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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Several metabotropic glutamate receptor (mGluR) subtypes have been identified in the cerebellar cortex that are targeted to different compartments in cerebellar cells. In this study, preembedding immunocytochemical methods for electron microscopy were used to investigate the subcellular distribution of the mGluR1b splice variant in the rat cerebellar cortex. Dendritic spines of Purkinje cells receiving parallel fiber synaptic terminals were immunoreactive for mGluR1b. With a preembedding immunogold method, ~25% of the mGluR1b immunolabeling was observed perisynaptically within 60 nm from the edge of the postsynaptic densities. Values of extrasynaptic gold particles beyond the first 60 nm were maintained at between 10 and 18% along the whole intracellular surface of the dentritic spine membranes of Purkinje cells. For comparison, the distribution of mGluR1a was studied. A predominant (~37%) perisynaptic localization of mGluR1a was seen in dendritic spines of Purkinje cells, dropping the extrasynaptic labeling to 15% in the 60-120-nm bin from the edge of the postsynaptic specialization. Our results reveal that mGluR1b and mGluR1a are localized to the same subcellular compartments in Purkinje cells but that the densities of the perisynaptic and extrasynaptic pools were different for both isoforms. The compartmentalization of mGluR1b and mGluR1a might serve distinct requirements in cerebellar neurotransmission.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Journal of medicinal chemistry 38 (1995), S. 1417-1426 
    ISSN: 1520-4804
    Source: ACS Legacy Archives
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We investigated the effects of oxygen (O2)/glucose deprivation on intracellular sodium concentration ([Na+]i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O2/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na+]i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists ( d-amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na+/Ca2+-exchanger, affected these responses to O2/glucose. The present results demonstrate that, in cortical neurons, O2/glucose deprivation induces an early rise in [Na+]i which cannot be ascribed to the activity of voltage gated Na+-channels, glutamate receptors or of the Na+/Ca2+-exchanger.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: A depolarization-induced, slowly decaying inward current was examined in slice-cultured CA3 pyramidal cells by voltage-clamp techniques and microfluorometric measurements of cytosolic free Ca2+ concentration ([Ca2+]i). Action potentials elicited by intracellular injection of short-lasting (50 – 100 ms) depolarizing current pulses were followed by a slowly decaying afterhyperpolarization (AHP). After switching to voltage-clamp mode, short-lasting (50 – 100 ms) depolarizing voltage jumps from –60 mV to between –30 and 0 mV induced a slowly decaying outward aftercurrent (IAHP) which was depressed by bath application of muscarine (0.5 μM). In the presence of muscarine, the same depolarizations induced a slowly decaying afterdepolarization (ADP) or inward aftercurrent (IADP)in voltage-clamp mode. This current was also induced in the presence of trans(±)-1-aminc-1,3-cyclopenta-nedicarboxylic acid (t-ACPD, 5 μM), an agonist of metabotropic glutamate receptors, but not in the presence of noradrenalin (5 μM), while both of these agonists depressed IAHP. IADP was depressed by reducing the external Ca2+ concentration from 3.8 to 0.5 mM, by external Co2+ (1 mM) and by external Cd2+ (10 – 100 μM). Combined voltage-clamp recordings and microfluorometric measurements of [Ca2+]i using the Ca2+ indicator fura-2 revealed that the amplitude of IADP was correlated with the amplitude of depolarization-induced Ca2+ influx, IADP was absent at membrane potentials 〈 –90 mV, and reached maximal amplitudes at ∼–55 mV. Raising the extracellular K+ concentration from 2.7 to 13.5 mM increased the amplitude of IADP and resulted in a positively directed shift of the apparent reversal potential of IADP. When the external Na+ concentration was reduced from 157 to 33 or 18 mM the current reversed at more negative potentials and was reduced to 40 and 21%, respectively, of control amplitude. Lowering the external Cl- concentration from 159 to 20 mM did not affect IADP. We conclude that IADP most likely represents a Ca2+-activated cation current, rather than a Ca2+ tail current, or an electrogenic Ca2+ extrusion current.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Genetically encoded fluorescent Ca2+ indicator proteins (FCIPs) are promising tools to study Ca2+ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well-defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5′ regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca2+ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation-induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse-induced presynaptic Ca2+ transients of parallel fibers are not affected by blockade of N-methyl-d-aspartate receptors.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    European journal of neuroscience 13 (2001), S. 0 
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Optical imaging of electrical activity has been suggested as a promising approach to investigate the multineuronal representation of information processing in brain tissue. While considerable progress has been made in the development of instrumentation suitable for high-speed imaging, intrinsic or extrinsic dye-mediated optical signals are often of limited use due to their slow response dynamics, low effective sensitivity, toxicity or undefined cellular origin. Protein-based and DNA-encoded voltage sensors could overcome these limitations. Here we report the design and generation of a voltage-sensitive fluorescent protein (VSFP) consisting of a voltage sensing domain of a potassium channel and a pair of cyan and yellow emitting mutants of green fluorescent protein (GFP). In response to a change in transmembrane voltage, the voltage sensor alters the amount of fluorescence resonance energy transfer (FRET) between the pair of GFP mutants. The optical signals respond in the millisecond time-scale of fast electrical signalling and are large enough to allow monitoring of voltage changes at the single cell level.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Double-mutant mice (DKO) lacking the two voltage-gated K+ channels Kv3.1 and Kv3.3 display a series of phenotypic alterations that include ataxia, myoclonus, tremor and alcohol hypersensitivity. The prominent cerebellar expression of mRNAs encoding Kv3.1 and Kv3.3 subunits raised the question as to whether altered electrical activity resulting from the lack of these K+ channels might be related to the dramatic motor changes. We used the tremorogenic agent harmaline to probe mutant mice lacking different K+ channel alleles for altered olivocerebellar circuit properties. Harmaline induced the characteristic 13-Hz tremor in wildtype mice (WT); however, no tremor was observed in DKO suggesting that the ensemble properties of the olivocerebellar circuitry are altered in the absence of Kv3.1 and Kv3.3 subunits. Harmaline induced tremor in Kv3.1-single mutants, but it was of smaller amplitude and at a lower frequency indicating the participation of Kv3.1 subunits in normal olivocerebellar system function. In contrast, harmaline tremor was virtually absent in Kv3.3-single mutants indicating an essential role for Kv3.3 subunits in tremor induction by harmaline. Immunohistochemical staining for Kv3.3 showed clear expression in the somata and proximal dendrites of Purkinje cells and in their axonal projections to the deep cerebellar nuclei (DCN). In DCN, both Kv3.1 and Kv3.3 subunits are expressed. Action potential duration is increased by ≈ 100% in Purkinje cells from Kv3.3-single mutants compared to WT or Kv3.1-single mutants. We conclude that Kv3.3 channel subunits are essential for the olivocerebellar system to generate and sustain normal harmaline tremor whereas Kv3.1 subunits influence tremor amplitude and frequency.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The homeostasis of intracellular Cl− concentration ([Cl−]i) is critical for neuronal function, including γ-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl−]i using a transgenetically expressed Cl−-sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 µm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl−-free solution, demonstrating that the EYFP signal represented an increase in [Cl−]i. Similar to glutamate, a rise in [Cl−]i was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K+], indicating that an increase in driving force for Cl− suffices to increase [Cl−]i. To elucidate the membrane mechanisms mediating the Cl− influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl−]i was resistant to furosemide, bumetanide and 4,4′-diisothiocyanato-stilbene-2,2′-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl−]i via several pathways involving NFA- and NPPB-sensitive anion channels and GABAA receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl− transporters. The present study highlights the vulnerability of [Cl−]i homeostasis after membrane depolarization in neurons.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl–, or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl– sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl– concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Pre-embedding immunogold histochemistry was combined with Phaseolus vulgaris leucoagglutinin anterograde tract tracing in order to analyse the relationship between the subcellular localization of the mGluR1a metabotropic glutamate receptors and the distribution of corticothalamic synapses in the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior nucleus (LP) of the rat. The injection of the tracer into area 17 labelled two types of corticothalamic terminals: (i) the small boutons constituting the majority of the labelled fibres which form asymmetrical synapses both in the dLGN and LP; and (ii) the giant terminals typically participating in glomerulus-like synaptic arrangements and found exclusively in the lateral posterior nucleus. The small corticothalamic terminals often established synapses with mGluR1a-immunopositive dendrites, with immunometal particles concentrated at the periphery of their postsynaptic membranes. In contrast, the synapses formed by giant boutons in the lateral posterior nucleus were always mGluR1a-immunonegative. We conclude that the corticothalamic fibres forming the small synaptic terminals are the most likely candidates for the postulated mGluR-mediated modulation of visual information flow by corticothalamic feedback mechanisms.
    Type of Medium: Electronic Resource
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