bluuebunny/crossref_metadata_2025_split huggingface dataset:
Localization of nAChR subunit mRNAs in the brain of Macaca mulatta
Authors: Han Zhi‐Yan , Le Novère Nicolas , Zoli Michele , Hill Joseph A. , Champtiaux Nicolas , Changeux Jean‐Pierre 
Published: March 2003

AbstractWe present here a systematic mapping of nAChR subunit mRNAs in Macaca mulatta brain. A fragment, from the transmembrane segments MIII to MIV of Macaca neuronal nAChR subunits was cloned, and shown to exhibit high identity (around 95%) to the corresponding human subunits. Then, specific oligodeoxynucleotides were synthesized for in situ hybridization experiments. Both α4 and β2 mRNA signals were widely distributed in the brain, being stronger in the thalamus and in the dopaminergic cells of the mesencephalon. Most brain nuclei displayed both α4 and β2 signals with the exception of some basal ganglia regions and the reticular thalamic nucleus which were devoid of α4 signal. α6 and β3 mRNA signals were selectively concentrated in the substantia nigra and the medial habenula. The strongest signals for α3 or β4 mRNAs were found in the epithalamus (medial habenula and pineal gland), whereas there were no specific α3 or β4 signals in mesencephalic dopaminergic nuclei. α5 and α7 mRNA signals were found in several brain areas, including cerebral cortex, thalamus and substantia nigra, although at a lower level than α4 and β2. The distribution of α3, α4, α5, α6, α7, β2, β3 and β4 subunit mRNAs in the monkey is substantially similar to that observed in rodent brain. Surprisingly, α2 mRNA signal was largely distributed in the Macaca brain, at levels comparable with those of α4 and β2. This observation represents the main difference between rodent and Macaca subunit mRNA distribution and suggests that, besides α4β2*, α2β2* nAChRs constitute a main nAChR isoform in primate brain.
Induction of VEGF and VEGF receptors in the spinal cord after mechanical spinal injury and prostaglandin administration
Authors: Sköld Mattias , Cullheim Staffan , Hammarberg Henrik , Piehl Fredrik , Suneson Anders , Lake Staffan , Sjögren AnnMargret , Walum Erik , Risling Mårten 
Published: March 2003

AbstractVascular endothelial growth factor (VEGF) is an angiogenetic factor that promotes endothelial cell proliferation during development and after injury to various types of tissue, including the central nervous system (CNS). Using immunohistochemical and in situ hybridization methods we have here demonstrated that VEGF and its receptors Flk‐1, Flt‐1 and Neuropilin‐1 mRNAs and proteins are induced after incisions in the rat spinal cord. The inducible enzyme for prostaglandin synthesis cyclooxygenase‐2 (COX‐2) is known to be upregulated after spinal injury, cerebral ischemia and to stimulate angiogenesis. To test the hypothesis that prostaglandins may be involved in the VEGF response after lesion we investigated whether intraspinal microinjections of prostaglandin F2α (PGF2α) alters VEGF expression in the spinal cord. Such treatment was followed by a strong upregulation of VEGF mRNA and protein in the injection area. Finally, by use of an in vitro model with cell cultures of meningeal fibroblast and astrocyte origin, resembling the lesion area cellular content after spinal cord injury but devoid of inflammatory cells, we showed that VEGF is expressed in this in vitro model cell system after treatment with PGF2α and prostaglandin E2 (PGE2). These data suggest that cells within a lesion area in the spinal cord are capable of expressing VEGF and its receptors in response to mechanical injury and that prostaglandins may induce VEGF expression in such cells, even in the absence of inflammatory cells.
Transient changes in cortical distribution of S100 proteins during reorganization of somatotopy in the primary motor cortex induced by facial nerve transection in adult rats
Authors: Landgrebe Michael , Laskawi Rainer , Wolff Joachim R. 
Published: March 2003

AbstractIn adult rats, the primary motor cortex (MI) comprises a somatotopic map of muscle representations. This somatotopy is modified after transection of the facial nerve (N7x). Mapping with cortical stimulation revealed that the underlying cortical reorganization is biphasic. Primary changes cause a transient disinhibition of long cortico‐cortical connections in both hemispheres. While the first reaction vanishes within a few hours, short intra‐areal connections are disinhibited within MI contralateral to N7x. The resulting co‐operation between adjacent parts of MI persists as long as peripheral reinnervation is prevented. Cellular mechanisms underlying this cortical reorganization are largely unknown. Here, we utilized changes in immunoreactivity of S100 proteins (S100‐IR) known as a sensitive indicator of astroglial reactions during plastic reactions in the central nervous system. Within 1 h of N7x, zones with enhanced S100‐IR appeared in both hemispheres. Between 3.5 and 18 h, reaction patterns with changing topography were transiently prominent in many cortical areas including parts of MI which surrounded the facial muscle representation fields. After 24 h, the facial muscle representation contralateral to N7x became labelled while S100‐IR enhancement disappeared in most of the cortex. S100‐IR‐enhancement vanished completely during the next day of survival. Data presented suggest that (i) enhancement of S100‐IR labels cortical tissue during the functional reorganization that is induced by N7x, (ii) large parts of the cerebral cortex participate in the reorganization, before it is finally focused on the representation field of MI that corresponds with contralateral N7x, and (iii) temporo‐spatial patterns of astrocytic reactions apparently play a role in the underlying plasticity reaction.
Age‐dependent pre‐ and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain
Authors: Fabian‐Fine Ruth , Volknandt Walter , Fine Alan , Stewart Michael G. 
Published: March 2003

AbstractOrganotypic slice cultures of rat hippocampus are widely used as experimental preparations for the study of synaptic plasticity, but their degree of correspondence with intact brain is not fully known. Here, using postembedding immunogold labelling, we describe the ultrastructural distribution of AMPA‐type glutamate receptors (GluR1–4) in CA3 stratum radiatum of organotypic hippocampal slice cultures at 10 days to 11 weeks in vitro and compare the labelling with intact brain of corresponding age. In both types of preparation, the 11‐week‐old samples contained the highest proportion of AMPA receptor‐like immunoreactive synapses. The incidence of labelled synapses, however, was higher in vivo (49%) than in vitro (24%). The intensity of labelling (number of gold particles per labelled synapse) also increased with age and was also higher in vivo than in vitro. In both organotypic cultures and intact brain, labelling was frequently found at presynaptic sites, often attached to vesicular structures. The specificity of these findings was supported both by light microscopic immunolabelling of GluR2/3 subunits and by electron microscopic double labelling of different epitopes of the GluR2 subunit. The vesicular localization of AMPA receptors was supported by Western blot analysis of subcellular fractions. Morphological evidence of presynaptic excitatory innervation of glutamatergic neurons supports a functional role for presynaptically located AMPA receptors. Our results therefore suggest that AMPA receptors occur in both pre‐ and postsynaptic profiles and that the distribution of AMPA receptors in cultured brain slices is fundamentally similar to intact brain, but that synaptic maturation may be retarded in vitro.
Two splice variants of the hypoxia‐inducible factor HIF‐1α as potential dimerization partners of ARNT2 in neurons
Authors: Drutel Guillaume , Kathmann Markus , Héron Anne , Gros Claude , Macé Séverine , Schwartz Jean‐Charles , Arrang Jean‐Michel 
Published: March 2003

AbstractThe hypoxia‐inducible factor (HIF‐1α), a basic helix‐loop‐helix transcription factor, is known to heterodimerize with ARNT1, a nuclear translocator, to trigger the overexpression in many cells of genes involved in resistance to hypoxia. Although HIF‐1α and ARNT1 are both expressed in brain, their cellular localization and function therein are unknown. Here, using in situ hybridization and immunocytochemistry, we show that HIF‐1α is expressed in normoxic cerebral neurons together with not only ARNT1 but also ARNT2, a cerebral translocator homologous to ARNT1 but displaying, unlike ARNT1, a selective neuronal expression. In contrast, other potential partners of the translocators, i.e. the aryl hydrocarbon receptor (AHR) and the single‐minded protein 2 (SIM2), are not expressed in the adult brain. We also identify two splice variants of HIF‐1α in brain, one of which dimerizes with ARNT2 even more avidly than with ARNT1. The resulting heterodimer, in contrast with the HIF‐1α/ARNT1 complex, does not recognize the HIF‐1‐binding site of the hypoxia‐induced erythropoietin (Epo) gene, suggesting that it controls transcription of a distinct set of genes. We therefore propose that HIF‐1α and ARNT2 function as preferential dimerization partners in neurons to control specific responses, some of which may not be triggered by hypoxia. In support of this proposal, in nonhypoxic PC12 cells constitutively coexpressing HIF‐1α, ARNT1 and ARNT2, downregulation of either HIF‐1α or ARNT2, obtained with selective antisense nucleotides, resulted in inhibition of [3H]thymidine incorporation.
Modulation of histamine H3 receptors in the brain of 6‐hydroxydopamine‐lesioned rats
Authors: Anichtchik Oleg V. , Huotari Marko , Peitsaro Nina , Haycock John W. , Männistö Pekka T. , Panula Pertti 
Published: March 2003

AbstractParkinson's disease is a major neurological disorder that primarily affects the nigral dopaminergic cells. Nigral histamine innervation is altered in human postmortem Parkinson's disease brains. However, it is not known if the altered innervation is a consequence of dopamine deficiency. The aim of the present study was to investigate possible changes in the H3 receptor system in a well‐characterized model of Parkinson's disease – the 6‐hydroxydopamine (6‐OHDA) lesioned rats. Histamine immunohistochemistry showed a minor increase of the fibre density index but we did not find any robust increase of histaminergic innervation in the ipsilateral substantia nigra on the lesioned side. In situ hybridization showed equal histidine decarboxylase mRNA expression on both sides in the posterior hypothalamus. H3 receptors were labelled with N‐alpha‐[3H]‐methyl histamine dihydrochloride ([3H] NAMH). Upregulation of binding to H3 receptors was found in the substantia nigra and ventral aspects of striatum on the ipsilateral side. An increase of GTP‐γ‐[35S] binding after H3 agonist activation was found in the striatum and substantia nigra on the lesioned side. In situ hybridization of H3 receptor mRNA demonstrated region‐specific mRNA expression and an increase of H3 receptor mRNA in ipsilateral striatum. Thus, the histaminergic system is involved in the pathological process after 6‐OHDA lesion of the rat brain at least through H3 receptor. On the later stages of the neurotoxic damage, less H3 receptors became functionally active. Increased H3 receptor mRNA expression and binding may, for example, modulate GABAergic neuronal activity in dopamine‐depleted striatum.
Development of NMDA and non‐NMDA receptor‐mediated excitatory synaptic transmission in geniculocortical and corticocortical connections in organotypic coculture preparations
Authors: Yamada Katsuya , Yamamoto Nobuhiko , Toyama Keisuke 
Published: March 2003

AbstractDevelopment of N‐methyl‐ d‐aspartate (NMDA) and non‐NMDA receptor‐mediated excitatory synaptic transmission was studied in the visual cortex using organotypic slice cocultures. A slice of visual cortex (VC) dissected from newborn rats was cocultured with either a chunk of embryonic lateral geniculate nucleus (LGN) or another VC. During 7–38 days in vitro (DIV), geniculocortical monosynaptic excitatory postsynaptic potentials (EPSPs) were recorded from layer IV neurons in response to stimulation of the LGN in cocultures of the VC with the LGN. Similarly, corticocortical monosynaptic EPSPs were recorded from layers II/III and V/VI neurons in cocultures of two VCs when stimulating the partner VC. The initial slopes of the non‐NMDA and NMDA receptor‐mediated components of the EPSPs, which were dissociated pharmacologically, were assessed and compared among three different culture stages, early (7–11 DIV), middle (12–15 DIV) and late (17–38 DIV). With progression of the culture stage, the non‐NMDA component tended to increase in both the geniculocortical and corticocortical connections. In contrast, the NMDA component exhibited distinct developmental changes. The NMDA component in layer IV neurons, which receive geniculate inputs, showed a transient increase in the middle stage. In the corticocortical connection, the magnitude of the NMDA component was large in the early stage and maintained through all culture stages in layer V/VI cells, whereas in layer II/III cells it decreased sharply by the late stage. Our results suggest that glutamatergic transmission in the visual cortex develops differently in the geniculocortical and corticocortical connections.
A quantitative analysis of l‐glutamate‐regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics
Authors: Chatton Jean‐Yves , Marquet Pierre , Magistretti Pierre J. 
Published: March 2003

AbstractThe mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate‐evoked Na+ increase was primarily due to Na+‐glutamate cotransport, as inhibition of non‐NMDA ionotropic receptors by 6‐cyano‐7‐nitroquinoxiline‐2,3‐dione (CNQX) only weakly diminished the response and d‐aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non‐NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non‐NMDA receptors do not contribute significantly to the glutamate‐evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady‐state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 μm already markedly increase the energetic demands of the astrocytes.
The lethal expression of the GluR2flip/GluR4flip AMPA receptor in HEK293 cells
Authors: Iizuka Masaki , Nishimura Seiichiro , Wakamori Minoru , Akiba Isamu , Imoto Keiji , Barsoumian Edward L. 
Published: March 2003

Abstractα‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) ‐type glutamate receptors play a critical role in excitotoxicity associated with cerebral hypoxia, ischaemia and other acute brain insults. AMPA receptors are composed of GluR1–GluR4 subunits in homomeric and heteromeric assemblies, forming nonselective cation channels. In addition, each subunit has alternative splice variants, flip and flop forms. Heterologous expression studies showed that the AMPA receptor channels exhibit diverse properties depending on subunit/variant composition. For example, the absence of the GluR2 subunit makes AMPA receptor assemblies Ca2+‐permeable. Excitotoxicity induced by activating AMPA receptor channels has been linked to excessive Ca2+ influx through the GluR2‐lacking channels. Here we demonstrate that coexpression of the AMPA receptor GluR2flip and GluR4flip subunits exerts a lethal effect on HEK293 cells, whereas no lethal activity is observed in other homomeric or heteromeric combinations of AMPA receptor subunits. Patch clamp recordings and Ca2+ imaging analyses have revealed that this GluR2flip/GluR4flip receptor exhibits a low Ca2+ permeability. This subunit combination, however, showed prolonged Na+ influx following AMPA stimulation, even in the absence of cyclothiazide, which attenuates AMPA receptor desensitization. Furthermore, the GluR2flip/GluR4flip‐mediated lethality was potentiated by the interruption of cellular Na+ extrusion mechanisms using ouabain or benzamil. These observations suggest that the GluR2flip/GluR4flip receptor‐mediated excitotoxicity is attributed to Na+ overload, but not Ca2+ influx.
Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons
Authors: Dumoulin A. , Lévi S. , Riveau B. , Gasnier B. , Triller A. 
Published: March 2003

AbstractIn the spinal cord, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine and/or GABAA receptors (GlyR and GABAAR, respectively). We have analysed here the sequence of events leading to inhibitory synapse formation during synaptogenesis of embryonic spinal cord neurons between 1 and 11 days in vitro (DIV). We used immunocytochemical methods to detect simultaneously an antigen specific to inhibitory terminals, the vesicular inhibitory amino acid transporter (VIAAT), and one of the following postsynaptic elements: GlyR, GABAAR or gephyrin, the anchoring protein of GlyR, which is also associated with GABAAR. Quantitative analysis revealed that until 5 DIV most gephyrin clusters were not adjacent to VIAAT‐positive profiles, but became associated with them at later stages. In contrast, GlyR and GABAAR clustered predominantly in front of VIAAT‐containing terminals at all stages. However, about 10% of receptor aggregates were detected at nonsynaptic loci. The two receptors colocalized in 66.2 ± 2.5% of the inhibitory postsynaptic domains after 11 DIV, while 30.3 ± 2.6% and 3.4 ± 0.8% of them contained only GlyR and GABAAR, respectively. Interestingly, at 3 DIV GABAAR clustered at a postsynaptic location prior to gephyrin and GlyR; GABAAR could thus be the initiating element in the construction of mixed glycine and GABAergic synapses. The late colocalization of gephyrin with GABAAR, and the demonstration by other groups that, in the absence of gephyrin, postsynaptic GABAAR is not detected, suggest that gephyrin is involved in the stabilization of GABAAR rather than in its initial accumulation at synaptic sites.
Reducing conditions significantly attenuate the neuroprotective efficacy of competitive, but not other NMDA receptor antagonists in vitro
Authors: Pringle Ashley K. , Self James , Eshak Marie , Iannotti Fausto 
Published: March 2003

AbstractInappropriate activation of NMDA receptors during a period of cerebral ischaemia is a crucial event in the pathway leading to neuronal degeneration. However, significant research has failed to deliver a clinically active NMDA receptor antagonist, and competitive NMDA antagonists are ineffective in many experimental models of ischaemia. The NMDA receptor itself has a number of modulatory sites which may affect receptor function under ischaemic conditions. Using rat organotypic hippocampal slice cultures we have investigated whether the redox modulatory site affects the neuroprotective efficacy of NMDA receptor antagonists against excitotoxicity and experimental ischaemia (OGD). NMDA toxicity was significantly enhanced in cultures pretreated with a reducing agent. The noncompetitive antagonist MK‐801 and a glycine‐site blocker were equally neuroprotective in both normal and reduced conditions, but there was a significant rightward shift in the dose–response curves of the competitive antagonists APV and CPP and the uncompetitive antagonist memantine. OGD produced neuronal damage predominantly in the CA1 region, which was prevented by MK‐801 and memantine, but not by APV or CPP. Inclusion of an oxidizing agent during the period of OGD had no effect alone, but significantly enhanced the neuroprotective potency of the competitive antagonists. These data clearly demonstrate that chemical reduction of the redox modulatory site of the NMDA receptor decreases the ability of competitive antagonists to block NMDA receptor‐mediated neuronal damage, and that the reducing conditions which occur during simulated ischaemia are sufficient to produce a similar effect. This may have important implications for the design of future neuroprotective agents.
Attenuation and augmentation of ischaemia‐related neuronal death by tumour necrosis factor‐α in vitro
Authors: Wilde Geraint J. C. , Pringle Ashley K. , Sundstrom Lars E. , Mann Derek A. , Iannotti Fausto 
Published: March 2003

AbstractUpregulation of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF) occurs rapidly in the brain following ischaemia, although it is unclear whether this represents a neurotoxic or neuroprotective response. We have investigated whether TNF has different actions in the pre‐ and postischaemic periods in a tissue culture model of cerebral ischaemia. Organotypic hippocampal slice cultures were prepared from 8–10‐day‐old rats and maintained in vitro for 14 days. Neuronal damage was induced by either 1 h oxygen–glucose deprivation or 3 h exposure to NMDA or the superoxide generator duroquinone, and assessed after 24 h by propidium iodide fluorescence. TNF pretreatment was neuroprotective against both oxygen–glucose deprivation and duroquinone. This effect was associated with an activation of the transcription factor NFκB and upregulation of manganese superoxide dismutase, and was prevented by a free radical scavenger. When addition of TNF was delayed until the postinsult period, an exacerbation of neurotoxicity occurred, which was also prevented by a free radical scavenger. The actions of TNF are determined by whether TNF is present before or after an ischaemia‐related insult. Both actions are mediated through the production of free radicals, and the response to TNF is determined by whether a cell is metabolically competent to respond by synthesis of antioxidant defences.