MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Neuroscience' source. Tue, 07 Feb 2012 07:05:47 +0100 http://www.medworm.com/index.php?rid=5484695&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_25 Schwann cells are one of the cellular candidates used in repair strategies following trauma and demyelination of the spinal cord. One of the major obstacles in the use of Schwann cells is their limited migratory ability within the astrocytic environment of the CNS and boundary formation between the Schwann cells of the graft and the host astrocytes. This boundary creates an abrupt obstacle for regenerating axons attempting to exit the Schwann cell graft back to the CNS. To facilitate the study of mechanisms underlying these interactions, in vitro coculture assays of Schwann–Astrocytes have been developed. In this chapter, we have described the methodology for two commonly used coculture systems known as the Schwann–Astrocyte boundary assay and the inverted coverslip migration a... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484695 Fri, 09 Dec 2011 01:09:10 +0100 5484695 http://www.medworm.com/index.php?rid=5484694&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_24 Microglia are the principal immune effector cells of the central nervous system (CNS). Under normal conditions, they occupy a quiescent surveillance phenotype, but following stimulation by microorganisms or inflammatory cytokines, microglia transform into highly activated migratory, phagocytic cells producing inflammatory cytokines and chemokines. Significantly, several studies have demonstrated that astrocytes attenuate microglial activation, reducing microglial adhesion, production of interleukin-12 (IL-12) and reactive oxygen species (ROS), and expression of inducible nitric oxide synthase (iNOS). In this chapter, we describe an astrocyte-microglia coculture system that can be used to investigate interactions between these two cell types. We also describe a flow cytometry approach to qu... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484694 Fri, 09 Dec 2011 01:09:10 +0100 5484694 http://www.medworm.com/index.php?rid=5484693&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_23 Astrocytes perform critical functions necessary for neuronal survival. Thus, examining the influence of astrocyte function on neuronal cell death during disease, including hypoxia/ischemia, has become an important avenue of investigation. In this chapter we detail the methodology and potential pitfalls for establishing cocultures of astrocytes and cortical neurons for studying hypoxia-induced neuronal death. In brief, astrocyte cultures are first established until they reach confluence. The medium is exchanged from a medium that supports astrocyte growth to a medium that supports neuronal viability 24 h before adding neurons to the astrocyte monolayer. After the neurons mature, the cultures are exposed to severe hypoxia and neuronal death is quantified 1–2 days later. (Source: Spring... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484693 Fri, 09 Dec 2011 01:09:10 +0100 5484693 http://www.medworm.com/index.php?rid=5484692&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_22 Astrocytes secrete factors that promote neuron survival, synapse formation, and plasticity. Understanding how these factors perform these roles requires a robust in vitro system that can effectively assess the impact of individual glial factors on neuronal properties. A classical approach to studying neuron-glial interactions in vitro uses a system where dissociated embryonic rat neurons are suspended over a feeder layer of rat astrocytes. Here, we describe a useful “sandwich” co-culture system where postnatal mouse hippocampal neurons are grown suspended above a feeder layer of mouse hippocampal astrocytes. We demonstrate that neurons in these cultures remain healthy beyond 3 weeks in vitro and develop more synapses compared to neurons grown without astrocytes. An advantage of... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484692 Fri, 09 Dec 2011 01:09:10 +0100 5484692 http://www.medworm.com/index.php?rid=5484691&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_21 Astrogliosis, whereby astrocytes in the central nervous system (CNS) become reactive in response to tissue damage, is a prominent process leading to the formation of the glial scar that inhibits axon regeneration after CNS injury. Upon becoming reactive, astrocytes undergo various molecular and morphological changes including upregulating their expression of GFAP and chondroitin sulfate proteoglycans (CSPGs) as well as other molecules that are inhibitory to axon growth. We have developed an in vitro model of reactive astrogliosis as a result of treating cultured astrocytes with transforming growth factor-ß (TGF-ß), which induces increased expression as well as secretion of CSPGs. These reactive astrocytes show inhibitory effects on neuron growth both in neuron-astrocyte cocultu... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484691 Fri, 09 Dec 2011 01:09:10 +0100 5484691 http://www.medworm.com/index.php?rid=5484690&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_20 Protocols are described for measurement in primary cultures of astrocytes of unidirectional fluxes of glutamate (influx and efflux), glutamate metabolism to glutamine or CO2, glucose influx, glycolysis, pyruvate dehydrogenation, oxidative metabolism of glucose, pyruvate carboxylation, glycogen synthesis, and glycogenolysis. References are made to the in vivo situation, and the importance of using metabolically competent cultures is emphasized. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484690 Fri, 09 Dec 2011 01:09:10 +0100 5484690 http://www.medworm.com/index.php?rid=5484689&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_19 A typical feature of astrocytes is their high level of connexin expression. These membrane proteins constitute the molecular basis of two types of channels: gap junction channels that allow direct cytoplasm-to-cytoplasm communication and hemichannels that provide a pathway for exchanges between the intra- and extracellular media. An unusual property of these channels is their permeability for ions but also for small signaling molecules. They support intercellular communication that contribute to dynamic neuroglial interaction and interplay with neuronal activity and survival. Here, we describe multiple techniques based either on electrophysiological approaches or the monitoring of dye intercellular diffusion and uptake that permits an investigation of the properties of gap junction channel... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484689 Fri, 09 Dec 2011 01:09:10 +0100 5484689 http://www.medworm.com/index.php?rid=5484688&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_18 Electrophysiologically, astrocytes are characterized by a high K+ resting conductance and a hyperpolarized resting membrane potential. Both features are due to the activity of astrocytic potassium channels. Astrocytes express a variety of voltage-dependent and leak potassium channels on the plasma membrane that contribute to the hyperpolarized resting membrane potential and other cellular processes. This chapter focuses on measuring K+ channel function in astrocytes, focusing on Kir4.1, an inwardly rectifying potassium channel. We and others have demonstrated that Kir4.1 contributes significantly to the high-resting K+ conductance and the hyperpolarized resting membrane potential. This channel is also implicated in channel-mediated regulation of extracellular potassium. (Source: Springer p... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484688 Fri, 09 Dec 2011 01:09:10 +0100 5484688 http://www.medworm.com/index.php?rid=5484687&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_17 Astrocytes produce numerous mediators under conditions of inflammation in the central nervous system. One such mediator is nitric oxide (NO) derived from nitric oxide synthase-2 (NOS-2), the high output, inducible NOS isoform. Expression of NOS-2 and production of NO can be stimulated in astrocyte cultures by combinations of cytokines and lipopolysaccharide, a gram-negative bacterial endotoxin. This chapter details methods to induce and analyze NOS-2 expression and NO production in astrocyte cultures. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484687 Fri, 09 Dec 2011 01:09:10 +0100 5484687 http://www.medworm.com/index.php?rid=5484686&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_16 Astrocytes are intimately involved in immunological and inflammatory events occurring in the central nervous system (CNS), due to their ability to secrete and respond to a large number of immunoregulatory cytokines/chemokines such as IL-1ß, IL-6, IL-8, IL-10, IL-17, IL-27, TNF-a, TGF-ß, IFN-?, IFN-ß, CCL2, CCL3, CCL5, CXCL10, and CXCL12. Although expression of cytokines and chemokines is limited in the normal CNS, elevated expression of these proteins, as seen in disease entities such as multiple sclerosis (MS), HIV-1 associated neurocognitive disorders (HAND), Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS), contributes to the development of inflammation and neuronal demise in these diseases. As a potent source of cy... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484686 Fri, 09 Dec 2011 01:09:10 +0100 5484686 http://www.medworm.com/index.php?rid=5484685&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_15 We describe the methodologies for the sensitive quantitative development of the inactive and active forms of both MMP-2 and MMP-9 from tissues and cells, by means of lysis of the collagen substrate in collagen-impregnated gel electropheresis by the zymogen and active gelatinases. These methodologies are a refinement of those used commonly, with instructions to increase sensitivity. Serious and often overlooked issues regarding sources of sample contamination and elements confounding the MMP band development and their interpretation are discussed. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484685 Fri, 09 Dec 2011 01:09:10 +0100 5484685 http://www.medworm.com/index.php?rid=5484684&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_14 Protocols are presented describing a unique in vitro injury model and how to culture and mature mouse, rat, and human astrocytes for its use. This injury model produces widespread injury and astrocyte reactivity that enable quantitative measurements of morphological, biochemical, and functional changes in rodent and human reactive astrocytes. To investigate structural and molecular mechanisms of reactivity in vitro, cultured astrocytes need to be purified and then in vitro “matured” to reach a highly differentiated state. This is achieved by culturing astrocytes on deformable collagen-coated membranes in the presence of adult-derived horse serum (HS), followed by its stepwise withdrawal. These in vitro matured, process-bearing, quiescent astrocytes are then subjected to mechani... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484684 Fri, 09 Dec 2011 01:09:10 +0100 5484684 http://www.medworm.com/index.php?rid=5484683&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_13 Astrocytes are highly polarized cells. This is manifest not only during development and in the adult brain, but also following injury. In response to a wound, astrocytes extend processes that participate in formation of a glial scar, which walls off lesions in the brain or spinal cord. Similarly, astrocytes in culture polarize dramatically and extend processes towards a scrape wound. This simple assay has allowed much progress in understanding the cellular events and molecular pathways in astrocyte polarization (1). Cell adhesion is essential for the early response to the wound, both with respect to process extension and cell polarization. This is evident in the involvement of members of the integrin family of cell adhesion molecules at the leading edge of the wounded astrocyte. Understand... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484683 Fri, 09 Dec 2011 01:09:10 +0100 5484683 http://www.medworm.com/index.php?rid=5484682&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_9 We report how tissue origin, developmental stages, and culture medium conditions influence cell differentiation and the prevalence of glial cells vs. neurons. We compare explants from early, middle, and late stages of development and two different fetal calf serum concentrations (1 and 10%) to identify the best conditions to obtain and grow viable astrocytes in culture. In addition, we describe how to cryopreserve and obtain viable cortical astrocytes from frozen fetal bovine brain samples. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484682 Fri, 09 Dec 2011 01:09:10 +0100 5484682 http://www.medworm.com/index.php?rid=5484681&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_8 Detailed methods for the preparation of optic nerve head (ONH) astrocytes from human donor eyes and retinal astrocytes from rat eyes are described. Included is the immunopanning method used for ONH astrocyte isolation as well as cell characterization. The isolation of purified retinal astrocytes is outlined as a method applicable to rodent and other mammalian retinas. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484681 Fri, 09 Dec 2011 01:09:10 +0100 5484681 http://www.medworm.com/index.php?rid=5484680&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_7 Astrocytes are possibly the most numerous cells of the vertebrate central nervous system, yet a detailed characterization of their functions is still missing. One potential reason for the obscurity of astrocytic function is that they represent a diverse population of cells that all share some critical characteristics. In the CNS, astrocytes have been proposed to perform many functions. For example, they are supportive cells that provide guidance to newly formed migrating neurons and axons. They regulate the functions of endothelial cells at the blood brain barrier, provide nutrients, and maintain homeostasis including ionic balance within the CNS. More recently, dissecting the central role of astrocytes in mediating injury responses in the CNS, particularly the spinal cord, has become an a... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484680 Fri, 09 Dec 2011 01:09:10 +0100 5484680 http://www.medworm.com/index.php?rid=5484679&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_6 Astrocytes play fundamental roles in the establishment and maintenance of tissue homeostasis in the central nervous system (CNS). To examine these different functions in vitro, it is important to be able to generate pure astrocyte cultures. While many “enriched” astrocyte cultures have been described, these are complicated by the presence of other contaminating cell types, such as microglia. In this chapter, we describe a method in which microglia-free astrocyte cultures are generated from neurospheres and also include an immunocytochemical approach to demonstrate the purity of these cultures. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484679 Fri, 09 Dec 2011 01:09:10 +0100 5484679 http://www.medworm.com/index.php?rid=5484678&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_5 Microglia, resident phagocytic cells of the central nervous system, are frequent contaminants of astrocyte cultures. Unfortunately and not always fully appreciated, contamination by microglia can confound results of studies designed to elucidate the molecular mechanisms underlying astrocyte-specific responses. The paradigm described herein employs the mitotic inhibitor, cytosine ß-d-arabinofuranoside, followed by the lysosomotropic agent, leucine methylester, to maximally deplete microglia, thereby generating highly enriched astrocyte monolayers that remain viable and functional. Successful removal of microglia from confluent monolayers of primary astrocyte cultures is achieved without the need for cell passage and successful reduction is confirmed by depletion of microglial-specific... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484678 Fri, 09 Dec 2011 01:09:10 +0100 5484678 http://www.medworm.com/index.php?rid=5484677&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_4 We describe a method to prepare postnatal rat brain primary cell cultures composed of astrocytes, oligodendrocytes, and microglia. After 1 week in vitro, the mixed glial cell cultures are free of neurons, meningeal cells and fibroblasts. We developed a simple procedure to selectively harvest enriched populations of each of the three major glial cell types. Because these cells are at a progenitor/immature stage, each can be further cultured separately in serum or serum-free media to yield large quantities of the desired glial cell subpopulations with a high degree of purity in the range of 96–99%. These cell culture models have been used extensively for performing biochemical, molecular, and pharmacological studies using standard assays and obtain sound quantitative data. These studie... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484677 Fri, 09 Dec 2011 01:09:10 +0100 5484677 http://www.medworm.com/index.php?rid=5484676&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_12 We describe the combined genetic and fluorescence microscopy approaches for identification of C. elegans glial cells in culture and assessment of their cytosolic Ca2+ dynamics. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484676 Fri, 09 Dec 2011 01:09:10 +0100 5484676 http://www.medworm.com/index.php?rid=5484675&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_11 Although rodent models have been essential to unveil the emerging functions of astrocytes, the existence of interspecies differences calls for caution in extrapolating data from rodent to human astrocytes. We have developed highly enriched primary astrocyte cultures from human fetuses and adult cerebro-cortical biopsies from neurosurgery patients. Immunocytochemical characterization shows that cultures are composed of more than 95% of cells expressing in vitro astrocytic markers. Examination of the morphological and proliferative properties of cultures derived from the cerebral cortex and the hypothalamus both in untreated conditions and after treatment with EGF-related ligands illustrates the high plasticity of human astrocytes and their functional heterogeneity according to the cerebral ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484675 Fri, 09 Dec 2011 01:09:10 +0100 5484675 http://www.medworm.com/index.php?rid=5484674&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_10 A procedure for the isolation and cultivation of astrocytes from swine is described. More specifically, the donor animals are adolescent minipigs about 3 months in age and 10 kg in weight. About 20 g of cerebral tissue can be isolated from the piglet, yielding enough astrocytes of homogeneous genetics for experimentation after only one passage in culture. The astrocyte isolation procedure includes mechanical and enzymatic digestion of the brain tissue followed by separation of the brain fragments, based on size and density. Astrocytes are further purified from any residual nonastrocytes by differential attachment during the first passage. The resulting culture is purely astrocytes (>98%) based upon their appearance in phase-contrast microscopy and their uniform expression of glial fibri... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484674 Fri, 09 Dec 2011 01:09:10 +0100 5484674 http://www.medworm.com/index.php?rid=5484673&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_3 Astrocytes participate in all essential CNS functions, including blood flow regulation, energy metabolism, ion and water homeostasis, immune defence, neurotransmission, and adult neurogenesis. It is thus not surprising that astrocytic morphology and function differ between regions, and that different subclasses of astrocytes exist within the same brain region. Recent lines of work also show that the complexity of protoplasmic astrocytes increases during evolution. Human astrocytes are structurally more complex, larger, and propagate calcium signals significantly faster than rodent astrocytes. In this chapter, we review the diversity of astrocytic form and function, while considering the markedly expanded roles of astrocytes with phylogenetic evolution. We also define major challenges for t... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484673 Fri, 09 Dec 2011 01:09:10 +0100 5484673 http://www.medworm.com/index.php?rid=5484672&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_2 Cells with certain attributes of very immature astroglial cells and their radial precursors can act as stem and/or progenitor cells during developmental and persistent neurogenesis. Neural stem/progenitor cells both express and are affected by a variety of developmentally regulated macromolecules and growth factors, and such signaling or recognition molecules are being uncovered through extensive genomic and proteomic studies, as well as tested using in vitro/in vivo cell growth bioassays. Glycosylated molecules are appreciated as distinct signaling molecules during morphogenesis in a variety of tissues and organs, with glycoconjugates (glycoproteins, glycolipids, and glycosaminoglycans) serving as mediators for the interactions of cells with each other and their substrates, to confer grow... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484672 Fri, 09 Dec 2011 01:09:09 +0100 5484672 http://www.medworm.com/index.php?rid=5484671&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-452-0_1 Astrocytes contribute to virtually every aspect of brain function, including ionic homeostasis, energy metabolism, and synaptic signaling. The varied and important roles of astrocytes have evolved to allow increasingly complex nervous systems to operate efficiently and with high fidelity. For example, astrocytes figure prominently in glutamatergic synaptic transmission, an elemental event of brain function: high-affinity glutamate uptake into astrocytes improves the temporal and spatial fidelity of glutamatergic signaling and astrocytes subsequently shuttle glutamine back to neurons for the synthesis of more glutamate. The important and dynamic contributions of astrocytes to normal brain function demand that the interactions between neurons and astrocytes be viewed as a “partnership,... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5484671 Fri, 09 Dec 2011 01:09:09 +0100 5484671 http://www.medworm.com/index.php?rid=5466678&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_25 Calcium is a ubiquitous intracellular messenger that has important functions in normal neuronal function. The pathology of Alzheimer’s disease has been shown to alter calcium homeostasis in neurons and astrocytes. Several calcium dye indicators are available to measure intracellular calcium within cells, including Oregon Green BAPTA-1 (OGB-1). Using fluorescence lifetime imaging microscopy, we adapted this single wavelength calcium dye into a ratiometric dye to allow quantitative imaging of cellular calcium. We used this approach for in vitro calibrations, single-cell microscopy, high-throughput imaging in automated plate readers, and in single cells in the intact living brain. While OGB is a commonly used fluorescent dye for imaging calcium qualitatively, there are distinct advantag... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466678 Fri, 11 Nov 2011 05:00:00 +0100 5466678 http://www.medworm.com/index.php?rid=5466677&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_24 Optical imaging is a valuable tool for investigating alterations in membrane turnover and vesicle trafficking. Established techniques can easily be adapted to study the mechanisms of synaptic dysfunction in models of neuropsychiatric disorders and neurodegenerative diseases, such as drug addiction, Parkinsonism, and Huntington’s disease. Fluorescent endocytic tracers, including FM1-43, have been used to optically monitor synaptic vesicle fusion and measure synaptic function in various preparations, including chromaffin cells, dissociated cell cultures, and brain slices. In this chapter, we describe a technique that provides a direct measure of pathway-specific exocytosis from glutamatergic corticostriatal terminals. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466677 Fri, 11 Nov 2011 05:00:00 +0100 5466677 http://www.medworm.com/index.php?rid=5466676&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_23 The voltage-gated sodium channel (Nav1) plays an important role in initiating and propagating action potentials in neuronal cells. We and others have recently found that the Alzheimer’s disease-related secretases BACE1 and presenilin (PS)/?-secretase regulate Nav1 function by cleaving auxiliary subunits of the channel complex. We have also shown that elevated BACE1 activity significantly decreases sodium current densities in neuroblastoma cells and acutely dissociated adult hippocampal neurons. For detailed molecular studies of sodium channel regulation, biochemical methods are now complementing classical electrophysiology. To understand how BACE1 regulates sodium current densities in our studies, we setup conditions to analyze surface levels of the pore-forming Nav1 a-subunits. By u... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466676 Fri, 11 Nov 2011 05:00:00 +0100 5466676 http://www.medworm.com/index.php?rid=5466675&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_22 Alzheimer’s disease, the most common neurodegenerative disease, is characterized by a progressive loss of synapses and accumulation of amyloid-beta (Aß) peptides in the brain. Previous studies demonstrated that acute increase in synaptic activity in cultured hippocampal slices and mouse brains (Cirrito et al. Neuron 48: 913–922, 2005; Kamenetz et al. Neuron 37: 925–937, 2003) enhanced secretion of Aß. Since synaptic activity promotes Aß secretion, it could also affect the trafficking and processing of its precursor, the amyloid precursor protein (APP). Here, we describe a method to investigate the effect of acute synaptic activation on APP trafficking within dendrites. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466675 Fri, 11 Nov 2011 05:00:00 +0100 5466675 http://www.medworm.com/index.php?rid=5466674&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_21 Proper control of mitochondrial turnover is critical for maintenance of cellular energetics under basal and stressed conditions, and for prevention of endogenous oxidative stress. Whole organelle turnover is mediated through macroautophagy, a process by which autophagosomes deliver mitochondria to the lysosome for hydrolytic degradation. While mitochondrial autophagy can occur as part of a nonselective upregulation of autophagy, selective degradation of damaged or unneeded mitochondria (mitophagy) is a rapidly growing area in development, cancer, and neurodegeneration, particularly with regard to Parkinson’s disease. Due to its dynamic nature, and the potential for regulatory perturbation by disease processes, no single technique is sufficient to evaluate mitophagy. Here, we describe... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466674 Fri, 11 Nov 2011 05:00:00 +0100 5466674 http://www.medworm.com/index.php?rid=5466673&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_20 Recent discoveries have brought mitochondria functions in focus of the neuroscience research community and greatly stimulated the demand for approaches to study mitochondria dysfunction in neurodegenerative diseases. Many mouse disease models have been generated, but studying mitochondria isolated from individual mouse brain regions is a challenge because of small amount of the available brain tissue. Conventional techniques for isolation and purification of mitochondria from mouse brain subregions, such as ventral midbrain, hippocampus, or striatum, require pooling brain tissue from six to nine animals for a single mitochondrial preparation. Working with pooled tissue significantly decreases the quality of data because of the time required to dissect several brains. It also greatly increa... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466673 Fri, 11 Nov 2011 05:00:00 +0100 5466673 http://www.medworm.com/index.php?rid=5466672&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_19 Changes in intracellular calcium concentration play a major role both in signal transduction and in cell death. In particular, mitochondrial Ca2+ overload is critically important as a determinant of irreversible cell injury. When accumulated above a threshold, matrix Ca2+ triggers opening of the mitochondrial permeability transition pore (mPTP), initiating ATP depletion and cell death via necrosis or by promoting cytochrome c release and initiating the apoptotic cascade. Measurement of mitochondrial Ca2+ uptake capacity (or the threshold for mPTP opening) is, therefore, important for understanding the mechanisms of pathophysiology in a variety of disease models and also for testing neuro- or cardioprotective drugs. We have, therefore, devised an approach that delivers Ca2+ directly to the ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466672 Fri, 11 Nov 2011 05:00:00 +0100 5466672 http://www.medworm.com/index.php?rid=5466671&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_18 The ubiquitin/proteasome pathway (UPP) is the major proteolytic quality control system in cells and involves tightly regulated removal of unwanted proteins and retention of those that are essential. In addition to its function in normal protein degradation, the UPP plays a critical role in the quality control process by degrading mutated or abnormally folded proteins. The proteolytic component of the UPP is a multiprotein complex known as the proteasome. Many factors, including the aging process, can cause proteasome impairment leading to formation of abnormal ubiquitin-protein aggregates that are found in most progressive neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. In this chapter, we describe protocols to measure proteasome activity, evaluate i... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466671 Fri, 11 Nov 2011 05:00:00 +0100 5466671 http://www.medworm.com/index.php?rid=5466670&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_17 Protein aggregates/inclusions are pathological hallmarks of a wide spectrum of neurodegenerative ­diseases. These aggregates have different shapes, sizes, distribution, and protein composition, which are unique features used for pathological diagnosis. The aggregates per se are also used as molecular targets for designing therapeutic approaches. Detection of these aggregates is generally achieved by using immunostaining methods, most often by immunohistochemistry. In clinical and pathologic practice, the neurologic tissues to be examined are generally fixed with formalin and processed to paraffin-embedded tissue blocks. These treatments result in covalent cross-linking of the protein molecules and preserve the tissue morphology, but dramatically mask the antigens, making it often diffi... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466670 Fri, 11 Nov 2011 05:00:00 +0100 5466670 http://www.medworm.com/index.php?rid=5466669&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_16 The extracellular accumulation of insoluble fibrillar peptides in brain parenchyma and vessel walls as amyloid is the hallmark of neurodegenerative diseases, such as Alzheimer’s disease and Prion diseases. Regardless their amino acid sequences, all amyloid peptides adopt an insoluble, highly ordered beta sheet structure when aggregated. Amyloid is homogeneous and eosinophilic and, common to most cross-beta-type structures; it is generally identified by apple-green birefringence when stained with Congo red and seen under polarized light. Amyloid can also be identified by an apple green color when stained with thioflavine-S and seen under a fluorescence microscope. By electron microscopy, the typical fibrillar ultrastructure of amyloid deposits is revealed. The biochemical nature of th... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466669 Fri, 11 Nov 2011 05:00:00 +0100 5466669 http://www.medworm.com/index.php?rid=5466668&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_9 Ongoing investigations into causes and cures for human movement disorders are important toward the elucidation of diseases, such as Parkinson’s disease (PD). The use of animal model systems can provide links to susceptibility factors as well as therapeutic interventions. In this regard, the nematode roundworm, Caenorhabditis elegans, is ideal for age-dependent neurodegenerative disease studies. It is genetically tractable, has a short life span, and a well-defined nervous system. Fluorescent markers, like GFP, are readily visualized in C. elegans as it is a transparent organism; thus the nervous system, and factors that alter the viability of neurons, can be directly examined in vivo. Through expression of the human disease protein, alpha-synuclein, in the worm dopamine neurons, neur... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466668 Fri, 11 Nov 2011 05:00:00 +0100 5466668 http://www.medworm.com/index.php?rid=5466667&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_8 Neurodegenerative diseases represent one of the most devastating types of diseases in older populations in our time. Significant efforts have been made over the last 20 years to understand the molecular, biochemical, and physiological alterations underlying these diseases. However, in most cases, little is known about their pathological mechanisms due to their high complexity and involvement of a multiplicity of cellular pathways. To gain insight into this group of disorders and to devise potential therapeutic approaches, cellular and animal models of neurodegenerative proteinopathies have been created. Among them, the yeast Saccharomyces cerevisiae has been one of the most popular model organisms due to the degree of conservation of many biological pathways from yeast to human as well as ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466667 Fri, 11 Nov 2011 05:00:00 +0100 5466667 http://www.medworm.com/index.php?rid=5466666&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_15 Neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), Huntington’s (HD), Parkinson’s (PD) and Alzheimer’s diseases (AD), are characterized by the loss of structure and function of specific neuronal circuitry in the brain. As a result of this loss, behavioral symptoms occur progressively. Understanding the causes of neurodegeneration is fundamental for the development of new therapeutic targets. For this purpose, several animal models of neurodegenerative disorders have been generated and characterized. During the characterization, behavioral science plays a crucial role by identifying specific symptoms in these animal models of human disorders. Later on, it also allows scientists to verify the efficacy of new treatments. This chapter describes some of ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466666 Fri, 11 Nov 2011 05:00:00 +0100 5466666 http://www.medworm.com/index.php?rid=5466665&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_14 Parkinson’s disease (PD) is one of the most common neurodegenerative disorders. Despite the substantial progress that has been achieved, the precise mechanisms involved in the development of this disease are still not fully understood. The most common concepts relate to the genetic background and environmental/toxic effects. A number of model systems have been introduced, which mimic the human disease to varying extents. In this chapter, we introduce some of the most widely accepted protocols of the pharmacological models of Parkinson’s disease. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466665 Fri, 11 Nov 2011 05:00:00 +0100 5466665 http://www.medworm.com/index.php?rid=5466664&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_13 Ischemic stroke is among the leading causes of mortality and long-term disability in the western world. Despite enormous research activities in the last decades, current therapeutic options for acute stroke patients are still very limited. Reliable and realistic in vivo animal models represent sine qua non for ­successful translation from bench to bedside. To date, several animal models of focal and global cerebral ischemia have been developed to mimic the clinical situation in humans as accurately as possible. This chapter focuses on models of focal cerebral ischemia, in particular on the most commonly used model: the intraluminal filament model of middle cerebral artery occlusion. The main objective is to provide a detailed instruction manual for researchers interested in learning th... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466664 Fri, 11 Nov 2011 05:00:00 +0100 5466664 http://www.medworm.com/index.php?rid=5466663&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_12 In recent years, the rapid advances in human genetics have identified many genes that are responsible for inherited diseases. In the effort to understand pathogenic pathways and develop therapeutics, researchers have developed genetic animal models as critical tools for the purpose of neurodegenerative disease study. In this overview chapter, we do not intend to cover all the transgenic models for particular diseases, their phenotypical analyses, and their specific usage in this research field. Instead, we outline methods of genetic manipulation and their usage in disease modeling, explain the underlying principles of these methods, and compare the advantages and pitfalls to these different transgenic methods. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466663 Fri, 11 Nov 2011 05:00:00 +0100 5466663 http://www.medworm.com/index.php?rid=5466662&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_11 Although the zebrafish (Danio rerio) has been used extensively for many years in neurodevelopmental studies, use of this teleost to study neurological diseases has evolved only recently. Being a vertebrate, this animal offers advantages for the study of human disease over other small animals, such as the fly or worm. Genetic, as well as nongenetic, disorders can be modeled in both the adult organism and the embryo. Genetic manipulation of the embryo to generate stable and transiently expressing transgenic fish, and to knockdown genes to study loss of their function, can be easily achieved. Because of large offspring numbers screening studies can also be readily performed. Here, we describe some of the protocols useful for modeling neurodegenerative disease in zebrafish embryos, with partic... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466662 Fri, 11 Nov 2011 05:00:00 +0100 5466662 http://www.medworm.com/index.php?rid=5466661&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_10 Most neurodegenerative disorders are associated with aggregates of ubiquitinated proteins, such as Lewy bodies in Parkinson’s disease and neurofibrillary tangles in Alzheimer’s disease. Although the etiology of the sporadic forms of these disorders remains elusive, these observations support our idea that proteasome impairment is an important risk factor in neurodegeneration. Proteasome dysfunction is, thus, expected to be a pivotal link between environmental and genetic factors that are implicated in triggering neurodegeneration. Here, we discuss the rationale for the use of Drosophila as a model system for the study of neurodegeneration. As an example of a specific application of this model system, we provide experimental methodology for the assessment of proteasome function ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466661 Fri, 11 Nov 2011 05:00:00 +0100 5466661 http://www.medworm.com/index.php?rid=5466660&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_7 The great potential of induced pluripotent cells (iPS) cells is that it allows the possibility of deriving pluripotent stem cells from any human patient. Generation of patient-derived stem cells serves as a great source for developing cell replacement therapies and also for creating human cellular model systems of specific diseases or disorders. This is only of benefit if there are well-established differentiation assay systems to generate the cell types of interest. This chapter describes robust and well-characterized protocols for differentiating iPS cells to neural progenitors, neurons, glia and neural crest cells. These established assays can be applied to iPS cell lines derived from patients with neurodegenerative disorders to study cellular mechanisms associated with neurodegeneratio... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466660 Fri, 11 Nov 2011 05:00:00 +0100 5466660 http://www.medworm.com/index.php?rid=5466659&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_6 Human embryonic stem cells (hESCs) and the related induced pluripotent stem cells (hiPSCs) have attracted considerable attention since they can provide an unlimited source of many different tissue types. One challenge of using pluripotent cells is directing their broad differentiation potential into one specific tissue or cell fate. The cell fate choices of extraembryonic, endoderm, mesoderm, and ectoderm (including neural) lineages represent the earliest decisions. We found that pluripotent cells efficiently neuralize by blocking the signaling pathways required for alternative cell fate decisions. In this chapter, we detail methods to direct hESCs or hiPSCs into early neural cells and subsequently postmitotic neurons. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466659 Fri, 11 Nov 2011 05:00:00 +0100 5466659 http://www.medworm.com/index.php?rid=5466658&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_5 Neuronal cultures, including motoneuron (MN) cultures, are established from embryonic animals. These approaches have provided novel insights into developmental and possibly disease mechanisms mediating cell survival or death. Motoneurons isolated from mouse models of disease, such as the SOD1G93A mouse, demonstrate subtle abnormalities that may contribute to pathology. Nonetheless, in the animal model, pathological events become more prominent as the animal matures, but the ability to isolate individual cells to investigate these events is limited. Here, we describe a protocol derived and modified from previously published protocols to isolate motoneurons from mature animals. While the yield of cells is low, the ability to examine mature motoneurons provides a new platform to investigate p... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466658 Fri, 11 Nov 2011 05:00:00 +0100 5466658 http://www.medworm.com/index.php?rid=5466657&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_4 Many experimental animal models of human neurodegenerative diseases have been developed to understand the events leading toward neuronal dysfunction and death. However, definitive comprehension of the molecular and cellular mechanisms in these animal models is problematic because of the complexity of the intact nervous tissue. Primary neuronal cultures prepared from rodent nervous tissues represent a powerful tool not only to study the individual contribution of different cell types (such as neurons or glia) to disease progression, but also to investigate the role of neuron–glia interactions during development and pathogenesis of disease. Here, we describe a method to isolate and culture neurons and astrocytes from the mouse cerebral cortex, and we also present a practical applicatio... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466657 Fri, 11 Nov 2011 05:00:00 +0100 5466657 http://www.medworm.com/index.php?rid=5466656&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_3 The application of electron microscopic immunolabeling techniques to the identification and analysis of degenerating processes in neural tissue has greatly enhanced the ability of researchers to examine apoptosis and other degenerative disease mechanisms. This is particularly true for the early stages of such mechanisms. Traditionally, degenerating processes could only be identified at the ultrastructural level after significant cellular atrophy had occurred, when subcellular detail was obscured and synaptic relationships altered. Using immunocytochemical labeling procedures, degenerating neural and glial processes are first identified through the use of antibodies directed against a variety of degenerative markers, such as proapoptotic effectors (i.e., cytoplasmic cytochrome c), pathologi... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466656 Fri, 11 Nov 2011 05:00:00 +0100 5466656 http://www.medworm.com/index.php?rid=5466655&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_2 Mitochondria are key regulators of cellular death. The mitochondrial membranes contain essential enzyme complexes for maintaining metabolic homeostasis and meeting the energy requirements of the cell (Tait and Green, Nat Rev Mol Cell Biol 11:621–632, 2010 and Galluzzi et al., Apoptosis 12:803–813, 2007). Thus, any perturbation of outer or inner mitochondrial membranes can lead to disruptions in the normal fluxes of key ions and metabolic proteins (i.e., ADP/ATP exchange), leading to eventual cellular death. In addition to maintaining cellular viability, mitochondria play a critical role in the initiation of programmed cell death. As initiators of the cell death process, key mitochondrial proteins [Cytochrome C (Cyt C) one of the most well-studied among them] are released from t... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466655 Fri, 11 Nov 2011 05:00:00 +0100 5466655 http://www.medworm.com/index.php?rid=5466654&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-328-8_1 Neurodegenerative disorders are common diseases that afflict our society with tremendous medical and financial burdens. As a whole, neurodegeneration strikes individuals of all ages, but becomes increasingly frequent with age, coming to affect a very large share of our elderly population. Due to the very complex nature of these diseases, which often result from combined genetic and environmental pathogenic factors, the scientific community that researches the causes and the therapy of neurodegeneration faces remarkable challenges, requiring constant technological advancements. This book describes a collection of experimental protocols used to study various aspects of neurodegeneration. In this introduction, we provide an overview of the field and the comprehensive technological approaches ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5466654 Fri, 11 Nov 2011 05:00:00 +0100 5466654 http://www.medworm.com/index.php?rid=5235420&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_9 Neuropeptide–plasma membrane interactions in the absence of a corresponding specific receptor may result in neuropeptide translocation into the cell. Translocation across the plasma membrane may represent a previously unknown mechanism by which neuropeptides can signal information to the cell interior. We introduce here two complementary optical methods with single-molecule sensitivity, fluorescence imaging with avalanche photodiode detectors (APD imaging) and fluorescence correlation spectroscopy (FCS), and demonstrate how they may be applied for the analysis of neuropeptide ability to penetrate into live cells in real time. APD imaging enables us to visualize fluorescently labeled neuropeptide molecules at very low, physiologically relevant concentrations, whereas FCS enables us to... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235420 Tue, 20 Sep 2011 19:47:28 +0100 5235420 http://www.medworm.com/index.php?rid=5235419&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_8 We examined the effect of overexpression of exogenous peptides comprising of the cytoplasmic tail sequence of vesicular carboxypeptidase E (CPE), proposed to be involved in the mechanism of trafficking of peptidergic secretory vesicles, in live hippocampal neurons. E16 rat hippocampal neurons were transfected with the peptidergic vesicle markers, CPE C-terminally tagged with red or green fluorescent protein, or brain-derived neurotrophic factor (BDNF) tagged with green fluorescent protein, and grown on dishes specialized for real-time live cell visualization. Movements of peptidergic vesicles were imaged in a temperature-controlled chamber on a confocal inverted microscope and analyzed with respect to their velocity, displacement distance, and processivity. (Source: Springer protocols feed... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235419 Tue, 20 Sep 2011 19:47:28 +0100 5235419 http://www.medworm.com/index.php?rid=5235418&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_7 Laser capture microdissection (LCM) provides an efficient and precise method for the sampling of single cells or subgroups of cells in heterogeneous tissues such as the brain. We have recently applied LCM coupled with microarray analysis for the examination of gene expression in the hypothalamic arcuate nucleus of free fed versus fasted rats. We successfully used QPCR analysis to validate our findings and to confirm the regulation of several known neuropeptides. We show that the combination of LCM and QPCR analysis provides a reliable, fast, and efficient alternative to semiquantitative in situ hybridization analysis of gene expression. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235418 Tue, 20 Sep 2011 19:47:28 +0100 5235418 http://www.medworm.com/index.php?rid=5235417&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_6 In situ polymerase chain reaction (PCR) is a histological technique that exploits the advantages of PCR for detection of mRNA directly in tissue sections. It somehow conjugates together PCR and in situ hybridization that is more traditionally employed for mRNA localization in cell organelles, intact cells, or tissue sections. This chapter describes the application of in situ PCR for neuropeptide mRNA localization. We provide here a detailed protocol for direct in situ reverse transcription (RT) PCR (RT-PCR) with nonradioactive probes after fixation and paraffin embedding or cryosectioning. Digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) are incorporated in the PCR product after RT and subsequently detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase. The pr... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235417 Tue, 20 Sep 2011 19:47:28 +0100 5235417 http://www.medworm.com/index.php?rid=5235416&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_5 Measurements of changes in pre-mRNA levels by intron-specific probes are generally accepted as more closely reflecting changes in gene transcription rates than are measurements of mRNA levels by exonic probes. This is, in part, because the pre-mRNAs, which include the primary transcript and various splicing intermediates located in the nucleus (also referred to as heteronuclear RNAs, or hnRNAs), are processed rapidly (with half-lives <60 min) as compared to neuropeptide mRNAs, which are then transferred to the cytoplasm and which have much longer half-lives (often over days). In this chapter, we describe the use of exon-and intron-specific probes to evaluate oxytocin (OT) and vasopressin (VP) neuropeptide gene expression by analyses of their mRNAs and hnRNAs by quantitative in situ hybr... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235416 Tue, 20 Sep 2011 19:47:28 +0100 5235416 http://www.medworm.com/index.php?rid=5235415&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_4 In situ hybridization has become a routine technique to provide insights into RNA localization. However, different protocols exist for multiple purposes, and it is, therefore, important to clearly define specific needs to choose the most suitable one(s). For instance, in situ hybridization can target different types of RNA, including mRNA or small noncoding RNA such as micro RNA (miRNA). Detection protocols are developed for light or electron microscopy and can be combined with immunocytochemistry to study RNA coexpression with proteins or peptides. In this chapter, we present some protocols to illustrate the diversity of in situ hybridization methods. We focus on the detection of mRNA or miRNA and show that the protocols are quite similar but use dedicated probe types, namely, oligo- or r... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235415 Tue, 20 Sep 2011 19:47:28 +0100 5235415 http://www.medworm.com/index.php?rid=5235414&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_3 The study of neuronal connections and neuron to neuron (or neuron to glia) communication is of fundamental importance in understanding brain structure and function. Therefore, ultrastructural investigation by the use of immunocytochemical techniques is a really precious tool to obtain an exact map of the localization of neurotransmitters (neuropeptides) and their receptors at different types of synapses. However, in immunocytochemical procedures one has always to search for the optimal compromise between structural preservation and retention of antigenicity. This is often made difficult by the need to localize not only small transmitter molecules, as in the case of transmitter amino acids and neuropeptides, but also their specific receptors that are usually large proteins very sensitive to... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235414 Tue, 20 Sep 2011 19:47:28 +0100 5235414 http://www.medworm.com/index.php?rid=5235413&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_2 Neuropeptides are particularly suited to comparative and evolutionary studies, since they have been highly conserved during evolution. Based on primary amino-acid structure, neuropeptides can be arranged into families and synthesized as multiple molecular variants. They may play different functional roles in different organs or tissues of the same species, but also among species and classes. Immunohistochemistry (IHC) is powerful technique for localizing the molecular expression of proteins in tissues and cells of different classes of vertebrates and has been fully exploited in the study of the mammalian brain. The present chapter provides a detailed description of the protocols routinely used in our laboratory to analyze the presence and distribution of neuropeptides in nonmammalian verte... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235413 Tue, 20 Sep 2011 19:47:28 +0100 5235413 http://www.medworm.com/index.php?rid=5235412&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_25 G protein-coupled receptors (GPCRs) comprise one of the largest families of transmembrane proteins involved in signal transduction of diverse external stimuli and represent the most successful target class in drug discovery. The availability of genome sequences in the postgenomic era has paved the way for in silico identification of novel GPCR family members based upon sequence similarity. Consequently, newly discovered receptors are by definition orphan GPCRs with no known ligand, and their functional characterization now poses a major challenge. Over the years, advances in understanding of GPCR biology have led to the development of cell-based assay systems that link orphan GPCRs to their activating ligand(s) in high-throughput format (reverse pharmacology). Many of these technologies mo... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235412 Tue, 20 Sep 2011 19:47:28 +0100 5235412 http://www.medworm.com/index.php?rid=5235411&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_24 Recombinant adeno-associated viral (rAAV) vectors can be used to locally or systemically enhance or silence gene expression. They are relatively nonimmunogenic and can transduce dividing and nondividing cells, and different rAAV serotypes may transduce diverse cell types. Therefore, rAAV vectors are excellent tools to study the function of neuropeptides in local brain areas. In this chapter, we describe a protocol to produce high-titer, in vivo grade, rAAV vector stocks. The protocol includes an Iodixanol gradient, an anion exchange column and a desalting/concentration step and can be used for every serotype. In addition, a short protocol for rAAV injections into the brain and directions on how to detect and localize transduced cells are given. (Source: Springer protocols feed by Neuroscie... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235411 Tue, 20 Sep 2011 19:47:28 +0100 5235411 http://www.medworm.com/index.php?rid=5235410&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_23 This paper describes an experimental approach based on nanotechnology for assessing the chronic actions of short-lived neuropeptides at specific sites of the brain. This methodology combines the advantages of two different techniques: the microinjection of a suspension of peptide-containing liposomes into a specific site of the brain, and the use of liposomes as a local and sustained release nanosystem of the peptide. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235410 Tue, 20 Sep 2011 19:47:28 +0100 5235410 http://www.medworm.com/index.php?rid=5235409&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_22 Phosphorothioate oligodeoxynucleotides (PODNs) can act as antisense molecules, knocking down proteins. Many PODNs have the unusual characteristic of being transported across the blood–brain barrier by a saturable system. This means that PODNs injected intravenously can accumulate in the central nervous system in quantities sufficient to knock down proteins in brain and the blood–brain barrier. A critical step in the development of PODNs that can be administered peripherally and knockdown proteins in the central nervous system is to determine the relation to the blood–brain barrier, specifically, does the PODN cross the blood–brain barrier and, if so, how fast and to what degree. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235409 Tue, 20 Sep 2011 19:47:28 +0100 5235409 http://www.medworm.com/index.php?rid=5235408&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_21 The blood–brain barrier (BBB) represents multiple barriers for drug delivery from the circulation. Peptides potentially useful to treat maladies of the brain are especially limited in their ability to cross the BBB due to several shortcomings. Specific delivery strategies have been conceived to outwit the BBB to target neuropeptides into the brain. It should be noted, however, that no unified method is possible for true brain-targeting of these fascinating biomolecules due to their structural features, properties, and intricate interplays among factors governing their entrance into and retention within the brain. In most brain-targeting prodrug approaches, a lipophilic and bioreversible moiety(ies) is covalently attached to the peptide that results in the complete loss of the innate ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235408 Tue, 20 Sep 2011 19:47:28 +0100 5235408 http://www.medworm.com/index.php?rid=5235407&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_20 A major barrier to entry of neuropeptides into the brain is low bioavailability and presence of the blood–brain barrier. Intranasal delivery of neuropeptides provides a potentially promising alternative to other routes of administration, since a direct pathway exists between the olfactory neuroepithelium and the brain. Use of the rat as an animal model in nose to brain delivery of neuropeptides allows for several advantages, including a large surface area within the nasal cavity dedicated to olfactory epithelium and robust neuronal pathways extending to and from most areas of the brain from the nose via the olfactory cortex. A major disadvantage to using rats for nose to brain delivery is the difficulty in selectively targeting the posterior olfactory epithelium (which facilitates de... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235407 Tue, 20 Sep 2011 19:47:28 +0100 5235407 http://www.medworm.com/index.php?rid=5235406&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_1 We know neuropeptides now for over 40 years as chemical signals in the brain. The discovery of neuropeptides is founded on groundbreaking research in physiology, endocrinology, and biochemistry during the last century and has been built on three seminal notions: (1) peptide hormones are chemical signals in the endocrine system; (2) neurosecretion of peptides is a general principle in the nervous system; and (3) the nervous system is responsive to peptide signals. These historical lines have contributed to how neuropeptides can be defined today: “Neuropeptides are small proteinaceous substances produced and released by neurons through the regulated secretory route and acting on neural substrates.” Thus, neuropeptides are the most diverse class of signaling molecules in the brain... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235406 Tue, 20 Sep 2011 19:47:28 +0100 5235406 http://www.medworm.com/index.php?rid=5235405&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_19 This protocol describes an ELISA-based method for assaying serum levels of autoantibodies reactive with neuropeptides. The method allows for measuring relative amounts of free and bound, i.e., those present in immune complexes, autoantibodies using two types of sample buffers providing normal and dissociative conditions, respectively. This method can be applied to measure autoantibody levels directed against other than neuropeptide molecules and in a variety of biological fluids. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235405 Tue, 20 Sep 2011 19:47:28 +0100 5235405 http://www.medworm.com/index.php?rid=5235404&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_18 The control of neuropeptide function is partially accomplished by aminopeptidases (neuropeptidases), which are the most abundant proteolytic enzymes in brain. Their analysis represents an important and quick tool to reflect the functional status of their endogenous substrates. Here, we describe an improved fluorometric method for the determination of neuropeptidase activities based on the fluorescence produced by ß-naphthylamine when released from the artificial substrates aminoacyl-ß-naphthylamides (arylamides) under the hydrolytic action of these enzymes. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235404 Tue, 20 Sep 2011 19:47:28 +0100 5235404 http://www.medworm.com/index.php?rid=5235403&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_17 Antibody-coated microprobes have been demonstrated to be useful for detecting the release of neuropeptide transmitters from discrete sites in the central nervous system (CNS). This technique uses glass micropipettes taken through a series of chemical coatings, starting with a ?-aminopropyltriethoxysilane solution and ending with the antibody specific to the peptide transmitter of interest. The key to the reliability and repeatability of the technique is a uniform, even coating of the siloxane polymer to the glass micropipette. The microprobes, as they are called following the completion of the coating process, are inserted stereotaxically into a specific area of the CNS and the physiological intervention is performed. Tip diameters are around 5–10 µm and, depending on the lengt... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235403 Tue, 20 Sep 2011 19:47:28 +0100 5235403 http://www.medworm.com/index.php?rid=5235402&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_16 Microdialysis is a technique that collects extracellular fluid through a semipermeable membrane. Various compounds are obtained with this technique in vivo in free-moving animals. Originally, this technique was developed to measure several biogenic amines in rat brain, for example, dopamine from the striatum, acetylcholine from the cerebral cortex, and noradrenaline from the hypothalamus. Recently, the membrane quality and detection limit of many substances have been improved; thus, the microdialysis techniques are widely used to quantify large molecules in the brain. These molecules include neuropeptides and hormones. In this chapter, we describe a principle of the microdialysis technique, how to prepare a microdialysis system, and how to obtain samples from the brain of free-moving anima... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235402 Tue, 20 Sep 2011 19:47:28 +0100 5235402 http://www.medworm.com/index.php?rid=5235401&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_15 Comparing two RNA populations that differ from the effects of a single independent variable, such as a drug treatment or a specific genetic defect, can establish differences in the abundance of specific transcripts that vary in a population dependent manner. There are different methods for identifying differentially expressed genes. These methods include microarray, Serial Analysis of Gene Expression (SAGE), and quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR). Herein, the protocol describes an easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under examination. It is specifically relevant when low levels of RNA starting material are available. This protocol describes the use of Switching Mechanism At RNA Termini Polymeras... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235401 Tue, 20 Sep 2011 19:47:28 +0100 5235401 http://www.medworm.com/index.php?rid=5235400&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_14 Neuropeptidomics refers to a global characterization approach for the investigation of neuropeptides, often under specific physiological conditions. Neuropeptides comprise a complex set of signaling molecules that are involved in regulatory functions and behavioral control in the nervous system. Neuropeptidomics is inherently challenging because neuropeptides are spatially, temporally, and chemically heterogeneous, making them difficult to predict in silico from genomic information. Mature neuropeptides are produced from intricate enzymatic processing of precursor proteins/prohormones via a range of posttranslational modifications, resulting in multiple final peptide products from each prohormone gene. Although there are several methods for targeted peptide studies, mass spectrometry (MS),... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235400 Tue, 20 Sep 2011 19:47:28 +0100 5235400 http://www.medworm.com/index.php?rid=5235399&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_13 Peptide study and analysis widely involve liquid chromatography. Among the different strategies available, reversed-phase liquid chromatography (RP-HPLC) is one of the methods of choice to separate species in a nontargeted approach. The compounds are sorted according to their hydrophobicity, even though the experimental order of elution could change according to the nature of the mobile phase and the stationary phase. In our work, we have developed protocols to resolve hundred of peptidic species. To overcome the limitations of peak capacity of RP-HPLC alone, it has been coupled downstream to tandem mass spectrometry using two different ionization modes. To overcome the limitations of peak capacity of RP-HPLC MS/MS, it has been coupled upstream to strong cation exchange liquid chromatograp... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235399 Tue, 20 Sep 2011 19:47:28 +0100 5235399 http://www.medworm.com/index.php?rid=5235398&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_12 The past half-century has seen an enormous development in the area of biomedical science. This includes also research related to neuroactive peptides. These compounds have been the subject for extensive studies in many cases resulting in knowledge opening for new therapeutic strategies for the management of various neurological disorders. In this research the radioimmunoassay has represented an invaluable tool. This method, first introduced for the assessment of serum and plasma levels of various hormones, meant a transition from bioassay to a more sensitive and precise technique for protein and peptide quantification in samples of clinical relevance. It accounts for an approach which became one of the most widely used methods in routine and research at many clinical and basic laboratories... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235398 Tue, 20 Sep 2011 19:47:28 +0100 5235398 http://www.medworm.com/index.php?rid=5235397&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_11 Electrophysiological recording techniques have been widely applied to study the functional role of neuropeptides. The present chapter focuses on the very often used techniques including whole-cell patch-clamp recording, sharp electrode intracellular recording, and extracellular field potential recording in slice preparation as well as in vivo animal recording. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235397 Tue, 20 Sep 2011 19:47:28 +0100 5235397 http://www.medworm.com/index.php?rid=5235396&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-310-3_10 Nerve fibers innervate every organ of the body and are involved in monitoring changes of the external and internal environment. Innervation directly controls a variety of physiological responses in an adaptive manner. Today, many lines of research indicate that also the immunological response is influenced by the nervous system and that nerve and immune cells directly interact through intercellular signal transduction by cytokines, neurotransmitters, and neuropeptides. For instance, mast cells are often found in close proximity of nerve fibers containing substance P and calcitonin gene-related peptide, two widely studied sensory neuropeptides, in a variety of tissues. To investigate the molecular mechanism of the direct functional interplay between nerve and immune cells, we have studied t... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5235396 Tue, 20 Sep 2011 19:47:27 +0100 5235396 http://www.medworm.com/index.php?rid=5018904&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_20 The phenomenon of antagonist-induced receptor upregulation is common to many G protein-coupled receptors (GPCRs) such as adrenergic, muscarinic, opioid, cannabinoid, histamine, GABA(B), serotonin, and dopamine receptors. This chapter reviews data that support antagonist-induced upregulation specifically of opioid receptors but many of the principles apply to other GPCRs as well. It is well documented that chronic exposure to opioid receptor antagonists reliably produces increases in binding to opioid receptors when the antagonists are administered in vivo or applied in vitro to cell culture systems. Antagonist exposure increases receptor number and is associated with functional supersensitivity to subsequent agonist administration. For example, the analgesic potency of morphine is increase... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018904 Wed, 13 Jul 2011 00:37:29 +0100 5018904 http://www.medworm.com/index.php?rid=5018903&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_19 Technologies developed to interfere with gene transcripts were developed back in the 1980. However, it was not before the last decade that light was shed on the underlying mechanisms of what is now known as RNA interference. From then, RNAi was propelled to the forefront as a revolutionizing approach for basic research and clinical therapy. In the present chapter, we will present an overview of RNAi and its mechanisms with a focus on GPCRs applied to neuroscience. We will briefly detail the steps to move from the in vitro assessment to the in vivo proof-of-principle to further ensure appropriate clinical transfer. Examples of successful experiments will be given in each section. Advances, drawbacks, and future directions will be discussed with RNAi as an exciting new technology that can be... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018903 Wed, 13 Jul 2011 00:37:28 +0100 5018903 http://www.medworm.com/index.php?rid=5018902&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_18 We present here two methods with single-molecule sensitivity, confocal laser scanning microscopy with avalanche photodiode (APD) detectors (APD imaging) and fluorescence correlation spectroscopy (FCS) suitable for nondestructive study of molecular interactions and intracellular transporting processes in living cells. These high-resolution methods provide functional readouts, giving measures of concentration, mobility and affinity, for the investigated molecules and enable us to monitor changes in these properties in living cells in real time. We have used these methods to study early events in opioid receptor activation with specific and nonspecific ligands, and discuss the new insights obtained by these approaches. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018902 Wed, 13 Jul 2011 00:37:28 +0100 5018902 http://www.medworm.com/index.php?rid=5018901&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_17 G protein-coupled receptors (GPCRs) are the major sites of actions for the body’s endogenous hormones and neurotransmitters which make them ideal targets for pharmaceutical development with the goal of either mimicking the normal transmitter response or tempering it. In recent years, targeting GPCRs has become more complicated as we realize that drug action at receptors is “context dependent” such that activation and inhibition is limited to the response evaluated and agonist and antagonist become terms that reflect a particular condition of the experimental or physiological output. Therefore, the composition of the receptor’s immediate environment may determine activation profiles as posttranslational modifications of the receptor or of the binding partners can ult... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018901 Wed, 13 Jul 2011 00:37:28 +0100 5018901 http://www.medworm.com/index.php?rid=5018900&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_16 In pharmacology, a central tenet of receptor theory has been that different agonists acting at a particular G protein-coupled receptor subtype produce the same profile of cellular responses. In recent years, advances in molecular pharmacology and the availability of diverse cell signaling assays have indicated that this idea is not sufficient to explain all the data obtained, and that agonists can produce different response profiles ­following binding to a receptor subtype in a cell. This has been termed biased agonism or functional selectivity­, and is thought to be due to the ability of agonists to stabilize different active conformations of the receptor. Logically, there is no reason why this idea cannot also be extended to receptor regulatory mechanisms, since different recepto... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018900 Wed, 13 Jul 2011 00:37:28 +0100 5018900 http://www.medworm.com/index.php?rid=5018899&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_15 With the rise of interest in receptor–receptor interaction research, optimization of experimental designs to study the nature of interactions has become increasingly important. In this chapter, traditional experimental designs and their associated statistics are reviewed, and their theoretical and practical limitations are described. The logic and theory behind isobolographic analysis is explained and their practical use is also described. As more and more receptor–receptor interactions are recognized with the use of two or more GPCR drugs, advances in the analysis of the type presented here will become more significant and important for clinical pharmacotherapy. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018899 Wed, 13 Jul 2011 00:37:27 +0100 5018899 http://www.medworm.com/index.php?rid=5018898&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_14 In this chapter, methods for the analysis of allosterism in equilibrium binding and functional assays are described. The functional response to activation of a G protein-coupled receptor is usually measured at a point downstream from receptor activation in the signaling pathway, and the effects of allosteric modulation on the response can be attributed to scalar changes in the observed affinity (a) and intrinsic efficacy (ß) of the agonist-receptor complex. If the concentration-response curve of the agonist is measured in the absence and presence of a range of concentrations of the allosteric modulator, then it is always possible to estimate the affinity constant of the modulator (K 2) and the product (?) of the modulatory changes in the observed affinity and efficacy of the ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018898 Wed, 13 Jul 2011 00:37:27 +0100 5018898 http://www.medworm.com/index.php?rid=5018897&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_13 Phosphorylation of G protein-coupled receptors (GPCRs) occurs within seconds of agonist stimulation and is one of the most prevalent mechanisms through which signalling of this super receptor family is regulated. Although traditionally associated with receptor desensitisation and internalisation, there is an increasing body of evidence that suggests that GPCRs employ phosphorylation as a mechanism of coupling the receptor to non-G protein signalling pathways. Recently, it has become clear that GPCRs can be differentially phosphorylated in different cell types or tissues, possibly by tissue- or cell type-specific employment of a variety of receptor kinases to generate specific “phosphorylation patterns” that might encode signalling properties on the receptor. Although hampered b... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018897 Wed, 13 Jul 2011 00:37:27 +0100 5018897 http://www.medworm.com/index.php?rid=5018896&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_12 The development of new therapeutic drugs acting at G protein-coupled receptors (GPCRs) whose ligand specificity is known is of great importance to the pharmaceutical industry and to the population at large. It is also vital that surrogate ligands can be identified for GPCRs at which the endogenous ligand(s) remain unknown so that their potential as drug targets can be assessed. It is against this background that we consider a selection of technologies that are emerging to meet these challenges. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018896 Wed, 13 Jul 2011 00:37:27 +0100 5018896 http://www.medworm.com/index.php?rid=5018895&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_11 A remarkable potential exists for current and future development of therapeutic drugs acting at GPCRs. As one of the initial steps in GPCR drug development, in vitro assays are required to characterize the pharmacology of new ligands acting at distinct GPCRs. This is routinely accomplished by first employing cellular models to establish high affinity, selectivity, and efficacy of a test compound for a specific GPCR involved in a disease process of interest. However, several limitations are encountered when native cell lines or isolated tissues expressing low levels of endogenous GCPRs are employed for receptor characterization. To overcome many of these issues, cells are routinely transfected with cDNA of a desired GPCR to create cell lines stably expressing a sufficient receptor density t... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018895 Wed, 13 Jul 2011 00:37:27 +0100 5018895 http://www.medworm.com/index.php?rid=5018894&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_9 G protein-coupled receptor (GPCR) responsiveness is dynamically regulated by various mechanisms, allowing fine-tuning of cell signaling. Modulation of GPCR plasma membrane density, via their release from intracellular compartments, constitutes a recently identified important process in this context. This phenomenon requires a complex network of interactions between GPCRs, “private” chaperones and escort proteins, and gatekeepers, which are directly involved in the retention of GPCRs in the intracellular compartments. The molecular and functional characterization of the players in this game is at its very beginning and requires appropriate quantitative methods of investigation to unravel the mechanisms that are involved. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018894 Wed, 13 Jul 2011 00:37:26 +0100 5018894 http://www.medworm.com/index.php?rid=5018893&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_8 Similar to many other plasma membrane proteins, G protein-coupled receptors (GPCRs) are synthesized in the endoplasmic reticulum (ER). After proper assembly and folding, the receptors are transported from the ER through the Golgi to the cell surface. As the first step in the anterograde trafficking, exit from the ER of nascent GPCRs plays a crucial role in receptor biosynthesis and controls receptor expression at the cell surface, which in turn dictates the functionality of the receptors. Recent studies have revealed that GPCRs possess linear motifs that are required for their export from the ER. In this chapter, we will discuss experimental approaches to identify the GPCR motifs which regulate anterograde transport, specifically exit from the ER, by focusing on a2B-adrenergic receptor. (S... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018893 Wed, 13 Jul 2011 00:37:26 +0100 5018893 http://www.medworm.com/index.php?rid=5018892&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_7 The G protein-coupled receptors (GPCRs) are a superfamily of transmembrane receptors that ­structurally possess an extracellular amino terminus, seven transmembrane domains linked by extracellular and intracellular loops, and a cytoplasmic carboxyl terminus. They are synthesized by ribosomes and enter into the endoplasmic reticulum (ER), from which they are transported to Golgi apparatus and the trans-Golgi network (TGN) and finally move to the plasma membrane. At the plasma membrane, GPCRs receive environmental stimuli and relay the message to the cells. During these processes, GPCRs undergo posttranslational modifications that regulate their maturation, their function at the cell surface and even the ultimate fate of the internalized receptor after agonist treatment. There are four m... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018892 Wed, 13 Jul 2011 00:37:26 +0100 5018892 http://www.medworm.com/index.php?rid=5018891&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_6 We report our first observations using this tool, and exemplify its usefulness at the level of receptor anatomy, function, and adaptations to drugs, with a particular focus on pain processes. This approach is potentially applicable to any GPCR, using an increasing choice among fluorescent reporter proteins, and offers unprecedented perspectives toward understanding GPCR biology and developing novel drugs of therapeutic interest. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018891 Wed, 13 Jul 2011 00:37:25 +0100 5018891 http://www.medworm.com/index.php?rid=5018890&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_10 A new approach for characterizing the plasma membrane delivery of G protein-coupled receptors is described in this chapter. This approach uses an existing technology, the regulated secretion/aggregation technology (RPD™), to cause the accumulation of G protein-coupled receptors in the ER of cells. The trafficking of accumulated receptor from the ER to the plasma membrane of cells can be initiated by a small, membrane permeable ligand. The plasma membrane delivery of receptors can be monitored using radioligand binding or microscopy or both techniques. Using this new approach it should be possible to determine the rate of plasma membrane delivery of a broad range of G protein-coupled receptors and thus facilitate learning more about their anterograde trafficking mechanisms. (Source: S... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018890 Wed, 13 Jul 2011 00:37:25 +0100 5018890 http://www.medworm.com/index.php?rid=5018889&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_5 G protein-coupled receptors (GPCRs) are ancestrally related membrane proteins on cells that mediate the pharmacological effect of most drugs and neurotransmitters. GPCRs are the largest group of membrane receptor proteins encoded in the human genome. Using the case study of vertebrate opioid receptors, this chapter introduces an evolutionary approach to understanding pharmacological selectivity, predicted from sequence analysis, and confirmed by experimental studies. The same approach can be used to examine receptor function and applies to other families of GPCRs besides the opioid receptor family. Opioid receptors consist of a family of four closely related proteins expressed in all vertebrates examined. The three types of opioid receptors shown unequivocally to mediate analgesia in anima... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018889 Wed, 13 Jul 2011 00:37:24 +0100 5018889 http://www.medworm.com/index.php?rid=5018888&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_4 G protein-coupled receptors (GPCRs) participate in a variety of physiological functions and are major targets of pharmaceutical drugs. More than 600 GPCRs have been identified in the human genome. Although GPCRs are expressed in multiple tissues and individual tissues express multiple GPCRs, many have exclusive or increased expression within the central nervous system (CNS). These unique and diverse expression patterns raise fundamental questions as to the molecular mechanisms underlying the tissue/cell-specific distribution of GPCRs as well as the means by which their expression is altered in response to stimuli. Gene expression in mammals involves both transcriptional and posttranscriptional mechanisms. In this chapter, we provide an overview of the transcriptional regulation of GPCRs an... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018888 Wed, 13 Jul 2011 00:37:24 +0100 5018888 http://www.medworm.com/index.php?rid=5018887&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_3 The genes for G protein-coupled receptors (GPCRs) including those encoding the classical mu, delta, and kappa opioid receptors (MOR, DOR, and KOR); cannabinoid receptors (CB1); ACTH receptor (melanocortin receptor type 2, MC2R); and serotonin receptors (5HT1B) have been a focus of the studies of our group for a number of years since these receptors are involved in specific addictions. Genetic variants of GPCR genes have been associated with vulnerability to stress, anxiety, depression, and predisposition to develop drug addiction. To study these variants including single nucleotide polymorphisms (SNPs) and their allocation on alleles (haplotypes), our group developed special techniques (genotyping assays using polyacrylamide gel pad technology, molecular haplotyping assays based on the use... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018887 Wed, 13 Jul 2011 00:37:24 +0100 5018887 http://www.medworm.com/index.php?rid=5018886&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_2 Alternative pre-mRNA splicing involves editing of a gene to generate a number of different mRNAs and proteins. It provides a mechanism for only 20,000 genes to generate hundreds of thousands of proteins. Like other proteins, it is estimated that 50% of G protein-coupled receptors undergo alternative splicing. While most commonly involving either the N-terminus or C-terminus, some variants have modifications in the interior of the receptor. Alternative splicing generates functionally distinct variants, due to an intrinsic difference in transduction or location. These features are well illustrated by the mu opioid receptor gene, OPRM1, which undergoes extensive alternative splicing. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018886 Wed, 13 Jul 2011 00:37:24 +0100 5018886 http://www.medworm.com/index.php?rid=5018885&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-179-6_1 The G protein-coupled receptors (GPCRs) form the largest and most multi-functional protein ­superfamilies known. From a drug discovery and pharmaceutical industry perspective, the GPCRs are among the most commercially and economically important groups of proteins yet identified, since they have so many vital metabolic functions and interact with such a diversity of ligands. Many distinct methodologies have been proposed to classify the GPCRs: motif-based techniques, machine learning, and several alignment-free techniques have all been used successful in this regard. This chapter reviews the available methodologies for classifying GPCRs. In particular, we allude to several innate problems in developing such approaches, such as the lack of sequence similarity between the six GPCR classes... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=5018885 Wed, 13 Jul 2011 00:37:23 +0100 5018885 http://www.medworm.com/index.php?rid=4911159&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_9 Disrupted-in-Schizophrenia 1 (DISC1) is a strong candidate gene for schizophrenia and major mental disorders. After its discovery in the Scottish chromosomal translocation, DISC1 has gained considerable attention in neuropsychiatric research. Recent studies have implicated DISC1 in fundamental processes of neurodevelopment and adulthood neuroplasticity. To get more insights into the functions of DISC1 in vivo, several mouse DISC1 models have been generated based on different approaches, including constitutive and inducible over-expression of different fragments of DISC1, targeted mutagenesis, and viral vector knockdown. Each model has provided important information regarding DISC1 functions and helped in elucidating the molecular pathways underlying behavioral disorders. The existing model... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911159 Thu, 09 Jun 2011 01:33:17 +0100 4911159 http://www.medworm.com/index.php?rid=4911158&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_8 Accumulating evidence indicates that the genetic architecture of psychiatric disorders does not strictly conform to the common disease/common allele hypothesis. The contribution of common genetic variants, while likely, may be fundamentally different from those of rare genetic variants. It is possible that common alleles do not increase disease risk per se but are disease modifiers sculpting the psychopathological landscape produced by rare alleles. Unlike common alleles, the statistical association of rare alleles is usually more robust and their functional effects more translatable into etiologically valid animal models. Although rare alleles may not be shared across individuals with the same diagnosis, the comparison of multiple animal models of rare risk alleles can identify common pat... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911158 Thu, 09 Jun 2011 01:33:17 +0100 4911158 http://www.medworm.com/index.php?rid=4911157&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_7 Evidence of altered antioxidant systems and signs of elevated oxidative stress are reported in peripheral tissue and brain of schizophrenic patients, including low levels of glutathione (GSH), a major thiol antioxidant and redox buffer. Functional and genetic data indicate that an impaired regulation of GSH synthesis is a vulnerability factor for the disease. Impaired GSH synthesis from a genetic origin combined with environmental risk factors generating oxidative stress (e.g., malnutrition, exposure to toxins, maternal infection and diabetes, obstetrical complications, and psychological stress) could lead to redox dysregulation. This could subsequently perturb normal brain development and maturation with delayed functional consequences emerging in early adulthood. Depending on the nature ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911157 Thu, 09 Jun 2011 01:33:17 +0100 4911157 http://www.medworm.com/index.php?rid=4911156&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_6 Evidence obtained from schizophrenia post-mortem brain studies have pointed to deficiencies in inhibitory systems, in particular of the fast-spiking parvalbumin (PV)-positive inhibitory interneurons, as responsible for several aspects of schizophrenia pathophysiology. This hypothesis has been confirmed in pharmacological as well as genetic models of the disease, but when and how this dysfunction occurs is still unknown. Exposure to NMDA receptor antagonists is one of the most used pharmacological models for the study of schizophrenia, due to its capacity to produce a psychotic syndrome in humans and to produce an outbreak in schizophrenia patients. Using this model, we and others have shown that dysfunction of the PV-inhibitory system is most probably responsible for the neural network alt... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911156 Thu, 09 Jun 2011 01:33:17 +0100 4911156 http://www.medworm.com/index.php?rid=4911155&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_5 It is now widely acknowledged that exposure to adverse environmental factors in utero may not only affect how the brain develops but have long-lasting consequences for later brain function in the adult offspring. This idea has gained particular prominence amongst researchers interested in the etiology of neurodevelopmental disorders such as schizophrenia and autism. Approximately 10 years ago we proposed that developmental vitamin D (DVD) deficiency may explain several epidemiological features of this disease, most noticeably the winter/spring season of birth effect. In 2003 we published results from our first study indicating there were structural changes in how the brain develops in these offspring. Since then we have firmly established that DVD deficiency not only affects brain cell dif... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911155 Thu, 09 Jun 2011 01:33:17 +0100 4911155 http://www.medworm.com/index.php?rid=4911154&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_4 This study investigated the effects of chronic neonatal hypoxia in rats as an animal model of schizophrenia. Methods: (1) After chronic neonatal hypoxia between postnatal day (PD) 4 and 8, half of the pups were fostered by normally treated nurse animals to control for possible maternal effects and (2) tested on PD 36, 86, 120, and 150 using three different behavioral tests: prepulse inhibition (PPI), social interaction and recognition, and motor activity in an open field. (3) Before the PD 150 test, 50% of the animals had been chronically treated with the antipsychotic drug clozapine (45 mg/kg/day). (4) At PD 155, different brain regions have been used for expression profiling of synaptic genes on cDNA microarrays (“glutamate chip”) with qRT-PCR confirmation. Additionally,... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911154 Thu, 09 Jun 2011 01:33:17 +0100 4911154 http://www.medworm.com/index.php?rid=4911153&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_3 An increasing number of epidemiologic studies have implicated in utero exposure to infection in the etiopathogenesis of schizophrenia. Recent work has capitalized on the use of prospectively acquired data on infection based on maternal biomarkers. These studies suggest that several maternal infections, including rubella, influenza, toxoplasmosis, herpes simplex virus/other genital-reproductive infections, and elevations in the cytokines interleukin-8 and TNF-a, are associated with increased schizophrenia risk among offspring. Animal models of in utero infection offer the potential to corroborate these findings under controlled conditions and address etiopathogenic mechanisms. Models of maternal immune activation (MIA) and behavioral and brain anomalies in schizophrenia have primarily emplo... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911153 Thu, 09 Jun 2011 01:33:17 +0100 4911153 http://www.medworm.com/index.php?rid=4911152&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_2 This chapter provides an overview on exposure to methylazoxymethanol (MAM) at embryonic day 17 as a promising animal model for schizophrenia that mimics behavioral abnormalities and deficits in prefrontal cortex networks. This early insult produces in adult offspring from E17 MAM-treated dams the following: (1) behavioral changes including spontaneous hyperactivity and hypersensitivity to psychotomimetic drugs that are reminiscent of positive symptoms of schizophrenia and associated with a temporal pattern of expression; (2) impaired social interaction similar to that observed in schizophrenic patients existing prior to the onset of disease; (3) cognitive deficits in a variety of domain: working and reference memory, behavioral flexibility, attentional functioning, object recognition, and ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911152 Thu, 09 Jun 2011 01:33:16 +0100 4911152 http://www.medworm.com/index.php?rid=4911151&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_1 The neonatal ventral hippocampal lesion (NVHL) rat model of schizophrenia has demonstrated broad heuristic utility as an investigative platform encompassing many of the behavioral, neurobiological, and developmental aspects of this devastating neuropsychiatric illness affecting 1% of all human beings. This chapter serves as an essential description of materials and methods for generating and verifying the NVHL model in rats, which continues to hold significant potential in helping us understand schizophrenia, co-morbid disorders, and their neurodevelopmental dynamics. Many of the approaches described here can be modified or adapted for producing other types of neurodevelopmental models of behavioral disorders. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911151 Thu, 09 Jun 2011 01:33:16 +0100 4911151 http://www.medworm.com/index.php?rid=4911150&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_12 Schizophrenia, a most prevalent brain disorder, remains to be one of the least understood. Unlike Alzheimer’s disease or Parkinson’s disease, schizophrenia lacks clear pathological lesions, which has made it difficult to model in animals. However, genetic studies have recently identified many susceptibility genes of this disorder including neuregulin 1 (NRG1) and its receptor ErbB4. Arguably, relevant mutant mice have provided a unique opportunity to model “schizophrenia” in animals where expression or function of susceptibility genes is altered. This review summarizes recent studies of NRG1 and ErbB4 mutant mice and their implication in schizophrenic pathology. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911150 Thu, 09 Jun 2011 01:33:16 +0100 4911150 http://www.medworm.com/index.php?rid=4911149&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_11 There is substantial evidence that psychosis is characterized by GABAergic gene promoters that are in a closed chromatin state, leading to reduced transcription of proteins essential for GABAergic and synaptic function in the forebrain. Two critical genes that are downregulated in cortical and hippocampal interneurons by 30–50% in psychotic postmortem brain are GAD1, the gene for GAD67, and RELN, the gene for reelin. The promoter region of these genes is vulnerable to cytosine methylation by DNA methyltransferases, enzymes that in psychosis are elevated in GABAergic interneurons. An alteration in histone deacetylase or methyltransferase expression provides further evidence for an underlying epigenetic etiology. Animal models aimed at studying the epigenetic basis for key phenotypic c... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911149 Thu, 09 Jun 2011 01:33:16 +0100 4911149 http://www.medworm.com/index.php?rid=4911148&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-157-4_10 NMDA receptor (NMDAR) hypofunction theory of schizophrenia has been assessed in rodents with pharmacological intervention and global knockout strategy of NMDAR blockade. However, these manipulations of NMDAR function have been relatively coarse, affecting all NMDA receptors throughout the brain. Here we tested the effects of eliminating NMDA receptors in about half the interneurons located in the cortex and the hippocampus in early postnatal development by engineering “conditional knockout” mice. Remarkably, the mutant mice produced a variety of schizophrenia-related phenotypes. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4911148 Thu, 09 Jun 2011 01:33:16 +0100 4911148 http://www.medworm.com/index.php?rid=4559965&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_10 In studies on pluripotency and differentiation, teratocarcinoma cell lines have been used as alternatives to mouse embryonic stem cell lines. Teratocarcinoma cell lines cost less to maintain and they are easier to genetically manipulate. Their disadvantage, of course, is being derived from a tumor, which prevents their use in certain types of studies such as in implantation and proliferation. The P19 cell line is one of the better studied teratocarcinoma cell lines, being first introduced in 1982. The cells express the same transcription factors for pluripotency maintenance as embryonic stem cells but do not require specialized media or feeder cells. Dimethylsulfoxide induces P19 cells to smooth muscle cells and beating cardiomyocytes, whereas, retinoic acid causes P19 cells to differentia... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559965 Tue, 08 Mar 2011 22:36:49 +0100 4559965 http://www.medworm.com/index.php?rid=4559964&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_11 Signal transduction is a key process to transmit information from the extracellular milieu, and to elicit changes in the biological activity of target cells. Several cell signaling pathways can be targeted by neurotoxicants and developmental neurotoxicants. This chapter focuses on the interactions of ethanol, a known human developmental neurotoxicant, with signal transduction pathways stimulated by acetylcholine through activation of muscarinic receptors. It shows how initial observations in vivo, upon developmental exposure to ethanol, have been followed-up by a series of studies in cell culture systems which have allowed the discoveries that ethanol, by interfering with muscarinic signaling in astroglial cells, inhibits their proliferation and their ability to foster neuronal differentia... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559964 Tue, 08 Mar 2011 22:36:48 +0100 4559964 http://www.medworm.com/index.php?rid=4559963&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_12 Neurotoxicological testing is mainly based on experimental animal models, but several cell and tissue culture models have been developed to study the mechanisms of neurotoxicity. In general, cells of human origin are attractive alternatives to the animal models for extrapolation of toxicity on humans. The characteristics, culture conditions and usefulness of the human neuroblastoma cell line SH-SY5Y as a model for axonopathy are presented in this chapter. It is described how the cell line can be differentiated to mature neurons by using serum-free “N2” medium supplemented with retinoic acid. Detailed protocols for the determination of neurite degeneration and general cytotoxicity are presented. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559963 Tue, 08 Mar 2011 22:36:47 +0100 4559963 http://www.medworm.com/index.php?rid=4559962&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_13 A major goal of our cellular toxicology research has been the identification of novel targets of organophosphorous compounds (OPs), to which end we have studied the effects of sub-lethal concentrations of OPs on morphological and molecular end points in differentiating mammalian cell lines. This chapter describes the key practical approaches that we use to monitor OP toxicity in mouse N2a neuroblastoma and rat C6 glioma cell lines. These cell lines are easy to maintain and are useful for the study of short-term, potentially reversible, effects of OPs on the outgrowth and maintenance of neurites and associated regulatory proteins. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559962 Tue, 08 Mar 2011 22:36:47 +0100 4559962 http://www.medworm.com/index.php?rid=4559961&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_14 Cataract formation during preclinical drug safety assessment studies can be a devastating safety finding during drug development based on the stage at which these findings usually occur. The lens explant culture models offer an extremely versatile and simple in vitro model to screen compound for such toxic liabilities or to study possible mechanisms of toxicity. The following chapter is designed to highlight numerous examples of cataract formation during the drug development process, techniques used to prepare the model and some approaches used to understand drug-induced cataract formation in vitro from a mechanistic, screening and species sensitivity perspective. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559961 Tue, 08 Mar 2011 22:36:47 +0100 4559961 http://www.medworm.com/index.php?rid=4559960&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_15 Most neurodegenerative diseases culminate in cell death, although it is not uncommon for signs of dysfunction to precede cell death in humans and animal models. There is considerable evidence that neuronal and glial cell death during development occurs through apoptosis but whether apoptosis occurs during degeneration is still a contentious issue. Apoptosis is a well-defined process with key steps that mark the progress of the process in individual cells. Necrosis and autophagy as primary causes of death are less well defined. Two issues need particular clarification: (1) when is necrosis deemed to be the death mechanism rather than a secondary outcome of another death mechanism, and (2) whether the autophagic process, or its deficiency, causes death, or whether autophagy is a bystander (o... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559960 Tue, 08 Mar 2011 22:36:47 +0100 4559960 http://www.medworm.com/index.php?rid=4559959&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_16 Inflammation contributes to a wide variety of brain pathologies. In this chapter methods are described for using microglia and astrocytes in culture to investigate inflammatory processes and inflammatory neurodegeneration, including the use of neuronal/glial co-cultures and transwells. Methods for detecting and characterising inflammatory activation in culture include changes in microglial phenotype, microglial phagocytosis and expression of pro-inflammatory cytokines. The protocols for how to determine cellular production of superoxide, hydrogen peroxide, peroxynitrite and nitric oxide in culture are described. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559959 Tue, 08 Mar 2011 22:36:47 +0100 4559959 http://www.medworm.com/index.php?rid=4559958&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_17 Oxidative stress is implicated as one of the major underlying mechanisms in a variety of human diseases. Reactive radicals, derived primarily from molecular oxygen, readily attack a variety of critical biological molecules, including DNA, cellular proteins, and lipids. Since lipid peroxidation is a central feature of cerebral oxidant injury, measurements of peroxidation products, such as F2-isoprostanes (F2-IsoPs) and F4-neuroprostanes (F4-NeuroPs), represent the most accurate approaches to identify oxidative injury in vivo. Our laboratory uses a gas chromatography/mass spectrometry negative ion chemical ionization with select ion monitoring for quantification of these biomarkers of oxidative stress in a plethora of biological media. This review summarizes state-of-the-art methodology for ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559958 Tue, 08 Mar 2011 22:36:47 +0100 4559958 http://www.medworm.com/index.php?rid=4559957&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_18 Carbonyl group formation on protein side chains is a common biochemical marker of oxidative stress and is frequently observed in a variety of acute and chronic neurological diseases including stroke, Alzheimer’s disease, and Parkinson’s disease. Given that proteins are often the immediate targets of cellular oxidative stress, it is of utmost importance to determine how adductions by reactive electrophiles and other oxidative reactions can irreversibly alter protein structure and function. Previously, protein adduction was thought to be a random process, but recently it has become increasingly clear that these protein modifications are specific and selective. In this work, two methodological approaches are presented which allow for the detection of protein carbonyl groups. While... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559957 Tue, 08 Mar 2011 22:36:46 +0100 4559957 http://www.medworm.com/index.php?rid=4559956&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_19 The selective loss of melanin-containing dopaminergic neurons in the substantia nigra pars compacta and locus coeruleus has motivated a large number of preclinical studies aimed at understanding the molecular mechanisms involved in the neurodegeneration of dopaminergic neurons in Parkinson’s disease. To perform these preclinical studies with catecholaminergic neurotoxins such as 6-hydroxydopamine, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), rotenon, paraquat, aminochrome, etc. requires to select cell model that expresses dopaminergic features such as dopamine transport, formation of neuromelanin, release of dopamine, and monoamine oxidase. The most used model cell lines are PC12 and SH-SY5Y cell lines due to their catecholaminergic properties, but we review several other cel... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559956 Tue, 08 Mar 2011 22:36:46 +0100 4559956 http://www.medworm.com/index.php?rid=4559955&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_1 The use of various in vitro systems is expanding dramatically not only in basic research, but also to meet regulatory requirements for chemicals and products of various kinds. Further significant developments are certain to result from the use of in vitro systems for high-throughput screening in pharmacology and toxicology. Because the maintenance of high standards is fundamental to all good scientific practice and is essential for maximising the reproducibility, reliability, credibility, acceptance and proper application of any results produced, guidelines have been developed to define minimum standards in cell and tissue culture to be called as Good Cell Culture Practice (GCCP). The scope of this chapter has been broadly defined to include systems based on cells and tissues obtained from... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559955 Tue, 08 Mar 2011 22:36:46 +0100 4559955 http://www.medworm.com/index.php?rid=4559954&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_20 The use of 13C and 15N labeled precursors in combination with adequate analytical tools makes it possible to study metabolic pathways in cultured neural cells. The most commonly used precursors are 13C labeled glucose, lactate, glutamate and acetate. For a dynamic evaluation of intermediary metabolism of cell cultures, incubation with 13C containing substrates followed by nuclear magnetic resonance spectroscopy (NMRS) and mass spectrometry (MS) is excellent. NMRS can be used on cell extracts or living cells if a sufficient quantity of labeled atoms is present. MS is the more sensitive of the two methods but often it requires derivatization and separation of the components before analysis. The review provides descriptions of the basic and practical aspects of culturing neural cells, incubat... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559954 Tue, 08 Mar 2011 22:36:46 +0100 4559954 http://www.medworm.com/index.php?rid=4559953&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_21 Glutamine (Gln) plays an important role in satisfying brain metabolic demands and as a precursor for the synthesis of glutamate and ?-aminobutyric acid (GABA). In vitro cultured cell studies have shown that carrier-mediated Gln transport between astrocytes and neurons represents a key factor in the glutamate–GABA–glutamine cycle. Gln transport in astrocytes involves the following systems: sodium-dependent: system N; system ASC; system A and sodium-independent: system L, whereas in neurons only systems A and L are active. Gln-specific carriers primarily mediate not only inward transport, but can also largely contribute to outwardly transport. Therefore, both uptake and release studies are important for the investigation of Gln transport and metabolism. In this unit, methods are ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559953 Tue, 08 Mar 2011 22:36:45 +0100 4559953 http://www.medworm.com/index.php?rid=4559952&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_22 Excitatory and inhibitory neurotransmission mediated by glutamate and GABA, respectively, plays a major role in generation of seizures. So far, emphasis has been placed on the GABA system in attempts to develop antiepileptic drugs. Tiagabine, a selective inhibitor of GABA transporter 1 (GAT1), is marketed for treatment of certain seizure types and serves as a proof of principle that inhibitors of GABA transport may be interesting in this context. The chapter describes the methodology available to investigate in detail the pharmacology of GABA transporters and design of studies leading to identification of drug candidates. Emphasis is placed on a possible role of extrasynaptic GABA transporters in seizure control. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559952 Tue, 08 Mar 2011 22:36:45 +0100 4559952 http://www.medworm.com/index.php?rid=4559951&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_24 Neuronal networks cultured on microelectrode array (MEA) neurochips provide a testing platform for neuroactive compounds such as neurotoxicants and neuropharmaceuticals. Electrical network activity is recorded, quantified, characterized and classified at the level of spike and burst patterns, yielding concentration–response curves of a multitude of activity-describing parameters for monitoring the effects on defined network activity states. The system allows highly complex studies of cellular behavior which can give new insight into the molecular mechanisms of drug or toxicant action. The MEA neurochip technology is suitable to study network electrical activity maturation (weeks 1–4), the long-term functioning of the active networks (many months), and give a precise characteriz... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559951 Tue, 08 Mar 2011 22:36:45 +0100 4559951 http://www.medworm.com/index.php?rid=4559950&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_23 Studies of ion channel pharmacology have witnessed considerable developments during the past half a century. Whereas voltage clamp techniques were applied to neuropharmacology for the studies of some chemicals in the late 1950s, it was not until 1960s that cellular neuropharmacological studies started flourishing when tetrodotoxin was discovered to exert a selective and potent block of the sodium channels. The progress in this field was greatly accelerated when patch clamp techniques developed in the early 1980s. Incorporation of molecular biology into this area further promoted the development, and cellular neuropharmacology has now become one of the most important biomedical sciences. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559950 Tue, 08 Mar 2011 22:36:45 +0100 4559950 http://www.medworm.com/index.php?rid=4559949&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_25 GABAA receptor (GABAAR) constitutes the main inhibitory receptor of the central nervous system. Due to the wide distribution and activity of its main neurotransmitter agonist, the ?-aminobutyric acid (GABA), its pharmacology has been thoroughly studied, given rise to the development of numerous drugs and of neuroactive compounds, some of the latest inducing neurotoxic effects. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559949 Tue, 08 Mar 2011 22:36:45 +0100 4559949 http://www.medworm.com/index.php?rid=4559948&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_2 This chapter describes the materials and methods necessary to generate human induced pluripotent stem cells (iPSCs) from primary human fibroblasts and direct their differentiation into neural progenitor cells. Application of such methods is an emerging model for the study of neurotoxicity focused on human neurons and glia derived from specific patients. The techniques described here include primary human fibroblast culture, lentiviral/retroviral-mediated iPSC inductions, iPSC clonal expansion and maintenance, validation of pluripotency markers, and neuronal differentiation of iPSCs. Methods and applications using iPSCs are rapidly changing: here we describe the current methods used in our laboratories. The iPSC induction method featured in this chapter is based on a two-step viral transduc... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559948 Tue, 08 Mar 2011 22:36:44 +0100 4559948 http://www.medworm.com/index.php?rid=4559947&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_3 Cells can be defined as “stem cells” when able to self-renew and differentiate into tissue-characteristic cells. Neural stem cells (NSCs) derived from the nervous system are able to generate neuronal and glial cells and are present not only in the developing nervous system, but also in specific regions of the adult brain. While embryonic NSCs play an essential role in the development and maturation of the nervous system, adult NSCs may have a role in the normal functions of the brain, including learning, memory, and emotional responses. Growing evidence points to developmental exposure to chemicals as a possible cause of nervous system disorders. The developing nervous system is particularly vulnerable to toxic agents, which may cause long-lasting effects that may be manifested... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559947 Tue, 08 Mar 2011 22:36:44 +0100 4559947 http://www.medworm.com/index.php?rid=4559946&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_5 The nervous system is a frequent target of industrial chemicals, pharmaceuticals, and environmental pollutants. To screen large numbers of compounds for their neurotoxic potential, in vitro systems are required which combine organ-specific traits with robustness and high reproducibility. These requirements are met by serum-free aggregating brain cell cultures derived from mechanically dissociated embryonic rat brain. The initial cell suspension, composed of neural stem cells, neural progenitor cells, immature postmitotic neurons, glioblasts, and microglial cells, is kept under continuous gyratory agitation. Spherical aggregates form spontaneously and are maintained in suspension culture for several weeks. Within the aggregates, the cells rearrange and mature, reproducing critical morphogen... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559946 Tue, 08 Mar 2011 22:36:44 +0100 4559946 http://www.medworm.com/index.php?rid=4559945&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_4 Cell cultures are widely used in biomedical research. Primary cultures are directly obtained from fresh tissue and reproduce during days or weeks the major characteristics of the original tissue cells. Primary cell culture systems have shown their usefulness for studying the specific activities and the underlying mechanisms of a variety of test compounds. Brain primary cultures have become a powerful tool to analyze the toxic effects and mechanisms of potentially harmful agents in the different brain cell types. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559945 Tue, 08 Mar 2011 22:36:44 +0100 4559945 http://www.medworm.com/index.php?rid=4559944&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_6 Movement disorders such as Parkinson’s Disease (PD) result, in part, from the selective loss of dopaminergic (DA) neurons. Many studies associated with DA neurodegeneration and neurotoxicity have successfully applied the model organism Caenorhabditis elegans to address PD-related questions. However, some pharmacological studies might be limited because the thick cuticle of the intact animal could preclude complete penetration of some chemicals. It is also difficult to examine neurons within the same individual nematode, day after day, for studies of aging and neurodegeneration. More recently, methods for culturing embryonic cells from C. elegans have proved to be an alternative resource for addressing the caveats associated with whole nematode studies. In this regard, cultured embryo... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559944 Tue, 08 Mar 2011 22:36:43 +0100 4559944 http://www.medworm.com/index.php?rid=4559943&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_7 Located at the level of brain capillaries, the blood–brain barrier (BBB) is a crucial component of the neurovascular unit, since its highly regulated properties are needed to maintain optimal conditions for proper neuronal and glial functions. Therefore, understanding BBB features and behaviours in physiological and pathological conditions is a key issue in neurobiology. Since the BBB controls the exchanges between the blood and brain compartments, it also represents a major hurdle for molecules to reach the brain parenchyma. Given the localisation and complexity of the BBB in vivo, modelling the BBB in vitro can help to investigate cellular and molecular mechanisms, as well as evaluate the ability of compounds to cross the BBB. In this chapter, three in vitro BBB models will be desc... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559943 Tue, 08 Mar 2011 22:36:43 +0100 4559943 http://www.medworm.com/index.php?rid=4559942&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_8 The choroid plexus epithelium forms the interface between the blood and the cerebrospinal fluid. In addition to its barrier function resulting from the presence of tight junctions sealing the epithelial cells together, the choroid plexus epithelium fulfills vectorial transport (influx and efflux), neuroprotective, antioxidant and secretory functions, all relevant to different aspects of neurotoxicological sciences. To investigate these choroidal functions without the interference of the blood–brain barrier proper and brain parenchyma, in vitro cellular models of the blood–cerebrospinal fluid barrier, retaining the differentiated phenotype of the choroidal epithelium, have been established, taking advantage of the advent of refined culture methods and availability of permeable m... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559942 Tue, 08 Mar 2011 22:36:42 +0100 4559942 http://www.medworm.com/index.php?rid=4559941&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-077-5_9 The objective of this chapter is to provide methodological strategies for gene introduction specifically into cultured neuronal cells. This approach has been used to study the role of specific proteins in neurodegenerative and neuroprotective events, as well as in neurotransmission, antioxidant defenses, energetic metabolism, and several other physiological phenomena related to the neuronal homeostasis. The chapter starts with a description of the most important vectors currently available for neuronal transfections. A particular emphasis is directed at plasmid vectors, and a simple but useful protocol to isolate plasmids from bacteria is presented. This is followed by a discussion on the fundamentals of gene manipulation emphasizing the basics on how to isolate a DNA fragment, as well as ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4559941 Tue, 08 Mar 2011 22:36:41 +0100 4559941 http://www.medworm.com/index.php?rid=4544758&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_11 Postoperative pain control remains difficult because the current treatments have limited efficacy; many patients experience moderate to severe pain after a variety of surgeries. Recognizing the gap between preclinical models of persistent pain and postsurgical pain, we and others have been interested in trying to better understand the mechanisms of pain caused by incisions, through the development of animal models. Plantar incision is one animal model for human postoperative pain. Models using hairy skin incision, gastrocnemius incision, and tail incision, and models for thoracotomy and abdominal surgery are reviewed. Relevant behaviors in relation to clinical postoperative pain are examined. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544758 Wed, 20 Oct 2010 00:00:00 +0100 4544758 http://www.medworm.com/index.php?rid=4544757&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_10 Painful distal sensory neuropathy is the most common neurological complication of HIV1 infection. There are several neuropathic pain syndromes associated with the disease; however, the most common is a sensory neuropathy called HIV sensory neuropathy (HIV-SN). HIV-SN can be subdivided into subacute or chronic distal sensory polyneuropathy (DSP) and subacute antiretroviral-induced toxic neuropathy (ATN). Both forms involve sensory loss and neuropathic pain. DSP occurs in up to 7–35% of HIV1-infected individuals and upwards of 34% of children infected with HIV1, while ATN develops following highly active antiretroviral therapy (HAART) treatment in up to 52% of patients. The mechanisms of HIV-SN remain unclear; however, the advent of several models of HIV1-associated peripheral neuropat... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544757 Wed, 20 Oct 2010 00:00:00 +0100 4544757 http://www.medworm.com/index.php?rid=4544756&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_1 The assessment of pain is of critical importance for mechanistic studies as well as for the validation of drug targets. The study of pain in awake animals raises ethical, philosophical, and technical problems. Philosophically, there is the problem that pain cannot be monitored directly in animals but can only be estimated by examining their responses to nociceptive stimuli; however, such responses do not necessarily mean that there is a concomitant sensation. In this chapter, I highlight several types of nociceptive stimuli (thermal, mechanical, or chemical), which have been used in different pain models such as acute pain, chronic pain, arthritis pain, inflammatory, and visceral pain. The monitored reactions are almost always motor responses ranging from spinal reflexes to complex behavio... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544756 Wed, 20 Oct 2010 00:00:00 +0100 4544756 http://www.medworm.com/index.php?rid=4544755&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_2 Animal models of inflammatory pain have been widely used to study the mechanisms of tissue injury-induced persistent pain. A variety of inflammatory agents or irritants, including complete Freund’s adjuvant, carrageenan, zymosan, mustard oil, formalin, capsaicin, bee venom, acidic saline, lipopolysaccharide, inflammatory cytokines, and sodium urate crystals, have been used to produce tissue injury and hyperalgesia in such structures as cutaneous/subcutaneous tissues, joints, and muscles. Additionally, models of pain hypersensitivity have also been established with injuries produced by burning, freezing, and ultra irradiation. Although these models do not simulate every aspect of chronic pain, they do model key features of human inflammatory pain. Studies in animals give insight into ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544755 Wed, 20 Oct 2010 00:00:00 +0100 4544755 http://www.medworm.com/index.php?rid=4544754&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_3 The study of visceral pain is of high clinical relevance and the findings more directly translational in the search for analgesic agents. Early studies of nerve recordings after acute visceral nerve activation with (1) mechanical distension of hollow organs such as the colon or esophagus, (2) chemical irritation, and/or (3) inflammation have provided relevant information about normal, typically silent visceral afferents and evoked behavioral responses. Clinically relevant information about visceral pain has been reported in studies utilizing intact animal models of inflammatory, diabetic, neuropathic, cancer, chemotherapy-induced and other injury-related visceral pain conditions. More recently, animal models designed to study mechanisms signaling the transitional stages from acute to chron... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544754 Wed, 20 Oct 2010 00:00:00 +0100 4544754 http://www.medworm.com/index.php?rid=4544753&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_4 Chronic pain can originate from injury or dysfunction in the peripheral nervous system, and currently available therapeutic methods are often ineffective. Despite the clinical significance, the mechanisms underlying the development and maintenance of chronic pain after peripheral nerve injury are obscure. During the last three decades, a number of animal models have been developed to study the chronic pain after peripheral nerve injury. Some of these animal models have been widely used in both academic research and pharmaceutical industry. The employment of animal models has greatly promoted our knowledge of the underlying mechanisms, suggested and tested new treatment strategies for chronic pain. This chapter reviewed the most important and widely used animal models of pain after peripher... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544753 Wed, 20 Oct 2010 00:00:00 +0100 4544753 http://www.medworm.com/index.php?rid=4544752&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_6 In this chapter, we describe a newly developed rodent model of chemogenic pain involving the inflammation of one or two lumbar sensory ganglia using the immune activator, zymosan. Using this model, we have investigated cellular, molecular, and ionic mechanisms of inflammatory responses within the dorsal root ganglion (DRG) and their contribution to the development of chemogenic pathological pain. DRG inflammation was induced by a single deposit of zymosan in the epidural space near the L5 DRG via a small hole drilled through the transverse process. After a single zymosan injection, rats developed bilateral mechanical hyperalgesia and allodynia which began by day 1 after surgery, peaked at days 3–7, and lasted up to 28 days. Robust satellite glial activation was observed in inflamed g... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544752 Wed, 20 Oct 2010 00:00:00 +0100 4544752 http://www.medworm.com/index.php?rid=4544751&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_5 Injury to dorsal root ganglion and/or the dorsal roots can lead to neuropathic pain. This chapter provides an overview of animal models that mimic ganglion compression by the implantation of a metal rod in the lumbar intervertebral foramen (CCD model) and dorsal root injury by loose ligation of the roots (DRC model) or partial dorsal rhizotomy (PDR model). (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544751 Wed, 20 Oct 2010 00:00:00 +0100 4544751 http://www.medworm.com/index.php?rid=4544750&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_7 Central neuropathic pain is triggered by trauma, neurological disease, or infection of the central nervous system. A number of powerful models of central pain produce consistent phenotypes of behavioral hypersensitivity and provide an understanding into the mechanisms underlying persistent changes in nociceptive processing. Here we discuss behavioral phenomenology, how changes in neuronal firing properties lead to system-wide modulation of information processing, and highlight relevant animal models associated with spinal cord injury, multiple sclerosis, and infection – key contributors to central pain. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544750 Wed, 20 Oct 2010 00:00:00 +0100 4544750 http://www.medworm.com/index.php?rid=4544749&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_8 The incidence of cancer pain is high in patients with advanced disease as well as in patients undergoing active treatment for solid tumors. Further, modern cancer therapies have significantly increased survival rates, making effective pain control critical as unrelieved pain significantly decreases the quality of life of such patients. Thus, the goal of pain management is to not only alleviate pain, but also maintain the patient’s normal quality of life. To meet this challenge, novel analgesics with greater efficacy but fewer side effects are needed for alleviating cancer-induced pain. Recent advances in understanding the mechanism(s) of cancer pain have been assisted by the development of several rodent models that have shown that there are unique tumor-induced central and periphera... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544749 Wed, 20 Oct 2010 00:00:00 +0100 4544749 http://www.medworm.com/index.php?rid=4544748&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-880-5_9 Pain is frequently the earliest and most problematic syndrome of distal peripheral neuropathy (DPN) in diabetic patients. The variety of time of onset, duration and progression, modalities, and severity of individual presentations of painful DPN makes classification and evaluation of mechanisms of pain in patients with diabetic neuropathy an outstandingly difficult task. One critical step to address this issue is the need for large-scale prospective studies that start in pain-free prediabetic or diabetic subjects and use questionnaires and neurological bedside and quantitative sensory tests standardized to assess progression of all possible modalities and types of pain. By their nature, however, even the best equipped and designed clinical studies remain mostly observational, and in-depth ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=4544748 Wed, 20 Oct 2010 00:00:00 +0100 4544748 http://www.medworm.com/index.php?rid=3287512&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-541-5_9 Glaucoma is a relatively common disease in which the pathological death of retinal ganglion cells causes progressive losses of sight, often leading to blindness. The diagnosis of glaucoma and the assessment of progression are based on a clinical quantification of the ocular characteristics of cupping of the optic nerve head, a loss of retinal nerve fiber layer thickness, and associated functional vision defects. Consequently, clinical tests are based on the quantification of these clinical characteristics of glaucomatous optic neuropathy. However, the basic neural and cellular pathophysiology that cause the characteristic signs of glaucoma cannot be studied in clinical patients and, therefore, animal models must be employed for basic research on glaucomatous optic neuropathy. For basic res... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=3287512 Fri, 15 Jan 2010 00:00:00 +0100 3287512 http://www.medworm.com/index.php?rid=3287511&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-541-5_8 The generation and advancement of animal models have contributed significantly to the advancement of glaucoma research. This chapter describes and summarizes major nonprimate animal models useful for the study of this disease. Rodent models, both rats and mice, have been popular for glaucoma studies, because of the relatively better-developed genetic and genomic tools and the similarity of the relevant ocular structures between human and these animals. The larger animals, e.g., rabbit, feline, canine, bovine, ovine, and porcine models, have also been successfully used and provided valuable information on various aspects of the disease. Some of the models depicted in this chapter involve a transient or chronic ocular hypertension. Others do not affect intraocular pressure, but instead addre... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=3287511 Fri, 15 Jan 2010 00:00:00 +0100 3287511 http://www.medworm.com/index.php?rid=3287510&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-541-5_7 Diabetic retinopathy threatens vision in millions of patients in the USA. Prolonged hyperglycemia causes irreversible pathological changes in the retina, leading to proliferative diabetic retinopathy with preretinal neovascularization and diabetic macular edema. Much of the disease progression appears similar between man and animal. Thus, animal models are essential in understanding the pathology of this disease and development of effective treatments. This chapter describes and discusses the use of the rat, mouse, and dog in diabetic retinopathy studies. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=3287510 Fri, 15 Jan 2010 00:00:00 +0100 3287510 http://www.medworm.com/index.php?rid=3287509&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-541-5_6 Retinopathy of prematurity (ROP), a condition affecting premature infants, is characterized by pathological angiogenesis, or neovascularization (NV), of the retina. Much of what is known about the development of the retinal vasculature and the progression of ROP has been learned through the use of animal models of oxygen-induced retinopathy (OIR), which approximate the human condition. Animal models of OIR have provided a wealth of information regarding the cellular and molecular pathogenesis of ROP. Moreover, this information has contributed to a better understanding of other, nonocular, neovascular conditions. This chapter describes the various animal models of OIR, and explores their contributions to the understanding and treatment of ROP. (Source: Springer protocols feed by Neuroscienc... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=3287509 Fri, 15 Jan 2010 00:00:00 +0100 3287509 http://www.medworm.com/index.php?rid=3287508&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-541-5_5 Age-related macular degeneration (AMD) has a number of characteristic features including late onset and accumulation of deposits (drusen) below the retinal pigment epithelium on Bruch’s membrane in the macula. A progressive increase in these deposits (in some individuals) leads to macular blindness, following either the local loss of the retinal pigment epithelium (geographic atrophy) or the hemorrhage of new blood vessels that originate in the choroid and invade the compartment between the photoreceptors and retinal pigment epithelium (choroidal neovascularization). Over the last few years a number of mouse models for AMD have been described that replicate some of the changes manifest in the human disease. This chapter begins with a description of the hallmarks of AMD, discusses som... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=3287508 Fri, 15 Jan 2010 00:00:00 +0100 3287508 http://www.medworm.com/index.php?rid=3287507&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-541-5_4 Retinal degeneration is often used to describe a category of human eye diseases, which are characterized by photoreceptor loss leading to severe visual impairment and blindness. An important, yet heterogeneous group of such diseases is called Retinitis Pigmentosa (RP). To understand the molecular mechanisms of disease induction and progression and to develop therapeutical strategies for the preservation of vision in RP patients, appropriate animal models are used in many research laboratories worldwide. The largest category of models consists of mutant (spontaneous and genetically engineered) mice. However, in recent years, zebrafish has been established as a highly valuable tool for the study of various biological problems, including retinal degeneration. In this review, we summarize the ... Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=3287507 Fri, 15 Jan 2010 00:00:00 +0100 3287507 http://www.medworm.com/index.php?rid=3287506&cid=s_37128_168_f&fid=37128&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-541-5_3 Here we review both established and emerging approaches for studying retinal diseases. We primarily focus on the use of the mouse as a genetic model, as it is a mammalian model with many resources and is amenable to a variety of genetic manipulations. Additionally, we highlight two other organisms, zebrafish and fruit fly that are emerging as valuable genetic tools to study retinal disease. We discuss the ways in which near-complete genome sequences of these three organisms are revolutionizing our ability to investigate the complex mechanisms involved in retinal diseases. (Source: Springer protocols feed by Neuroscience) Springer protocols feed by Neuroscience news http://www.medworm.com/rss/comments.php?id=3287506 Fri, 15 Jan 2010 00:00:00 +0100 3287506