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  • 1
    Keywords: Medicine ; Neurosciences ; Neurobiology ; Biology / Technique ; Biomedicine ; Neurosciences ; Neurobiology ; Biological Techniques ; Springer eBooks
    Description / Table of Contents: Preface -- Production of high-quality antibodies for the study of receptors and ion channels in brain -- Co-immunoprecipitation from brain -- Sub-synaptic membrane fractionation -- Investigation of ℗ neurotransmitter receptors in brain slices using cell surface biotinylation -- Single nanoparticle tracking of surface ion channels and receptors in brain cells -- Radioligand binding detection of receptors in brain membranes -- Recombinant Alphavirus-Mediated Expression of Ion Channels and Receptors in the Brain -- Fluorescent ligands and TR-FRET to study receptor-receptor interactions in the brain -- In situ Proximity Ligation Assay to study and understand the distribution and balance of GPCR homo- and heteroreceptor complexes in the brain -- Fluorescent in situ hybridization for sensitive and specific labeling -- Autoradiographic visualization of G protein-coupled receptors in brain -- Analysis of the expression profile and regional distribution of neurotransmitter receptors and ion channels in the central nervous system using histoblots -- Immunohistochemistry for ion channels and their interacting molecules: Tips for improving antibody accessibility -- Localization of GFP-tagged proteins at the electron microscope -- Pre-embedding methods in the localization of receptors and ion channels -- Post-embedding immunohistochemistry in the localization of receptors and ion channels -- High-Resolution localization of membrane proteins by SDS-digested freeze-fracture replica labelling (SDS-FRL) -- Application of virus vectors for anterograde tract-tracing and single-neuron labeling studies -- Analysis of synaptic connections using viral vectors at the electron microscopic level -- Morphological and neurochemical characterization of electrophysiologically identified cells -- Using electrophysiology to study synaptic and extrasynaptic ionotropic receptors in hippocampal neurons -- Biophysical methods to analyze direct G-protein regulation of neuronal voltage-gated calcium channels -- Electrophysiological recordings in behaving animals -- Voltammetry in behaving animals -- In vivo brain microdialysis of monoamines -- Calcium transients in single dendrites and spines of pyramidal neurons in vitro -- Dynamic recording of membrane potential from hippocampal neurons by using a FRET-based voltage biosensor -- Determination of GPCR-mediated cAMP accumulation in rat striatal synaptosomes -- GPCR-mediated MAPK/ERK cascade activation in brain slices -- Index
    Abstract: Receptor and Ion Channel Detection in the Brain provides state-of-the-art and up-to-date methodological information on molecular, neuroanatomical and functional techniques that are currently used to study neurotransmitter receptors and ion channels in the brain. The chapters have been contributed by world-wide recognized neuroscientists who explain in an easy and detailed way well established and tested protocols embracing molecular, cellular, subcellular, anatomical and electrophysiological aspects of the brain. This comprehensive and practical manual is presented in a simple, step-by-step manner for laboratory use, and also offers unambiguous detail and key implementation advice that proves essential for successful results and facilitate choosing the best method for the target proteins under study. This work serves as a useful guide for young researchers and students in training as well as for neurologists and established scientists who wish to extend their repertoire of techniques
    Pages: XVIII, 475 p. 84 illus., 57 illus. in color. : online resource.
    ISBN: 9781493930647
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  • 2
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are involved in the control of neuronal excitability and plasticity. In this study, we used immunoblotting and immunohistochemical techniques to reveal the developmental expression and subcellular distribution of the HCN1 subunit in the cerebellar cortex. During postnatal development, the spatio-temporal expression of HCN1 correlated well with the morphological events occurring during the ontogenesis of cerebellar interneurons. Using immunoblotting techniques, HCN1 was weakly detected during the first postnatal week and continued to increase throughout postnatal development, peaking at postnatal day (P)15. At the light-microscopic level, HCN1 immunoreactivity was very weak until P7 whereas from P10–12 to adulthood it was strongly detected in the lower third of the molecular layer and in the Purkinje cell layer. HCN1 was present in axons running through the molecular layer and in the pericellular basket around Purkinje cells at P12, but in the periaxonal plexus (the pinceau) surrounding their initial segment only after P15. Using immunofluorescence, HCN1 colocalized with GAD65 and synaptophysin, demonstrating that the subunit was present in inhibitory axons and axon terminals. At the electron-microscopic level, in adulthood, HCN1 immunoparticles were detected at postsynaptic sites in basket and Purkinje cells but most immunoparticles were found at presynaptic sites in basket cell axons and in terminals. In the axon terminals, the distribution of HCN1 was relatively uniform along the extrasynaptic plasma membrane; this was confirmed using quantitative techniques. The present findings suggest that HCN1 channels may provide a significant route for modulating co-ordinated cerebellar synaptic transmission through basket cells.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Among various types of low- and high-threshold calcium channels, the high voltage-activated P/Q-type channel is the most abundant in the cerebellum. These P/Q-type channels are involved in the regulation of neurotransmitter release and in the integration of dendritic inputs. We used an antibody specific for the α1A subunit of the P/Q-type channel in quantitative pre-embedding immunogold labelling combined with three-dimensional reconstruction to reveal the subcellular distribution of pre- and postsynaptic P/Q-type channels in the rat cerebellum. At the light microscopic level, immunoreactivity for the α1A protein was prevalent in the molecular layer, whereas immunostaining was moderate in the somata of Purkinje cells and weak in the granule cell layer. At the electron microscopic level, the most intense immunoreactivity for the α1A subunit was found in the presynaptic active zone of parallel fibre varicosities. The dendritic spines of Purkinje cells were also strongly labelled with the highest density of immunoparticles detected within 180 nm from the edge of the asymmetrical parallel fibre–Purkinje cell synapses. By contrast, the immunolabelling was sparse in climbing fibre varicosities and axon terminals of GABAergic cells, and weak and diffuse in dendritic shafts of Purkinje cells. The association of the α1A subunit with the glutamatergic parallel fibre–Purkinje cell synapses suggests that presynaptic channels have a major role in the mediation of excitatory neurotransmission, whereas postsynaptic channels are likely to be involved in depolarization-induced generation of local calcium transients in Purkinje cells.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Voltage-dependent ion channels have specific patterns of distribution along the neuronal plasma membrane of dendrites, cell bodies and axons, which need to be unravelled in order to understand their contribution to neuronal excitability and firing patterns. We have investigated the subcellular compartmentalization of Kv1.4, a transient, fast-inactivating potassium channel, in fusiform cells and related interneurons of the rat dorsal cochlear nucleus. A polyclonal antibody which binds to a region near the N-terminus domain of a Kv1.4 channel was raised in rabbits. Using a high-resolution combination of immunocytochemical methods, Kv1.4 was localized mainly in the apical dendritic trunks and cell bodies of fusiform cells, as well as in dendrites and cell bodies of interneurons of the dorsal cochlear nucleus, likely cartwheel cells. Quantitative immunogold immunocytochemistry revealed a pronounced distal to proximal gradient in the dendrosomatic distribution of Kv1.4. In plasma membrane localizations, Kv1.4 was preferentially present in dendritic spines, either in the spine neck or in perisynaptic locations, always away from the postsynaptic density. These findings indicate that Kv1.4 is largely distributed in dendritic compartments of fusiform and cartwheel cells of the dorsal cochlear nucleus. Its preferential localization in dendritic spines, where granule cell axons make powerful excitatory synapses, suggests a role for this voltage-dependent ion channel in the regulation of dendritic excitability and excitatory inputs.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Transient pioneer neurons in the neocortical marginal zone generate an early corticofugal axonal projection at E12–E16 (Meyer et al. 1998). We have analysed the functional activity of glutamate and GABA receptors in such cells by measuring changes in intracellular calcium concentrations ([Ca2+]i). The activation of GABAA receptors with muscimol, as well as bath application of glutamate, lead to increases in [Ca2+]i in pioneer neurons. The stimulatory action of glutamate is mostly produced through the NMDA-type of ionotropic receptors. Metabotropic glutamate receptor activation has no effect on [Ca2+]i. Consistent with such results, immunocytochemical studies showed a prominent expression of GABAA and NMDA receptors in pioneer neurons. The activation of such receptors may be implicated in the remodelling of pioneer neurons during development.
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  • 6
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Ionotropic and metabotropic (mGluR1a) glutamate receptors were reported to be segregated from each other within the postsynaptic membrane at individual synapses. In order to establish whether this pattern of distribution applies to the hippocampal principal cells and to other postsynaptic metabotropic glutamate receptors, the mGluR1a/b/c and mGluR5 subtypes were localized by immunocytochemistry. Principal cells in all hippocampal fields were reactive for mGluR5, the strata oriens and radiatum of the CA1 area being most strongly immunolabelled. Labelling for mGluR1b/c was strongest on some pyramids in the CA3 area, weaker on granule cells and absent on CA1 pyramids. Subpopulations of non-principal cells showed strong mGluR1 or mGluR5 immunoreactivity. Electron microscopic pre-embedding immunoperoxidase and both pre- and postembedding immunogold methods consistently revealed the extrasynaptic location of both mGluRs in the somatic and dendritic membrane of pyramidal and granule cells. The density of immunolabelling was highest on dendritic spines. At synapses, immunoparticles for both mGluR1 and mGluR5 were found always outside the postsynaptic membrane specializations. Receptors were particularly concentrated in a perisynaptic annulus around type I synaptic junctions, including the invaginations at ‘perforated’synapses. Measurements of immunolabelling on dendritic spines showed decreasing levels of receptor as a function of distance from the edge of the synaptic specialization. We propose that glutamatergic synapses with an irregular edge develop in order to increase the circumference of synaptic junctions leading to an increase in the metabotropic to ionotropic glutamate receptor ratio at glutamate release sites. The perisynaptic position of postsynaptic metabotropic glutamate receptors appears to be a general feature of glutamatergic synaptic organization and may apply to other G-protein-coupled receptors.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The release of neurotransmitters is modulated by presynaptic metabotropic glutamate receptors (mGluRs), which show a highly selective expression and subcellular location in glutamatergic terminals in the hippocampus. Using immunocytochemistry, we investigated whether one of the receptors, mGluR7, whose level of expression is governed by the postsynaptic target, was present in GABAergic terminals and whether such terminals targeted particular cells. A total of 165 interneuron dendritic profiles receiving 466 synapses (82% mGluR7a-positive) were analysed. The presynaptic active zones of most GAD-(77%) or GABA-positive (94%) synaptic boutons on interneurons innervated by mGluR7a-enriched glutamatergic terminals (mGluR7a-decorated) were immunopositive for mGluR7a. GABAergic terminals on pyramidal cells and most other interneurons in str. oriens were mGluR7a-immunonegative. The mGluR7a-decorated cells were mostly somatostatin- and mGluR1α-immunopositive neurons in str. oriens and the alveus. Their GABAergic input mainly originated from VIP-positive terminals, 90% of which expressed high levels of mGluR7a in the presynaptic active zone. Parvalbumin-positive synaptic terminals were rare on mGluR7a-decorated cells, but on these neurons 73% of them were mGluR7a-immunopositive. Some type II synapses innervating interneurons were immunopositive for mGluR7b, as were some type I synapses. Because not all target cells of VIP-positive neurons are known it has not been possible to determine whether mGluR7 is expressed in a target-cell-specific manner in the terminals of single GABAergic cells. The activation of mGluR7 may decrease GABA release to mGluR7-decorated cells at times of high pyramidal cell activity, which elevates extracellular glutamate levels. Alternatively, the presynaptic receptor may be activated by as yet unidentified endogenous ligands released by the GABAergic terminals or the postsynaptic dendrites.
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  • 8
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Glutamate receptors have been linked to the regulation of several developmental events in the CNS. By using cortical slices of early postnatal mice, we show that in layer I cells, glutamate produces intracellular calcium ([Ca2+]i) elevations mediated by ionotropic and metabotropic glutamate receptors (mGluRs). The contribution of mGluRs to these responses was demonstrated by application of tACPD, an agonist to groups I and II mGluRs, which evoked [Ca2+]i increases that could be reversibly blocked by MCPG, an antagonist to groups I and II mGluRs. In the absence of extracellular Ca2+, repetitive applications of tACPD or quisqualate, an agonist to group I mGluRs, elicited decreasing [Ca2+]i responses that were restored by refilling a thapsigargin-sensitive Ca2+ store. The use of specific group I mGluR agonists CHPG and DHPG indicated that the functional mGluR in layer I was of the mGluR1 subtype. Subtype specific antibodies confirmed the presence of mGlur1α, but not mGluR5, in Cajal-Retzius (Reelin-immunoreactive) neurons.
    Type of Medium: Electronic Resource
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