing. During earplugging, vGluT1 expression in the presynaptic terminal decreased and synaptic vesicles were smaller. Together, there was an increase in postsynaptic density (PSD) thickness and an upregulation of GluA3 AMPA receptor subunits on bushy cells. After earplug removal and a 10 d recovery period, the density of synaptic vesicles increased, vesicles were also larger, and the PSD of endbulb synapses was larger and thicker. The upregulation of the GluA3 AMPAR subunit observed during earplugging was maintained after the recovery period. This suggests that GluA3 plays a role in plasticity in the cochlear nucleus. Our study demonstrates that sound deprivation has long-lasting alterations on structural and molecular presynaptic and postsynaptic components at the level of the first auditory nerve synapse in the auditory brainstem. Despite being the second most prevalent form of hearing loss, conductive hearing loss and its effects on central synapses have received relatively little attention. Here, we show that 10 d of monaural conductive hearing loss leads to an increase in hearing thresholds, to an increased central gain upstream of the cochlear nucleus at the level of the lateral lemniscus, and to long-lasting presynaptic and postsynaptic structural and molecular effects at the endbulb of the Held synapse. Knowledge of the structural and molecular changes associated with decreased sensory experience, along with their potential reversibility, is important for the treatment of hearing deficits, such as hyperacusis and chronic otitis media with effusion, which is prevalent in young children with language acquisition or educational disabilities.

Vesicle Size Distribution as a Novel Nuclear Forensics Tool.

The first nuclear bomb detonation on Earth involved a plutonium implosion-type device exploded at the Trinity test site (33°40′38.28″N, 106°28′31.44″W), White Sands Proving Grounds, near Alamogordo, New Mexico. Melting and subsequent quenching of the local arkosic sand produced glassy material, designated “Trinitite”. In cross section, Trinitite comprises a thin (1–2 mm), primarily glassy surface above a lower zone (1–2 cm) of mixed melt and mineral fragments from the precursor sand. Multiple hypotheses have been put forward to explain these well-documented but heterogeneous textures. This study reports the first quantitative textural analysis of vesicles in Trinitite to constrain their physical and thermal history. Vesicle morphology and size distributions confirm the upper, glassy surface records a distinct processing history from the lower region, that is useful in determining the original sample surface orientation. Specifically, the glassy layer has lower vesicle density, with larger sizes and more rounded population in cross-section. This vertical stratigraphy is attributed to a two-stage evolution of Trinitite glass from quench cooling of the upper layer followed by prolonged heating of the subsurface. Defining the physical regime of post-melting processes constrains the potential for surface mixing and vesicle formation in a post-detonation environment.

3D analysis of synaptic vesicle density and distribution after acute foot-shock stress by using serial section transmission electron microscopy.

Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter-containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot-shock stress and (2) rats with sham stress as control group. Two-dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in-section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three-dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one-dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot-shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.

Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95

Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95(S295)) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin.

Different segments of the cerebral vasculature reveal specific endothelial specifications, while tight junction proteins appear equally distributed

The identification of the "paucity of transportation vesicles" and "belt-like" tight junctions (TJs) of endothelial cells as the "morphological correlate of a blood-brain barrier" (BBB) by Reese and Karnovsky (J Cell Biol 34:207-217, 1967) has become textbook knowledge, and countless studies have helped to further define the elements, functions, and dynamics of the BBB. Most work, however, has focused on parenchymal capillaries or less clearly defined "microvessels", while a systematic study on similarities and differences between BBB architecture along the vascular tree within the brain and the meninges has been lacking. Since astrocytes induce endothelial cells to display BBB-typical characteristics by sonic hedgehog and Wnt/β-catenin signaling, we hypothesized that BBB-typical features should be most pronounced in parenchymal capillaries, where endothelium and astrocytes are separated by a basement membrane only. In contrast, this intimate contact is absent in leptomeningeal vessels, thereby potentially affecting BBB architecture. However, here, we show that claudin-3, claudin-5, zonula occludens-1, and occludin as typical constitutes of BBB TJs are comparably distributed in all segments of the parenchymal and the meningeal vascular tree of C57Bl6 mice. While electron microscopy revealed equally occluded interendothelial clefts, arterial vessels of the brain parenchyma but not within the meninges exhibited significantly longer TJ overlaps compared to capillaries. The highest density of endothelial vesicles was found in arterial vessels. Thus, endothelial expression of BBB-typical TJ proteins is not reflected by the distance to surrounding astrocytes, but electron microscopy reveals significant differences of endothelial specification along different segments of the CNS vasculature.

Microtubule-associated protein 1B (MAP1B)-deficient neurons show structural presynaptic deficiencies in vitro and altered presynaptic physiology.

Microtubule-associated protein 1B (MAP1B) is expressed predominantly during the early stages of development of the nervous system, where it regulates processes such as axonal guidance and elongation. Nevertheless, MAP1B expression in the brain persists in adult stages, where it participates in the regulation of the structure and physiology of dendritic spines in glutamatergic synapses. Moreover, MAP1B expression is also found in presynaptic synaptosomal preparations. In this work, we describe a presynaptic phenotype in mature neurons derived from MAP1B knockout (MAP1B KO) mice. Mature neurons express MAP1B, and its deficiency does not alter the expression levels of a subgroup of other synaptic proteins. MAP1B KO neurons display a decrease in the density of presynaptic and postsynaptic terminals, which involves a reduction in the density of synaptic contacts, and an increased proportion of orphan presynaptic terminals. Accordingly, MAP1B KO neurons present altered synaptic vesicle fusion events, as shown by FM4-64 release assay, and a decrease in the density of both synaptic vesicles and dense core vesicles at presynaptic terminals. Finally, an increased proportion of excitatory immature symmetrical synaptic contacts in MAP1B KO neurons was detected. Altogether these results suggest a novel role for MAP1B in presynaptic structure and physiology regulation in vitro.

Trichocysts-Paramecium's Projectile-like Secretory Organelles: Reappraisal of their Biogenesis, Composition, Intracellular Transport, and Possible Functions.

This review summarizes biogenesis, composition, intracellular transport, and possible functions of trichocysts. Trichocyst release by Paramecium is the fastest dense core-secretory vesicle exocytosis known. This is enabled by the crystalline nature of the trichocyst "body" whose matrix proteins (tmp), upon contact with extracellular Ca2+ , undergo explosive recrystallization that propagates cooperatively throughout the organelle. Membrane fusion during stimulated trichocyst exocytosis involves Ca2+ mobilization from alveolar sacs and tightly coupled store-operated Ca2+ -influx, initiated by activation of ryanodine receptor-like Ca2+ -release channels. Particularly, aminoethyldextran perfectly mimics a physiological function of trichocysts, i.e. defense against predators, by vigorous, local trichocyst discharge. The tmp's contained in the main "body" of a trichocyst are arranged in a defined pattern, resulting in crossstriation, whose period expands upon expulsion. The second part of a trichocyst, the "tip", contains secretory lectins which diffuse upon discharge. Repulsion from predators may not be the only function of trichocysts. We consider ciliary reversal accompanying stimulated trichocyst exocytosis (also in mutants devoid of depolarization-activated Ca2+ channels) a second, automatically superimposed defense mechanism. A third defensive mechanism may be effectuated by the secretory lectins of the trichocyst tip; they may inhibit toxicyst exocytosis in Dileptus by crosslinking surface proteins (an effect mimicked in Paramecium by antibodies against cell surface components). Some of the proteins, body and tip, are glycosylated as visualized by binding of exogenous lectins. This reflects the biogenetic pathway, from the endoplasmic reticulum via the Golgi apparatus, which is also supported by details from molecular biology. There are fragile links connecting the matrix of a trichocyst with its membrane; these may signal the filling state, full or empty, before and after tmp release upon exocytosis, respectively. This is supported by experimentally produced "frustrated exocytosis", i.e. membrane fusion without contents release, followed by membrane resealing and entry in a new cycle of reattachment for stimulated exocytosis. There are some more puzzles to be solved: Considering the absence of any detectable Ca2+ and of acidity in the organelle, what causes the striking effects of silencing the genes of some specific Ca2+ -release channels and of subunits of the H+ -ATPase? What determines the inherent polarity of a trichocyst? What precisely causes the inability of trichocyst mutants to dock at the cell membrane? Many details now call for further experimental work to unravel more secrets about these fascinating organelles.

New ultrastructural observations of human oocyte smooth endoplasmic reticulum tubular aggregates and cortical reaction: update on the molecular mechanisms involved

IntroductionThe ultrastructure of the human mature metaphase-II oocyte before and after the cortical reaction has been previously described. However, we found a new vesicular aggregate associated with smooth endoplasmic reticulum tubular aggregates (aSERT) and new observations regarding the cortical reaction. ObjectivesTo describe at the ultrastructural level a novel vesicle aggregate associated with aSERT and new observations regarding the cortical reaction. Materials and methodsDonated, surplus mature oocytes were processed for transmission electron microscopy immediately after recover. Calcium detection was performed using the pyroantimonate technique. The cortical reaction was artificially induced by ionophore treatment. ResultsWe observed an accumulation of small vesicles at the periphery of aSERT. At high magnification these were composed by small pale vesicles coated by tiny vesicles, with associated dense materials, giving a rosette-like appearance. Adjacent to these there was another group of small dense vesicles incompletely coated by similar tiny vesicles. Using calcium detection at the ultrastructural level, antimonate deposits were observed in the tubules of aSERT and in the surrounding mitochondria but not in the rosette-like structures. Regarding cortical vesicles, although most previous studies described their contents as homogeneous dense, others referred the presence of another type of cortical vesicles whose contents were moderate dense. Using ionophore oocyte activation, we observed that moderate dense cortical vesicles may correspond to a progressive swelling of the dense cortical vesicles prior to exocytosis. ConclusionsWe describe a novel vesicle aggregate associated to aSERT and new observations on the remodeling of cortical vesicles before exocytosis.

Embryonic development of the saccular sensory epithelium in relation to otolith growth in the inner ear of the silver carp, Hypophthalmichthys molitrix (Valenciennes, 1844) (Teleostei: Cyprinidae): light and electron microscopic study

The development of the saccular sensory epithelium in the inner ear of the silver carp, Hypophthalmichthys molitrix, was studied from an early stage (2 days after fertilization), when the otic vesicle or otocyst first formed, to the post-larval stage (7 days after hatching), when the development of the inner ear approximates that of the juvenile stage. Light microscopy revealed: (1) the otic vesicle, the primordia of the saccular otolith and the stato-acoustic ganglion were observed by 2 days after fertilization; (2) the saccular macula overlain by a small round otolith is established at 3 days after fertilization, while the utricular macula with its otolith is first observed at 4 days after fertilization; (3) the saccular macula began to differentiate at 3 days after hatching and became well differentiated at 7 days after hatching. At 7 days after hatching, transmission electron microscopy (TEM) revealed: (1) the apical surface of each hair cell is covered with a ciliary bundle formed of a long kinocilium and a short bundle of stereocilia. The supporting cells are provided with microridges on their apical surface and seemed to be covered with small and large vesicles, suggesting that they have a secretory function beside the supporting one; (2) secretory materials such as multivesicular bodies, electron dense granules, empty vesicles and cytoplasmic extrusions are observed scattering over the saccular sensory epithelium, which probably contribute to the formation of the otolith and/or otolithic membrane. Using scanning electron microscopy (SEM), two types of hair bundles are distinguished: the first type consisted of numerous short stereocilia and a kinocilium; the second type has a small number of short stereocilia with a very long kinocilium. Most of these results are discussed with special regard to the environmental factors affecting the early development of the inner ear in teleost fishes.