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In response to different stress signals, the c-Jun NH2-terminal kinase (JNK) can trigger cell death. However, JNK also facilitates the survival and cell cycle progression of tumor cells by mechanisms that are poorly defined. Here, we show that schwannoma RN22 cells can survive and proliferate under serum-free conditions although serum withdrawal rapidly induces mitochondrial fission and swelling. Although the morphologic changes observed in the mitochondria did not trigger cytochrome c release, they were accompanied by an increase in the mitochondrial membrane potential (ΔΨM) and of immunoreactivity for active JNK in these organelles. Pharmacologic inhibition of JNK provoked a further increase of the ΔΨM, an increase in reactive oxygen species (ROS) production, and a sustained decrease in cell viability due to necrosis. This increase in necrosis was prevented by the presence of ROS scavengers. Immunoreactivity for active JNK was also observed in the mitochondria of neuroblastoma 1E-115 and neuroblastoma 2a neuroblastoma cell lines on serum withdrawal, whereas active JNK was barely detected in serum-deprived fibroblasts. Accordingly, the reduction in neural tumor cell viability induced by JNK inhibition was largely attenuated in serum-deprived fibroblasts. These data indicate that local activation of JNK in the mitochondria can protect against necrotic cell death associated with ROS production, facilitating the growth of neural tumor cells subjected to serum deprivation. (Mol Cancer Res 2007;5(1):47–60)

Supplementary Figures S1-S6 from Mitochondrial c-Jun NH2-Terminal Kinase Prevents the Accumulation of Reactive Oxygen Species and Reduces Necrotic Damage in Neural Tumor Cells that Lack Trophic Support

Determining the number of synapses that are present in different brain regions is crucial to understand brain connectivity as a whole. Membrane-associated guanylate kinases (MAGUKs) are a family of scaffolding proteins that are expressed in excitatory glutamatergic synapses. We used genetic labeling of two of these proteins (PSD95 and SAP102), and Spinning Disc confocal Microscopy (SDM), to estimate the number of fluorescent puncta in the CA1 area of the hippocampus. We also used FIB-SEM, a three-dimensional electron microscopy technique, to calculate the actual numbers of synapses in the same area. We then estimated the ratio between the three-dimensional densities obtained with FIB-SEM (synapses/µm) and the bi-dimensional densities obtained with SDM (puncta/100 µm). Given that it is impractical to use FIB-SEM brain-wide, we used previously available SDM data from other brain regions and we applied this ratio as a conversion factor to estimate the minimum density of synapses in those regions. We found the highest densities of synapses in the isocortex, olfactory areas, hippocampal formation and cortical subplate. Low densities were found in the pallidum, hypothalamus, brainstem and cerebellum. Finally, the striatum and thalamus showed a wide range of synapse densities., This work was supported by grants from the following entities: the Spanish “Ministerio de Ciencia, Innovación y Universidades” (Grant PGC2018-094307-B-I00 and the Cajal Blue Brain Project [C080020-09; the Spanish partner of the Blue Brain Project initiative from EPFL, Switzerland]; the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 785907 (Human Brain Project, SGA2); the Wellcome Trust (Technology Development Grant 202932); and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (695568 SYNNOVATE). L.T.-R. is a recipient of grants from the EMBO Long-term fellowship 2016–2018 and the IBRO-PERC InEurope grants programme.

Mitochondria play a key role in energy production and calcium buffering, among many other functions. They provide most of the energy required by neurons, and they are transported along axons and dendrites to the regions of higher energy demands. We have used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the somatosensory cortex of the juvenile rat. We have estimated the volume fraction occupied by mitochondria and their distribution between dendritic, axonal, and nonsynaptic processes. The volume fraction of mitochondria increased from layer I (4.59%) to reach its maximum in layer IV (7.74%) and decreased to its minimum in layer VI (4.03%). On average, 44% of mitochondrial volume was located in dendrites, 15% in axons and 41% in nonsynaptic elements. Given that dendrites, axons, and nonsynaptic elements occupied 38%, 23%, and 39% of the neuropil, respectively, it can be concluded that dendrites are proportionally richer in mitochondria with respect to axons, supporting the notion that most energy consumption takes place at the postsynaptic side. We also found a positive correlation between the volume fraction of mitochondria located in neuronal processes and the density of synapses., Grants from the following entities: the Spanish Ministerio de Economía y Competitividad (Grants SAF 2015-603-P and the Cajal Blue Brain Project, Spanish partner of the Blue Brain Project initiative from EPFL); the European Union Horizon 2020 research and innovation program under Grant agreement No. 785907 (Human Brain Project, SGA2); and Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066, Spain). Correspondence to: amerchan@fi.upm.es.

Knowing the proportions of asymmetric (excitatory) and symmetric (inhibitory) synapses in the neuropil is critical for understanding the design of cortical circuits. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the six layers of the juvenile rat (postnatal day 14) somatosensory cortex (hindlimb representation). We segmented in three-dimensions 6184 synaptic junctions and determined whether they were established on dendritic spines or dendritic shafts. Of all these synapses, 87–94% were asymmetric and 6–13% were symmetric. Asymmetric synapses were preferentially located on dendritic spines in all layers (80–91%) while symmetric synapses were mainly located on dendritic shafts (62–86%). Furthermore, we found that less than 6% of the dendritic spines establish more than one synapse. The vast majority of axospinous synapses were established on the spine head. Synapses on the spine neck were scarce, although they were more common when the dendritic spine established multiple synapses. This study provides a new large quantitative dataset that may contribute not only to the knowledge of the ultrastructure of the cortex, but also towards defining the connectivity patterns through all cortical layers. Electronic supplementary material The online version of this article (doi:10.1007/s00429-017-1470-7) contains supplementary material, which is available to authorized users.

Cellular ultrastructures for signal integration are unknown in any nervous system. The ellipsoid body (EB) of the Drosophila brain is thought to control locomotion upon integration of various modalities of sensory signals with the animal internal status. However, the expected excitatory and inhibitory input convergence that virtually all brain centres exhibit is not yet described in the EB. Based on the EB expression domains of genetic constructs from the choline acetyl transferase (Cha), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) genes, we identified a new set of neurons with the characteristic ring-shaped morphology (R neurons) which are presumably cholinergic, in addition to the existing GABA-expressing neurons. The R1 morphological subtype is represented in the Cha- and TH-expressing classes. In addition, using transmission electron microscopy, we identified a novel type of synapse in the EB, which exhibits the precise array of two independent active zones over the same postsynaptic dendritic domain, that we named 'agora'. This array is compatible with a coincidence detector role, and represents ~8% of all EB synapses in Drosophila. Presumably excitatory R neurons contribute to coincident synapses. Functional silencing of EB neurons by driving genetically tetanus toxin expression either reduces walking speed or alters movement orientation depending on the targeted R neuron subset, thus revealing functional specialisations in the EB for locomotion control.

In the cerebral cortex, most synapses are found in the neuropil, but relatively little is known about their 3-dimensional organization. Using an automated dual-beam electron microscope that combines focused ion beam milling and scanning electron microscopy, we have been able to obtain 10 three-dimensional samples with an average volume of 180 µm(3) from the neuropil of layer III of the young rat somatosensory cortex (hindlimb representation). We have used specific software tools to fully reconstruct 1695 synaptic junctions present in these samples and to accurately quantify the number of synapses per unit volume. These tools also allowed us to determine synapse position and to analyze their spatial distribution using spatial statistical methods. Our results indicate that the distribution of synaptic junctions in the neuropil is nearly random, only constrained by the fact that synapses cannot overlap in space. A theoretical model based on random sequential absorption, which closely reproduces the actual distribution of synapses, is also presented.

In response to different stress signals, the c-Jun NH2-terminal kinase (JNK) can trigger cell death. However, JNK also facilitates the survival and cell cycle progression of tumor cells by mechanisms that are poorly defined. Here, we show that schwannoma RN22 cells can survive and proliferate under serum-free conditions although serum withdrawal rapidly induces mitochondrial fission and swelling. Although the morphologic changes observed in the mitochondria did not trigger cytochrome c release, they were accompanied by an increase in the mitochondrial membrane potential (ΔΨM) and of immunoreactivity for active JNK in these organelles. Pharmacologic inhibition of JNK provoked a further increase of the ΔΨM, an increase in reactive oxygen species (ROS) production, and a sustained decrease in cell viability due to necrosis. This increase in necrosis was prevented by the presence of ROS scavengers. Immunoreactivity for active JNK was also observed in the mitochondria of neuroblastoma 1E-115 and neuroblastoma 2a neuroblastoma cell lines on serum withdrawal, whereas active JNK was barely detected in serum-deprived fibroblasts. Accordingly, the reduction in neural tumor cell viability induced by JNK inhibition was largely attenuated in serum-deprived fibroblasts. These data indicate that local activation of JNK in the mitochondria can protect against necrotic cell death associated with ROS production, facilitating the growth of neural tumor cells subjected to serum deprivation. (Mol Cancer Res 2007;5(1):47–60)

Este texto analiza las actitudes ante la muerte en la literatura testimonial de la PrimeraGuerra Mundial. La finalidad de esto es mostrar de qué manera la muerte como paso a laeternidad, significó dar la vida en aras de una buena causa, por lo que se consideraba unavoluntad nacional, con la cual se alcanzaba el recuerdo indeleble de sus compatriotas y elbien morir.

Synapses are specialized communication points between neurons, and their number is a major determinant of cognitive abilities. These dynamic structures undergo developmental- and activity-dependent changes. During brain aging and certain diseases, synapses are gradually lost, causing mental decline. It is, thus, critical to identify the molecular mechanisms controlling synapse number. We show here that the levels of phosphoinositide 3 kinase (PI3K) regulate synapse number in both Drosophila larval motor neurons and adult brain projection neurons. The supernumerary synapses induced by PI3K overexpression are functional and elicit changes in behavior. Remarkably, PI3K activation induces synaptogenesis in aged adult neurons as well. We demonstrate that persistent PI3K activity is necessary for synapse maintenance. We also report that PI3K controls the expression and localization of synaptic markers in human neuroblastoma cells, suggesting that PI3K synaptogenic activity is conserved in humans. Thus,wepropose that PI3K stimulation can be applied to prevent or delay synapse loss in normal aging and in neurological disorders. Copyright © 2006 Society for Neuroscience.

We present a first-draft digital reconstruction of the microcircuitry of somatosensory cortex of juvenile rat. The reconstruction uses cellular and synaptic organizing principles to algorithmically reconstruct detailed anatomy and physiology from sparse experimental data. An objective anatomical method defines a neocortical volume of 0.29 ± 0.01 mm3 containing ∼31,000 neurons, and patch-clamp studies identify 55 layer-specific morphological and 207 morpho-electrical neuron subtypes. When digitally reconstructed neurons are positioned in the volume and synapse formation is restricted to biological bouton densities and numbers of synapses per connection, their overlapping arbors form ∼8 million connections with ∼37 million synapses. Simulations reproduce an array of in vitro and in vivo experiments without parameter tuning. Additionally, we find a spectrum of network states with a sharp transition from synchronous to asynchronous activity, modulated by physiological mechanisms. The spectrum of network states, dynamically reconfigured around this transition, supports diverse information processing strategies., The work was supported by funding from the EPFL to the Laboratory of Neural Microcircuitry (LNMC) and funding from the ETH Domain for the Blue Brain Project (BBP). Additional support was provided by funding for the Human Brain Project from the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement no. 604102 (HBP). Further funding came from The Gatsby Charitable Foundation ; the Cajal Blue Brain Project, Ministerio de Economia y Competitividad Spanish Ministry of Education and Science ; and an EPFL-Hebrew University Collaborative Grant . In the years 2005–2009, the Blue Gene/L system was funded by the EPFL . Financial support for the subsequent CADMOS Blue Gene/P and Blue Gene/Q systems was provided by the Canton of Geneva, Canton of Vaud, Hans Wilsdorf Foundation , Louis-Jeantet Foundation , University of Geneva , University of Lausanne , and École Polytechnique Fédérale de Lausanne . The BlueBrain IV BlueGene/Q system is financed by ETH Board Funding to the Blue Brain Project as a National Research Infrastructure and hosted at the Swiss National Supercomputing Center (CSCS). A large proportion of the data used in this study was generated at the Weizmann Institute for Science, Israel between 1996 and 2002 through the support of Thomas McKenna from the Office of Naval Research, USA.

Summary. The in vivo productive infection by the ovine Visna/maedi lentivirus (VISNA) is restricted to cells of the monocyte/macrophage lineage. The basis for this restriction is not understood. Although the VISNA envelope (Env) glycoprotein is the main target for virus neutralization, studies on the role of this protein in virus infection are limited. A vaccinia virus recombinant (VV-env-MV) containing the entire VISNA env sequence was generated and shown to produce in infected cells a protein of about 165 kDa (referred to as gp150). During VV-envMV infection, expression of env caused extensive cell-to-cell fusion in cell lines of different origins. Pulse-chase and Western blot analyses revealed that gp150 is not cleaved in VV-env-MV infected cells. The glycoprotein gp150 formed oligomers held by disulfide bonding. Cell-to-cell fusion was prevented in the presence of the inhibitor of glycosilation, tunicamycin, but it was markedly enhanced by an inhibitor of proteoglycan synthesis, β-D-xyloside. These findings showed that the receptor for VISNA Env is widely distributed within cells, that fusion-from-within of cells can occur in the apparent absence of proteolytic cleavage of gp150, and that fusion require a glycosylated Env but not the addition of proteoglycan chains at the cell surface. This recombinant virus could have utility as a potential vaccine against VISNA.

El uso de la imagen y el testimonio como fuentes de investigación ha planteado nuevos retos y ha traído a discusión diversos problemas metodológicos y de análisis en el campo de las ciencias sociales. Bajo estas premisas, busca aproximarse al discurso audio-visual de los movimientos guerrilleros en América Latina, reuniendo dos vertientes novedosas e importantes en el campo de la historia: la historia oral y la historia gráfica. Reinterpretar estos acontecimientos forma parte de lo que falta por conocer de la historia reciente de América Latina a través de nuevas perspectivas.

Recent evidence indicates that 17β-estradiol may have neuroprotective and neuroregenerative properties. Estradiol is formed locally in neural tissue from precursor androgens. The expression of aromatase, the enzyme that catalyses the conversion of androgens to estrogens, is restricted, under normal circumstances, to specific neuronal populations. These neurons are located in brain areas in which local estrogen formation may be involved in neuroendocrine control and in the modulation of reproductive or sex dimorphic behaviours. In this study the distribution of aromatase immunoreactivity has been assessed in the brain of mice and rats after a neurotoxic lesion induced by the systemic administration of kainic acid. This treatment resulted in the induction of aromatase expression by reactive glia in the hippocampus and in other brain areas that are affected by kainic acid. The reactive glia were identified as astrocytes by co-localization of aromatase with glial fibrillary acidic protein and by ultrastructural analysis. No immunoreactive astrocytes were detected in control animals. The same result, the de novo induction of aromatase expression in reactive astrocytes on the hippocampus, was observed after a penetrating brain injury. Furthermore, using a 3H2O assay, aromatase activity was found to increase significantly in the injured hippocampus. These findings indicate that although astrocytes do not normally express aromatase, the enzyme expression is induced in these glial cells by different forms of brain injury. The results suggest a role for local astroglial estrogen formation in brain repair.

In the light of functional studies, it has been suggested that antibodies directed to α subunits of G-proteins delivered into cerebrospinal fluid (CSF) reached and blocked the function of neural transducer proteins. Current understanding indicates that IgGs do not move freely across plasma membranes. Therefore, to characterize the uptake of these antibodies by neural cells, anti-Gi2α IgGs were labeled with 125 I , fluorescein or with gold particles. The expression of Gα subunits was also reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Following intracerebroventricular (icv) injection of gold-conjugated anti-Gi2α IgGs, electrondense particles entered and became distributed in the cytoplasm and plasma membranes of neural cells. Scattered particles were also found in dendrites and nuclei. Unlabeled IgGs diminished cerebral signals of fluorescein-labeled anti-Gα IgGs, indicating that this uptake can be saturated. Cerebral radiostaining promoted by in vivo anti-Gi2α 125 I -IgGs was almost absent in Gi2α knocked-down mice, but not after decreasing the quantity of Gzα subunits. The immunosignals of CSF anti-Gα 125 I -IgGs, as well as the impairment of opioid-evoked antinociception, were increased by agonist-induced activation of G protein-coupled receptors. The impairing effect of the antibodies on opioid-evoked antinociception was prevented by agents blocking the cellular uptake of proteins, i.e., cytochalasin B, BSA, DMSO, H7, and by down regulation of protein kinase Cβ1 (PKCβ1). In mice treated with an ODN to PKCβ1 mRNA, 125 I -IgGs to Gi2α subunits remained bound to periventricular structures and did not spread to deeper areas of the CNS. These results indicate that IgGs delivered into the CSF show a saturable binding to Gα subunits that translocate to the external side of the neural membrane before being internalized by a PKCβ1-dependent mechanism.

Extensive studies on protective immunity to rodent malaria provided the basis for the current experiments in which mice were immunized with recombinant (re) influenza and vaccinia viruses expressing selected sequences of the circumsporozoite (CS) protein of the human malaria parasitePlasmodiumfalciparum. Mice of different H-2 haplotypes immunized with re influenza viruses expressing the immunodominant B cell epitope of this CS protein produced high titers of antibodies to the parasite. A cytotoxic T lymphocyte epitope of the CS protein ofP. falciparum, PF3, recognized by CD8+T cells of H-2kmice, was expressed in a re vaccinia virus (VacPf) and a re influenza virus (FluPf). Immunization of mice with either FluPf or VacPf elicited a modest CS-specific CD8+T cell response detected by interferon γ secretion of individual immune cells. Priming of mice with FluPf, followed by a booster with VacPf, resulted in a striking enhancement of this T cell response. The reverse protocol, i.e., priming with VacPf followed by a booster with FluPf, failed to enhance the primary response. VacPf also greatly enhanced the primary response of mice injected withP. falciparumsporozoites or with a lipopeptide containing PF3. A booster with FluPf also amplified the response of lipopeptide- or sporozoite-primed mice but less than a VacPf booster did. Although mice are not susceptible to infection byP. falciparumsporozoites, we demonstrated that administration of two distinct immunogens expressing PF3 elicited activated, extravasating CS-specific T cells that protected against an intracerebral VacPf challenge.

The quantification and measurement of synapses is a major goal in the study of brain organization in both health and disease. Serial section electron microscopy (EM) is the ideal method since it permits the direct quantification of crucial features such as the number of synapses per unit volume or the distribution and size of synapses. However, a major limitation is that obtaining long series of ultrathin sections is extremely time-consuming and difficult. Consequently, quantitative EM studies are scarce and the most common method employed to estimate synaptic density in the human brain is indirect, by counting at the light microscopic level immunoreactive puncta using synaptic markers. The recent development of automatic EM methods in experimental animals, such as the combination of focused ion beam milling and scanning electron microscopy (FIB/SEM), are opening new avenues. Here we explored the utility of FIB/SEM to examine the cerebral cortex of Alzheimer's disease patients. We found that FIB/SEM is an excellent tool to study in detail the ultrastructure and alterations of the synaptic organization of the human brain. Using this technology, it is possible to reconstruct different types of plaques and the surrounding neuropil to find new aspects of the pathological process associated with the disease, namely; to count the exact number and types of synapses in different regions of the plaques, to study the spatial distribution of synapses, and to analyze the morphology and nature of the various types of dystrophic neurites and amyloid deposits. © 2013 - IOS Press and the authors. All rights reserved.

This study has analysed by immunocytochemistry the expression pattern of histone H1 zero after the osmotically induced activation of transcription in supraoptic nucleus neurons of the rat. In control rats, histone H1 zero was constitutively expressed in neuronal and glial cell nuclei of supraoptic nucleus. After chronic neuronal stimulation by intermittent salt-loading, the majority of neuronal cell nuclei exhibited a marked reduction of immunostaining, which was confirmed by densitometric analysis of immunoreactivity. This effect was reversible, since optical density values returned to control levels when the stimulation of supraoptic neurons was suppressed by rehydration. Ultrastructural immunocytochemistry of histone H1 zero showed that immunogold particles specifically decorated chromatin fibers, with the highest accumulation of particles being on the condensed inactive chromatin. These results indicate that transcriptional activation in supraoptic neurons is accompanied by a depletion of the chromatin-associated histone H1 zero, and also suggest that this transcription-dependent expression of histone H1 zero may be involved in regulating chromatin condensation and gene expression in mature neurons that constitutively express this protein.

Neurotransmitters used by chemical synapses are stored in synaptic vesicles that accumulate in axon terminals. The number and position of these vesicles have been related to some important functional properties of the synapse. For this reason, an accurate mechanism for semi-automatically counting these small cellular structures will be of great help for neuroscientists. In this paper, we present a Differential Evolution algorithm that quantifies the number of synaptic vesicles in electron micrographs. The algorithm has been tested on several images that have been obtained from the somatosensory cortex of the rat and compared with some traditional approaches for detecting circular structures. Finally, the results have been validated by two independent expert anatomists.

The neurons in the cortical white matter (WM neurons) originate from the first set of postmitotic neurons that migrates from the ventricular zone. In particular, they arise in the subplate that contains the earliest cells generated in the telencephalon, prior to the appearance of neurons in gray matter cortical layers. These cortical WM neurons are very numerous during development, when they are thought to participate in transient synaptic networks, although many of these cells later die, and relatively few cells survive as WM neurons in the adult. We used light and electron microscopy to analyze the distribution and density of WM neurons in various areas of the adult human cerebral cortex. Furthermore, we examined the perisomatic innervation of these neurons and estimated the density of synapses in the white matter. Finally, we examined the distribution and neurochemical nature of interneurons that putatively innervate the somata of WM neurons. From the data obtained, we can draw three main conclusions: first, the density of WM neurons varies depending on the cortical areas; second, calretinin-immunoreactive neurons represent the major subpopulation of GABAergic WM neurons; and, third, the somata of WM neurons are surrounded by both glutamatergic and GABAergic axon terminals, although only symmetric axosomatic synapses were found. By contrast, both symmetric and asymmetric axodendritic synapses were observed in the neuropil. We discuss the possible functional implications of these findings in terms of cortical circuits. © 2010 Wiley-Liss, Inc.

One of the main pathological hallmarks of Alzheimer’s disease (AD) is the accumulation of plaques in the cerebral cortex, which may appear either in the neuropil or in direct association with neuronal somata. Since different axonal systems innervate the dendritic (mostly glutamatergic) and perisomatic (mostly GABAergic) regions of neurons, the accumulation of plaques in the neuropil or associated with the soma might produce different alterations to synaptic circuits. We have used a variety of conventional light, confocal and electron microscopy techniques to study their relationship with neuronal somata in the cerebral cortex from AD patients and APP/PS1 transgenic mice. The main finding was that the membrane surfaces of neurons (mainly pyramidal cells) in contact with plaques lack GABAergic perisomatic synapses. Since these perisomatic synapses are thought to exert a strong influence on the output of pyramidal cells, their loss may lead to the hyperactivity of the neurons in contact with plaques. These results suggest that plaques modify circuits in a more selective manner than previously thought.

The advent of transmission electron microscopy (TEM) in the 1950’s represented a fundamental step in the study of neuronal circuits. The application of this technique soon led to the realization that the number of synapses changes during the course of normal life, as well as under certain pathological or experimental circumstances. Since then, one of the main goals in neurosciences has been to define simple and accurate methods to estimate the magnitude of these changes. Contrary to analysing single sections, TEM reconstructions are extremely time-consuming and difficult. Therefore, most quantitative studies use stereological methods to define the three-dimensional characteristics of synaptic junctions that are studied in two dimensions. Here, to count the exact number of synapses per unit of volume we have applied a new three-dimensional reconstruction method that involves the combination of focused ion beam milling and scanning electron microscopy (FIB/SEM). We show that the images obtained with FIB/SEM are similar to those obtained with TEM, but with the advantage that FIB/SEM permits serial reconstructions of large volumes of tissue to be generated rapidly and automatically. Furthermore, we compared the estimates of the number of synapses obtained with stereological methods with the values obtained by FIB/SEM reconstructions. We concluded that FIB/SEM not only provides the actual number of synapses per volume but it is also much easier and faster to use than other currently available TEM methods. More importantly, it also avoids most of the errors introduced by stereological methods and overcomes the difficulties associated with these techniques.

Although pyramidal cells are the main excitatory neurons in the cerebral cortex, it has recently been reported that they can evoke inhibitory postsynaptic currents in neighboring pyramidal neurons. These inhibitory effects were proposed to be mediated by putative axo-axonic excitatory synapses between the axon terminals of pyramidal cells and perisomatic inhibitory axon terminals [Ren M, Yoshimura Y, Takada N, Horibe S, Komatsu Y (2007) Science 316:758-761]. However, the existence of this type of axo-axonic synapse was not found using serial section electron microscopy. Instead, we observed that inhibitory axon terminals synapsing on pyramidal cell bodies were frequently apposed by terminals that established excitatory synapses with neighbouring dendrites. We propose that a spillover of glutamate from these excitatory synapses can activate the adjacent inhibitory axo-somatic terminals.

Certain cognitive functions differ in men and women, although the anatomical and functional substrates underlying these differences remain unknown. Because neocortical activity is directly related with higher brain function, numerous studies have focused on the cerebral cortex when searching for possible structural correlates of cognitive gender differences. However, there are no studies on possible gender differences at the synaptic level. In the present work we have used stereological and correlative light and electron microscopy to show that men have a significantly higher synaptic density than women in all cortical layers of the temporal neocortex. These differences may represent a microanatomical substrate contributing to the functional gender differences in brain activity. © 2008 by The National Academy of Sciences of the USA.

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.

Background/Aim: The ultrasonic transit time is currently the best method for measuring the blood flow rate in the extracorporeal hemodialysis circuit. The purpose of this study was to analyze the differences between blood flow as indicated by the hemodialysis blood roller pump (prescribed blood flow) and by an ultrasonic flowmeter (delivered blood flow). Methods: The ultrasonic blood flow was measured in 20 patients on chronic hemodialysis who were dialyzed through an arteriovenous fistula. During each dialysis session the ultrasonic blood flow was measured at three different blood roller pump flow rates (300, 350, and 400 ml/min). In order to analyze the influence of inflow and outflow pressures on blood flow, this study was conducted during nine consecutive dialysis sessions during which needles of different sizes were used. Results: The ultrasonic flow was always lower than indicated by the blood roller pump: 265 ± 12, 304 ± 15, and 341 ± 19 ml/min for blood roller pump flow rates of 300, 350, and 400 ml/min, respectively (variability: –11.6, –13.1, and –14.8%, respectively). An univariate regression analysis showed that the reduction in flow recorded ultrasonically correlated with both venous blood line pressure (r = –0.2679, p < 0.001) and negative arterial blood line pressure (r = 0.6773, p < 0.001). By multivariate analysis, only the arterial blood line pressure has a predictive value. When all measurements were grouped by arterial blood line pressure ranges, the variability between ultrasonic blood flow and blood roller pump flow was found to be similar in those groups with the same arterial blood line pressure, regardless of the blood roller pump flow rate. Conclusions: The blood flow indicated by the dialysis blood roller pump is always greater than the delivered blood flow, and this difference is in turn conditioned by the negative pressure induced by the blood roller pump in the arterial blood line.

A study on long-term astrocytic responses (from 1 day to 20 months after lesioning in 4-month-old rats, and from 1 day to 6 months in 20-month-old rats) to diverse unilateral damage of the nucleus basalis (nbM) by injection of 40 nmol of ibotenic acid, or 50 or 100 nmols of quisqualic acid was performed using a histochemical method (immunoreactivity against the glial fibrillary acidic protein GFAP). Glial reactivity (i.e., isolated or clustered hypertrophic and/or hyper-reactive astrocytes) was evaluated in several ipsilateral and contralateral brain regions: the 'local response' within the damaged nbM region; the 'proximal response' (a new concept proposed by us) in the non-damaged structures neighbouring the nbM; and the 'remote response' in the ipsilateral brain cortex and in the contralateral cortex and nbM. In 4-month-old animals, the remote cortical glial responses, independent of the involution of cortical cholinergic activity and randomly located in layers I-V of motor and somatosensory cortical regions, were similar in appearance over a long period (13-20 months), with the highest reactivity 45 days after lesioning. The proximal response lasted from 1 day to 13 months and afterwards tended to disappear. Contralateral reactivity and ipsilateral cortical scars were observed. The local (nbM) glial response was maintained throughout the period studied. Subsets of astrocytes of different reactivities were observed, most of their elements being highly intermeshed. In 20-month-old animals, nbM lesions produced less positive, but similar, glial reactive patterns. This glial reactivity was superposed onto the glial reactivity of old age. All these results are discussed. The maintenance of reactive astrocytes many months after lesioning suggests the existence of cellular factors other than those produced by damaged nbM neurons. Taking into account the role of glial cells under pathological conditions, it is possible that these reactive astrocytes in humans could promote neurodegenerative processes, such as amyloid plaque formation and neurodegeneration (Alzheimer's disease). Along this line, nbM cholinergic involution could then originate cortical involution through induced reactive astrocytosis. Themes: Disorders of the nervous system. Topics: Degenerative diseases: Alzheimer's. Copyright (C) 2000 Elsevier Science B.V.

In mice whose Gi/o-protein function had been impaired by antisense 'knock-down' or pertussis toxin treatment, i.c.v. injection of myr+-Gi/o alpha subunits restored the effectiveness of beta-endorphin, morphine, DPDPE, clonidine and neurotensin to produce antinociception. Myr+-G alpha subunits of the class of G-proteins actually impaired were more effective than unlike but related myr+-G alpha subunits. Selectivity was noted in that only exogenous myr+-G alpha subunits affected (enhanced) the activity of agonists in G alpha-deficient signalling systems. This treatment had little effect on agonist potency when the impairment resided at the receptor level. The potential of the opioids, clonidine and R-PIA to increase G alpha-related in vitro hydrolysis of GTP was also re-established after injecting myr+-Gi2 alpha subunits into Gi2-knocked-down mice. Myr+-Gi2 alpha subunits pre-incubated with GTPgammaS or GDPbetaS before i.c.v. injection did not improve the activity of agonists in vivo (antinociception) or in vitro (regulation of low Km GTPase). After impairing the function of PKCbeta1 by antisense treatment or with the inhibitor H7, the effect of myr+-G alpha subunits on agonist potency was prevented. Electron microscope analysis showed the entry of gold-conjugated myr+-G alpha subunits into neural cells. These particles were found in the cytoplasm, associated with the plasma membranes of different neuronal processes and also in synaptic junctions. In cultured neurons and astrocytes myr+-Gi2 alpha-associated fluorescence was internalised in a dose-dependent manner and distributed in the plasma membrane and cytosol, as well as in nuclei of dividing astrocytes. Thus, G alpha subunits in CSF enter into neurons and functionally couple to the receptor-triggered signalling cascade. As G-proteins have been implicated in the pathophysiology of several neural disorders, this finding may be valuable in the therapy of such dysfunctions.

Synapses are key elements in the organization of nervous circuits. Obtaining serial images from large samples of nervous tissue in an automated manner is now possible through combined focused ion beam milling and scanning electron microscopy. However, the identification, 3D reconstruction and quantification of synapses within these samples are labor intensive procedures that require continuous user intervention. We have developed a software tool to automatically perform the segmentation of synapses in a reconstructed 3D volume of the cerebral cortex, thereby greatly facilitating and accelerating these processes. The tool is interactive, allowing the user to supervise the segmentation process, to modify the appropriate parameters and to validate the results. It is also modular to enable new functions to be implemented as needed. We have also ensured it has a user friendly interface and that it is portable, making it accessible to a wide range of potential users.

Changes in the size of the synaptic junction are thought to have significant functional consequences. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the six layers of the rat somatosensory cortex. We have segmented in 3D a large number of synapses (n= 6891) to analyze the size and shape of excitatory (asymmetric) and inhibitory (symmetric) synapses, using dedicated software. This study provided three main findings. Firstly, the mean synaptic sizes were smaller for asymmetric than for symmetric synapses in all cortical layers. In all cases, synaptic junction sizes followed a log-normal distribution. Secondly, most cortical synapses had disc-shaped postsynaptic densities (PSDs; 93%). A few were perforated (4.5%), while a smaller proportion (2.5%) showed a tortuous horseshoe-shaped perimeter. Thirdly, the curvature was larger for symmetric than for asymmetric synapses in all layers. However, there was no correlation between synaptic area and curvature.