bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of tumour‐suppressor phosphoprotein p53 in the apoptosis of cultured rat cerebellar neurones triggered by excitatory amino acids
Authors: Uberti Daniela , Belloni Marco , Grilli Mariagrazia , Spano PierFranco , Memo Maurizio
Published: March 2003

We found that primary cultures of rat cerebellar granule cells, although definitely postmitotic and terminally differentiated, express the tumour‐suppressor phosphoprotein p53. In particular, granule cells both expressed significant levels of p53 mRNA and positively reacted to an anti‐p53 antibody, from the first day of culturing. During neurone differentiation, p53 mRNA content did not significantly change, at least up to 12 days in vitro, while p53 immunoreactivity increased gradually. p53 expression appeared to be further modulable being upregulated after stimulation of glutamate ionotropic receptors by glutamate or kainate. Although qualitatively similar, p53 induction by glutamate and kainate differed in terms of intensity and time‐course. The glutamate increase of p53 immunoreactivity appeared within 30 min after the treatment and lasted for at least 2 h. Kainate‐induced increase of p53 immunoreactivity was delayed, becoming apparent within 2 h and lasting for at least 8 h. Both kainate‐ and glutamate‐induced increases of p53 immunoreactivity were prevented by the non‐competitive NMDA receptor antagonist MK 801. As shown by the electrophoretic mobility shift analysis, both glutamate and kainate induced increases of p53 DNA binding activity. Blockade of p53 induction by a specific p53 antisense oligonucleotide resulted in a partial reduction of excitotoxicity with a complete inhibition of the excitatory amino acids induced apoptosis.

Our data suggest that stimulation of ionotropic glutamate receptors in neurones results in a p53‐dependent apoptosis.
Compartments within human primary auditory cortex: evidence from cytochrome oxidase and acetylcholinesterase staining
Authors: Clarke Stephanie , Rivier François
Published: March 2003

The human primary auditory area (AI) corresponds to granular cortex located on Heschl’s gyrus. We studied its pattern of cytochrome oxidase and acetylcholinesterase activity in 10 normal human hemispheres. In cytochrome‐oxidase‐stained coronal sections layer IV was prominent by its dark staining. The overall staining intensity varied along the medio‐lateral extent of AI; a 2.0–2.5‐mm‐wide antero‐posterior dark band was present at mid‐AI. In acetylcholinesterase‐stained coronal sections a dark antero‐posterior band appeared at the same location, corresponding to the highly granular part of AI. In cytochrome‐oxidase‐stained tangential sections of flattened AI, ≈ 500‐μm thick alternating dark and light cytochrome oxidase stripes were present in layers III and IV. These stripes were perpendicular to the dark band. Comparison with tonotopic maps of human AI obtained by activation studies suggests that the cytochrome oxidase and acetylcholinesterase dark band is most likely parallel to isofrequency lines and may correspond to the representation of frequencies critical for speech comprehension. The narrow stripes may be related to particular binaural or ampliotopic domains, whose presence is suggested by evidence from electrophysiological recordings in cat AI and from magnetoencephalographic studies in humans.
MAP2d mRNA is expressed in identified neuronal populations in the developing and adult rat brain and its subcellular distribution differs from that of MAP2b in hippocampal neurones
Authors: Ferhat Lotfi , Represa Alfonso , Ferhat Walid , Ben‐Ari Yezekiel , Khrestchatisky Michel
Published: March 2003

The brain microtubule‐associated protein MAP2 family is composed of high‐molecular‐weight (MAP2a and MAP2b) and low‐molecular‐weight (MAP2c and MAP2d) isoforms. The common C‐terminal region of HMW MAP2 and MAP2c contains three repeated microtubule‐binding domains while MAP2d comprises four repeats. MAP2c mRNA is known to be expressed at high levels in the immature brain. We show that in the brains of rat pups, MAP2c mRNAs are indeed expressed at high levels compared with MAP2d. However, in adult rat brains, MAP2d mRNA levels are higher than MAP2c. In order to identify the neural cells expressing MAP2d, we used in situ hybridization. In vivo, we show that MAP2d mRNA is expressed in well‐identified neuronal populations of the brain. In primary cultures of hippocampal neurones, double‐labelling experiments confirm that MAP2d is clearly expressed in neurones. We also evaluated in this study the subcellular distribution of the MAP2d mRNAs in cultured hippocampal neurones and we report that in contrast with MAP2b mRNAs, mostly localized in dendrites, MAP2d mRNAs are essentially located in neuronal cell bodies.
Nongenomic effects of oestrogen: embryonic mouse midbrain neurones respond with a rapid release of calcium from intracellular stores
Authors: Beyer Cordian , Raab Heike
Published: March 2003

Evidence is emerging that oestrogen, besides acting via classical nuclear receptors, can rapidly influence the physiology of nerve cells through other mechanisms. Oestrogens have been shown to modulate the differentiation and function of embryonic midbrain dopaminergic neurones by stimulating neurite outgrowth, expression of tyrosine hydroxylase mRNA, dopamine uptake and release in spite of the fact that dopaminergic cells in the prenatal midbrain do not express the classical oestrogen receptor. This study therefore intended to unravel possible signal transduction pathways activated by oestrogen which might be associated with the above oestrogen effects. As a physiological second‐messenger mechanism, we studied the influence of oestrogen on fluctuations of intracellular Ca2+ levels [Ca2+]i by microspectrofluorimetry of the Ca2+‐sensitive indicator Fura‐2, in primary cultures from embryonic mouse midbrains. 17β‐estradiol (10 n
m–1 p
m) but not 17α‐estradiol increased [Ca2+]i within 1–3 s in a dose‐dependent way. Removal of extracellular Ca2+ abrogated K+‐stimulated Ca2+ rise but did not affect 17β‐estradiol stimulation. Pretreatment of cells with thapsigargin (1 μm, 10 min), an inhibitor of Ca2+‐pumping ATPases in the endoplasmic reticulum, abolished the 17β‐estradiol effect but not the K+‐stimulated [Ca2+]i rise. Oestrogen effects on [Ca2+]i were completely mimicked by using a membrane‐impermeant oestrogen‐BSA construct. In order to identify oestrogen‐sensitive cells, some cultures were subsequently immunostained for microtubule‐associated protein II, tyrosine hydroxylase, or GABA. All oestrogen‐sensitive cells were immunocytochemically characterized as neurones, and about half of these responsive neurones was found to be dopaminergic or GABAergic. These results demonstrate that 17β‐estradiol is capable of rapidly modulating physiological parameters of developing midbrain neurones by directly interacting with specific membrane binding sites coupled to a signal transduction mechanism that causes a calcium release from intracellular Ca2+ stores. It is suggested that oestrogen effects on differentiation and function of midbrain dopaminergic neurones are mediated by intracellular Ca2+ signalling.
In utero gene transfer reveals survival effects of nerve growth factor on rat brain cholinergic neurones during development
Authors: Martínez‐Serrano Alberto , Olsson Martin , Gates Monte A. , Björklund Anders
Published: March 2003

Nerve growth factor (NGF) is a maintenance factor for cholinergic neurones in the brain, but its properties as a developmental survival factor are largely unknown. The low accessibility of the developing mammalian brain to experimental manipulation makes it difficult to increase NGF levels during the early phases of brain development. In the present study we have used an in utero, ex‐vivo gene transfer approach to explore NGF actions during development of the cholinergic system in the rat brain. Significantly increased numbers of cholinergic neurones were found only in the mesopontine complex in animals receiving NGF‐secreting transplants, whereas the cholinergic neurones in the basal forebrain and striatum were not clearly affected. The present results suggest that overexpression of NGF during development may promote the survival of distinct populations of central cholinergic neurones into adulthood.
Nitric oxide sensitive depolarization‐induced hyperpolarization: a possible role for gap junctions during development
Authors: Strata Fabrizio , Atzori Marco , Molnar Margherita , Ugolini Gabriele , Berretta Nicola , Cherubini Enrico
Published: March 2003

Electrical coupling is a widespread feature of developing neuronal circuits and it contributes to the generation of patterned activity. In the developing rat hippocampus, release of GABA by coactive hilar interneurones generates widespread synchronized activity. Here it is shown that hilar interneurones strongly rectify in the outward direction when depolarized. This depolarization‐induced hyperpolarization, abolished by gap junction uncouplers, is modulated by nitric oxide. This phenomenon might represent a current‐shunting mechanism of the excess current by providing functional inhibition at a developmental stage when GABA is excitatory. Spatial buffering of the current might represent an osmotic mechanism for growth and differentiation.
GluR1 and GluR2/3 subunits of the AMPA‐type glutamate receptor are associated with particular types of neurone in laminae I–III of the spinal dorsal horn of the rat
Authors: Kerr R. C. Spike R. , Maxwell  , Todd
Published: March 2003

GluR1 and GluR2 subunits of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor are expressed at high levels by neurones in laminae I–III of rat spinal dorsal horn, an area which contains numerous, densely packed small neurones. In order to determine whether these subunits are expressed by inhibitory or excitatory neurones, we combined pre‐embedding immunocytochemistry with antibodies that recognize either GluR1, or an epitope common to GluR2 and 3, with postembedding detection of γ‐aminobutyric acid (GABA) and glycine. Most (78%) of the neurones with GluR1‐immunoreactivity were GABA‐immunoreactive, and some of these were also glycine‐immunoreactive, whereas nearly all (97%) of the GluR2/3‐immunoreactive neurones were not GABA‐ or glycine‐immunoreactive.

We carried out double‐immunofluorescence and confocal microscopy to provide further information on the neurochemistry of cells that express these subunits. As expected, all neurotensin‐ and virtually all somatostatin‐immunoreactive cells (which are thought to be excitatory interneurones) were GluR2/3‐ but not GluR1‐immunoreactive, whereas parvalbumin‐containing cells (most of which are GABAergic) possessed GluR1‐, but usually not GluR2/3‐immunoreactivity. Neurones that contained nitric oxide synthase (most of which are GABAergic) were more variable, with 57% GluR1‐immunoreactive and 41% GluR2/3‐immunoreactive. Cholinergic neurones in lamina III (which are also GABAergic) invariably showed each type of GluR‐immunoreactivity.

These results suggest that neuronal populations in laminae I–III have characteristic patterns of GluR expression: GluR1 is particularly associated with inhibitory neurones, and GluR2 with excitatory neurones. This makes it likely that some of the AMPA receptors present on the inhibitory interneurones lack the GluR2 subunit, and may therefore have significant Ca2+‐permeability.
In vitro and in vivo behaviour of NDF‐expanded monkey Schwann cells
Authors: Avellana‐Adalid  , Bachelin  , Lachapelle  , Escriou  , Ratzkin  , Van Evercooren A. Baron‐
Published: March 2003

Schwann cells, the myelin‐forming cells of the peripheral nervous system may play a major role in the regeneration and remyelination not only of the peripheral but also of the central nervous system. The discovery of the mitogenicity of human recombinant forms of neuregulins (glial growth factors) on primate Schwann cells allows us to envisage a considerable expansion of these cells in culture with a view to autologous transplantation in the central nervous system. To assay this possibility, we used human recombinant neu‐differentiation factor beta (NDFβ) to expand monkey Schwann cells derived from perinatal and adult nerve biopsies. We report that NDFβ containing the epidermal growth factor (EGF)‐like domain (residues 177–228) is a potent mitogen for monkey Schwann cells but is more effective on perinatal than adult Schwann cells. Moreover, continuous treatment with NDFβ, does not seem to prevent Schwann cells differentiation into myelin‐forming cells after their transplantation into the demyelinated mouse spinal cord. These observations, in addition to the close similarities of in vitro behaviour which exist between human and monkey Schwann cells, indicate that monkey Schwann cells could be an ideal tool to study the potential and limits of autologous transplantation in a non‐human primate model of central nervous system demyelination.
VAMP‐1 and VAMP‐2 gene expression in rat spinal motoneurones: differential regulation after neuronal injury
Authors: Jacobsson Gunilla , Piehl Fredrik , Meister Björn
Published: March 2003

Vesicle‐associated membrane protein (VAMP; synaptobrevin) is involved in the molecular regulation of transmitter release at the presynaptic plasma membrane. VAMP exists in two isoforms, VAMP‐1 and VAMP‐2, which are transcribed from two separate genes and differentially expressed in the nervous system. In situ hybridization was used to examine whether VAMP isoform mRNA expression may be altered by experimental manipulations. The effect of nerve injury on VAMP‐1 and VAMP‐2 mRNA levels in motoneurones of the rat lumbar spinal cord was compared with lesion‐induced changes in the expression of choline acetyl transferase (ChAT) and α‐calcitonin gene‐related peptide (α‐CGRP) mRNA. After unilateral sciatic nerve transection (axotomy), VAMP‐1 mRNA expression decreased significantly in parallel with a downregulation of ChAT mRNA in axotomized motoneurones compared with the corresponding motoneurones on the contralateral unlesioned side. There was a rapid decrease in VAMP‐1 and ChAT mRNA levels at 2 days after axotomy, and at 7 days there was a 65% decrease in VAMP‐1 mRNA and a 48% decrease in ChAT mRNA. VAMP‐1 mRNA levels continued to decrease at 14 and 21 days, while ChAT mRNA levels had returned to normal at this time. In contrast, VAMP‐2 and α‐CGRP mRNA levels were upregulated in axotomized motoneurones. A significant increase for both VAMP‐2 and α‐CGRP mRNA levels was present 2 days after axotomy, and a maximum was reached after 7 days for α‐CGRP mRNA (163%) and after 14 days for VAMP‐2 mRNA (587%). Immunohistochemical analysis did not reveal any detectable changes in VAMP‐1‐ or VAMP‐2‐like immunoreactivity in the motoneurone cell soma after axotomy. In the proximal end of the transected sciatic nerve, there was an increase in VAMP‐1‐ and VAMP‐2‐LI, which was most prominent at 2 days after lesion. The results show that, in axotomized spinal motoneurones, VAMP‐1 mRNA is downregulated and VAMP‐2 mRNA is upregulated, indicating differential regulation of the two separate VAMP genes and differential roles for the two VAMP isoforms in the regulation of exocytosis after nerve injury.
Two types of calcium currents of the mouse bipolar cells recorded in the retinal slice preparation
Authors: De La Villa Pedro , Vaquero Cecilia F. , Kaneko Akimichi
Published: March 2003

In the vertebrate retina, the bipolar cell makes reciprocal synapses with amacrine cells at the axon terminal. It has been postulated that amacrine cells may control the transmitter release from bipolar cells by modulating their calcium currents (ICa). To clarify this possibility calcium currents were studied in bipolar cells of the mouse retina using a slice preparation. ICa was identified by voltage clamp protocols, ionic substitution and pharmacological tools.

Depolarization to –30 mV from a holding voltage of –80 mV induced an inward current consisting of an initial transient and a long‐lasting sustained component. The transient component was inactivated by holding the membrane at more positive voltages. Addition of 100 μm nifedipine suppressed the sustained component, leaving the transient component almost intact. The sustained component was enhanced when external solution contained 0.1 μm Bay K 8644 or when the external Ca2+ was substituted by equimolar Ba2+. Omega‐conotoxin (10 μmω‐ctxn GVIA) did not alter either component. We concluded that the transient component is a low‐voltage activated T‐type ICa, while the sustained component is a high‐voltage activated L‐type ICa.

T‐type ICa was recorded in all cells tested, while L‐type ICa was found only in cells that retained axon terminals ramifying in the inner plexiform layer. Thus, it is highly likely that L‐type ICa is generated at the axon terminal and contributes to the transmitter release from the bipolar cell.

The present results confirm that in addition to the T‐type ICa that had been previously described, bipolar cells of the mammalian retina also contain L‐type ICa similar to the one that has been reported in bipolar cells of the goldfish. The use of retinal slice preparation allowed us to record this current that was not seen previously in the dissociated mouse bipolar cells.
VIP and PACAP potentiate the action of glutamate on BDNF expression in mouse cortical neurones
Authors: Pellegri Giovanni , Magistretti Pierre J. , Martin Jean‐Luc
Published: March 2003

In view of the neurotrophic effect of vasoactive intestinal peptide (VIP), the regulation of brain‐derived neurotrophic factor (BDNF) expression by VIP and the related peptide pituitary adenylate cyclase‐activating polypeptide (PACAP) was analysed by Northern blot in primary cultures of cortical neurones. Results reported in this article demonstrate that VIP and PACAP stimulate the expression of BDNF mRNA in primary cultures of cortical neurones and astrocytes. In primary cultures of cortical neurones, induction of BDNF mRNA by VIP and PACAP is completely inhibited by the N‐methyl‐d‐aspartate (NMDA) receptor antagonists MK‐801 and AP5, therefore indicating that VIP and PACAP do not stimulate BDNF expression directly but rather by potentiating the effect of glutamate tonically released by neurones and acting at NMDA receptors.

In addition to its neurotrophic effects, BDNF has been shown to be involved in neuronal plasticity and results reported here suggest that by stimulating BDNF expression, VIP and PACAP could modulate synaptic plasticity in the cerebral cortex.
Pre‐ and postsynaptic blockade of neuromuscular transmission by Miller–Fisher syndrome IgG at mouse motor nerve terminals
Authors: Buchwald Brigitte , Weishaupt Andreas , Toyka Klaus Viktor , Dudel Josef
Published: March 2003

Miller–Fisher syndrome, a variant of an acute inflammatory neuropathy is often associated with serum antibodies to the ganglioside GQ1b, but the pathogenic role of these antibodies and other serum factors is unclear. We here investigated the effect of highly purified immunoglobulin G (IgG) from patients with typical Miller–Fisher syndrome, recording quantal endplate currents by means of a perfused macro‐patch‐clamp electrode on hemidiaphragms of adult mice. The GQ1b‐positive and the GQ1b‐negative Miller–Fisher IgG as well as its monovalent Fab‐fragments depressed evoked quantal release in a fast and fully reversible, concentration and voltage dependent manner. The time‐course of quantal release was changed with the late releases becoming more frequent. The extent of depression of release followed a Michaelis–Menten kinetic and depended on the extracellular calcium concentration. In addition the amplitude of quanta was reduced postsynaptically. IgG and sera from healthy subjects had no effect. Our results indicate that in Miller–Fisher syndrome, IgG antibodies to an undetermined antigen depress the release process, most likely by interfering with the presynaptic Ca2+ inflow or by interacting with proteins of the exocytotic apparatus, and prevent the activation of postsynaptic channels. Antibodies thus seem to be one pathogenic factor for muscle weakness in Miller–Fisher syndrome and our findings may explain why muscle strength recovers rapidly after therapeutical plasmapheresis.
Long‐lasting enhanced expression in the rat hippocampus of NMDAR1 splice variants in a kainate model of epilepsy
Authors: Rafiki Amina , Ben‐Ari Yezekiel , Khrestchatisky Michel , Represa Alfonso
Published: March 2003

Chronic epilepsy is associated with increased excitability which may result from abnormal glutamatergic synaptic transmission involving altered properties of N‐methyl‐d‐aspartate (NMDA) receptors. To date two gene families encoding NMDA receptor subunits have been cloned, NR1 and NR2. Eight NR1 mRNAs are generated by alternative splicing of exons 5, 21 and 22; the NR1–1 to NR1–4 C‐terminal variants exist in the a or b version depending on the presence or absence of the domain encoded by exon 5. Epilepsy was induced in rats by unilateral intra‐amygdalar injection of kainate and animals were killed from 6 h to 4 months following the injection. Increased NR1 mRNA levels were observed during status epilepticus (6–24 h after the injection), both ipsilateral and contralateral, while a second wave of NMDAR1 mRNA increase occurred in chronic epileptic animals, between 21 days and 4 months following kainate injection. Our data show: (i) a permanent increase of the NR1–2a and NR1–2b mRNA species (containing exon 22) in all hippocampal fields, both ipsilateral and contralateral, and (ii) an increase of the NR1–3 (a and b) mRNAs (containing exon 21) in the ipsilateral CA1, and NR1–3a mRNA in the ipsilateral dentate gyrus. No long‐term changes were observed for the NR1–1 and NR1–4 splice variants. In the ipsilateral CA3 area a globally decreased mRNA expression was associated with neuronal loss. A possible contribution to the maintenance of the epileptic state by an increased expression of NMDA receptors is discussed.
Developmental regulation of T‐, N‐ and L‐type calcium currents in mouse embryonic sensory neurones
Authors: Desmadryl G. , Hilaire C. , Vigues S. , Diochot S. , Valmier J.
Published: March 2003

We investigated the development of a low (T‐type) and two high voltage‐activated (N‐ and L‐type) calcium channel currents in large diameter dorsal root ganglion neurones acutely isolated from embryonic mice using the whole‐cell patch‐clamp technique. The low and high voltage‐activated barium currents (LVA and HVA) were identified by their distinct threshold of activation and their sensitivity to pharmacological agents, dihydropyridines and ω‐conotoxin‐GVIA, at embryonic day 13 (E13), E15 and E17–18, respectively, before, during and after synaptogenesis. The amplitude and density of LVA currents, measured during a –40 mV pulse from a holding potential of –100 mV, increased significantly between E13 and E15, and remained constant between E15 and E17–18. The density of global HVA current, elicited by 0 mV pulse, increased between E13 and E15/E17–18.

The density of the N‐type current studied by the application of ω‐conotoxin‐GVIA (1 μm) increased significantly between E13 and E15/E17–18. The use of the dihydropyridine nitrendipine (1 μm) revealed that the density of L‐type current remained constant at each stage of development. Nevertheless, application of dihydropyridine Bay K 8644 (3 μm) demonstrated a significant slowing of the deactivation tail current between embryonic days 13 and 15, which may reflect a qualitative maturation of this class of calcium channel current.

The temporal relationship between the changes in calcium channel pattern and the period of target innervation suggests possible roles of T‐, N‐ and L‐type currents during developmental key events such as natural neurone death and onset of synapse formation.
Development and characterization of antibodies directed against the mouse D4 dopamine receptor
Authors: Mauger C. , Sivan B. , Brockhaus M. , Fuchs S. , Civelli O. , Monsma F.
Published: March 2003

Polyclonal antibodies against the mouse D4 dopamine receptor have been developed in order to investigate the anatomical localization of this receptor in the mouse brain. Two antibodies were generated against specific peptides corresponding to predicted extracellular and intracellular regions of the D4 protein. Specificity of these antibodies was demonstrated on human embryonic kidney 293 (HEK 293) cells transfected with different dopamine receptor subtypes; immunoreactivity was detected only in cells transfected with the mouse D4 dopamine receptor cDNA. Following in vitro transcription/translation of the mouse D4 cDNA, a single protein band of 36 kDa was selectively immunoprecipitated with the anti‐D4 antibodies. The antibodies also detected a single protein of 36 kDa in Western blot of HEK 293 cells transiently transfected with the mouse D4 receptor. These antibodies were able to detect the D4 receptor in several regions of the mouse brain. In the regions examined, D4 immunoreactivity was found in neurones located in layers II–VI of the frontal and piriform cortices, with the highest concentration in layer II; in scattered neurones in the caudate putamen and in larger neurones in the globus pallidus. In all experiments, both antibodies exhibit the same specificity, and all immunoreactivity could be abolished by preincubation with the corresponding peptide antigen.
High‐affinity copper block of GABAA receptor‐mediated currents in acutely isolated cerebellar Purkinje cells of the rat
Authors: Sharonova Irina N. , Vorobjev Vladimir S. , Haas Helmut L.
Published: March 2003

The actions of Cu2+ ions on GABAA receptor‐mediated currents in acutely isolated Purkinje cells from rat cerebellum were studied using the whole‐cell patch‐clamp technique and a rapid perfusion system. Bath application of Cu2+ reduced currents induced by 2 μmγ‐aminobutyric acid (GABA) in a concentration‐dependent manner with an IC50 of 35 n
m. The Cu2+‐induced block of GABA responses was not voltage‐dependent. Increasing the GABA concentration (from 2 to 50 μm) decreased the blocking effect of Cu2+. Dose–response analysis for activation of GABAA receptors revealed a twofold decrease in apparent affinity for GABA in the presence of 0.1 μm Cu2+. Recovery from the block required several minutes after removal of Cu2+ from the medium. The block was removed by histidine, which preferentially forms complexes with Cu2+, or by other chelating substances. Application of 10 μm histidine immediately before application of 2 μm GABA completely relieved the block of GABA responses produced by 0.1 μm Cu2+. The effect of histidine was concentration‐dependent with an EC50 of 0.75 μm. The results demonstrate that Cu2+ is a potent inhibitor of GABA‐evoked responses in rat Purkinje cells. Copper may be an endogenous synaptic modulating factor. Cu2+ toxicity, notably in Wilson’s disease, could result to some extent from chronic GABAA receptor blockade.
Reversible impairment of long‐term potentiation in transgenic Cu/Zn‐SOD mice
Authors: Gahtan E. , Auerbach J.M. , Groner Y. , Segal M.
Published: March 2003

Copper/zinc superoxide dismutase (CuZn‐SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene encoding CuZn‐SOD resides on human chromosome 21 and is overexpressed in Down syndrome (DS) patients. Overexpression of CuZn‐SOD in transgenic (Tg) mice and cultured cells creates chronic oxidative stress leading to enhanced susceptibility to degeneration and apoptotic cell death. We have now found that three lines of Tg‐CuZn‐SOD mice, one of which also overexpresses S100β, a glial calcium binding protein, are deficient in spatial memory. Furthermore, hippocampal slices taken from these mice have an apparently normal synaptic physiology, but are impaired in the ability to express long‐term potentiation (LTP). This effect on hippocampal LTP was abrogated by treatment of slices with the H2O2 scavenger catalase or the antioxidant N‐t‐butyl‐phenylnitrone (BPN). It is proposed that elevated CuZnSOD causes an increase in tetanic stimulation‐evoked formation of H2O2 which leads to diminished LTP and cognitive deficits in these mice.
CRE and TRE sequences of the rat tyrosine hydroxylase promoter are required for TH basal expression in adult mice but not in the embryo
Authors: Trocmé Caryn , Sarkis Chamsy , Hermel Jean‐Michel , Duchateau René , Harrison Stephen , Simonneau Michel , Al‐Shawi Raya , Mallet Jacques
Published: March 2003

Tyrosine hydroxylase (TH), the rate‐limiting enzyme in the biosynthesis of catecholamine neurotransmitters, is expressed in a restricted number of areas, and subject to numerous regulations during development and in adulthood. Two transcription factor binding sites present in the proximal region of the TH gene, the TPA‐responsive element (TRE) and the c‐AMP responsive element (CRE), have been shown to play important roles in TH gene regulation in vitro. In order to elucidate in vivo the role of these two sites, we produced transgenic mice bearing a 5.3‐kb fragment from the 5′ flanking sequence of the TH gene with mutations in either the CRE‐ or TRE‐sites. Using the intact 5.3‐kb fragment fused to two different reporter genes (HSV1‐tk and lacZ), we show that this promoter fragment is able to specifically direct expression in catecholaminergic tissues both in adult mice and embryos. Interestingly, the CRE‐ and TRE‐mutated transgenes were not expressed in adult mice, contrary to the situation in embryos where they were specifically expressed in catecholaminergic regions. These results demonstrate that the CRE and TRE play an essential role in basal TH expression in adult tissues in vivo. Moreover, they suggest that distinct transcription factors are involved in TH regulation in developing and adult tissues. In support of this, gel mobility shift experiments revealed a complex present only in embryonic tissues. Taken together, these data highlight the diversity of the mechanisms underlying the establishment and maintenance of the catecholaminergic phenotype.
Influence of automatic word reading on motor control
Authors: Gentilucci Maurizio , Gangitano Massimo
Published: March 2003

We investigated the possible influence of automatic word reading on processes of visuo‐motor transformation. Six subjects were required to reach and grasp a rod on whose visible face the word ‘long’ or ‘short’ was printed. Word reading was not explicitly required. In order to induce subjects to visually analyse the object trial by trial, object position and size were randomly varied during the experimental session. The kinematics of the reaching component was affected by word presentation. Peak acceleration, peak velocity, and peak deceleration of arm were higher for the word ‘long’ with respect to the word ‘short’. That is, during the initial movement phase subjects automatically associated the meaning of the word with the distance to be covered and activated a motor program for a farther and/or nearer object position. During the final movement phase, subjects modified the braking forces (deceleration) in order to correct the initial error. No effect of the words on the grasp component was observed. These results suggest a possible influence of cognitive functions on motor control and seem to contrast with the notion that the analyses executed in the ventral and dorsal cortical visual streams are different and independent.
Anticonvulsant properties of BIBP3226, a non‐peptide selective antagonist at neuropeptide Y Y1 receptors
Authors: Gariboldi Marco , Conti Mirco , Cavaleri Davide , Samanin Rosario , Vezzani Annamaria
Published: March 2003

Several lines of evidence indicate that neuropeptide Y (NPY)‐mediated neurotransmission in the hippocampus is altered by limbic seizures. The functional consequences of this change are still unresolved and clearly depend on the type of NPY receptors involved. We have investigated the role of NPY Y1 receptor subtypes, which are enriched in the dentate area of the hippocampus, on EEG seizures induced by a local injection of 0.04 μg kainic acid in rats.

Intrahippocampal administration of 10 μg BIBP3226 (N2‐ (diphenylacetyl)‐N‐[(4‐hydroxyphenyl)methyl]d‐arginamide), a non‐peptide selective antagonist at the NPY Y1 receptors, increased threefold on average (P < 0.01) the time to onset of seizures and reduced the number of seizures and the total time in seizures three‐ and fourfold, respectively (P < 0.01). Its inactive S‐enantiomer BIBP3435 was ineffective on seizure activity. One microgram [Leu31,Pro34]NPY, an agonist at Y1 receptors, did not modify per se the EEG sequelae induced by kainic acid but it antagonized the anticonvulsant effect of BIBP3226.

These results indicate that NPY Y1 receptors in the hippocampus are involved in epileptic phenomena and suggest that selective Y1 receptor antagonists may be of value for attenuating limbic seizures.
Modification of parallel activity elicited by propagating bursts in developing networks of rat cortical neurones
Authors: Maeda Eisaku , Kuroda Yoichiro , Robinson Hugh P. C. , Kawana Akio
Published: March 2003

Networks of cultured cortical neurones exhibit regular, synchronized, propagating bursts which are synaptically mediated, and which are hypothesized to play a part in activity‐dependent formation of connections during development in vivo. The relationship between the strength of synaptic connections and the characteristics of synchronized propagating bursting, however, is unclear. Modification of synchronized activity in cortical cultures in response to electrical stimulation was examined using multisite electrode array recording. By measuring the response of the network to weak, localized, test stimulation (TS), we observed a potentiation of activity following a relatively stronger inducing stimulation (IS). This potentiation was evident as an increased probability of eliciting bursts by TS, an increased frequency of spontaneous bursts and number of spikes per burst, and increased speed of burst propagation, and it lasted for at least 20 min. Changing the parameters of IS revealed that high frequency tetanic stimulation is not necessary to induce potentiation, while it is essential for IS to produce a regeneratively propagating burst. The results provide a direct demonstration of modification of both the spatial and temporal characteristics of synchronized network activity, and suggest an important physiological role for propagating synchronized bursting, as a mechanism for inducing plastic modifications in the developing cortex.
Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre‐recipient layer) of the mouse hippocampal CA3 subfield
Authors: Watanabe Masahiko , Fukaya Masahiro , Sakimura Kenji , Manabe Toshiya , Mishina Masayoshi , Inoue Yoshiro
Published: March 2003

Hippocampal synapses express two distinct forms of the long‐term potentiation (LTP), i.e. NMDA receptor‐dependent and ‐independent LTPs. To understand its molecular‐anatomical basis, we produced affinity‐purified antibodies against the GluRε1 (NR2A), GluRε2 (NR2B), and GluRζ1 (NR1) subunits of the N‐methyl‐d‐aspartate (NMDA) receptor channel, and determined their distributions in the mouse hippocampus. Using NMDA receptor subunit‐deficient mice as the specificity controls, section pretreatment with proteases (pepsin and proteinase K) was found to be very effective to detect authentic NMDA receptor subunits. As the result of modified immunohistochemistry, all three subunits were detected at the highest level in the strata oriens and radiatum of the CA1 subfield, and high levels were also seen in most other neuropil layers of the CA1 and CA3 subfields and of the dentate gyrus. However, the stratum lucidum, a mossy fibre‐recipient layer of the CA3 subfield, contained low levels of the GluRε1 and GluRζ1 subunits and almost excluded the GluRε2 subunit. Double immunofluorescence with the AMPA receptor GluRα1 (GluR1 or GluR‐A) subunit further demonstrated that the GluRε1 subunit was colocalized in a subset, not all, of GluRα1‐immunopositive structures in the stratum lucidum. Therefore, the selective scarcity of these NMDA receptor subunits in the stratum lucidum suggests that a different synaptic targeting mechanism exerts within a single CA3 pyramidal neurone in vivo, which would explain contrasting significance of the NMDA receptor channel in LTP induction mechanisms between the mossy fibre‐CA3 synapse and other hippocampal synapses.
Evidence for the involvement of corticotrophin‐releasing hormone in the pathogenesis of traumatic brain injury
Authors: Roe Susan Y. , McGowan Eileen M. , Rothwell Nancy J.
Published: March 2003

The aim of this study was to investigate the role of the neuropeptide corticotrophin‐releasing hormone (CRH) in neurodegeneration induced by traumatic brain injury, using a well characterized model of lateral fluid percussion injury in male, Sprague‐Dawley rats. In the first series of experiments, CRH gene expression was assessed by in situ hybridization after traumatic brain injury. A bilateral increase in CRH mRNA in the paraventricular nucleus was observed in rats subjected to traumatic brain injury compared with sham‐operated controls. A maximal (40%) increase in hybridization signal was detected 2 h after trauma compared with control rat brains. In addition, marked induction of CRH transcripts was found in the ipsilateral amygdala after trauma, but no increase was detected in the ipsilateral cortex around the area of damage. In a separate experiment, the effects of the CRH antagonist, D‐Phe CRH(12–41) (25 μg total dose), or appropriate vehicle injected intracerebroventricularly, was tested on infarct volume caused by brain injury. Repeated intracerebroventricular injection of D‐Phe CRH(12–41) significantly reduced, by 45%, the volume of cortical damage in injured rats compared with vehicle‐treated, trauma animals. The rapid upregulation of CRH gene expression in the paraventricular nucleus and amygdala following lateral fluid percussion injury and the marked neuroprotection achieved by inhibiting CRH action suggest that CRH is involved directly in the pathogenesis of traumatic brain injury. This observation may have important implications for the development of novel therapeutic strategies for treating the neurological consequences of brain trauma and related conditions.
Influence of acetylcholinesterase on embryonic spinal rat motoneurones growth in culture: a quantitative morphometric study
Authors: Bataillé Stephan , Portalier Paule , Coulon Patrice , Ternaux Jean‐Pierre
Published: March 2003

Rat spinal motoneurones sampled at day embryonic 15 were purified using a Nycodenz gradient and cultured in defined medium, during 7 days, on glass coverslips coated with poly‐l‐lysine and laminine. Purified acetylcholinesterase (AChE), ecothiopate, BW 284C51 and fasciculin II, inhibitors of either the catalytic or peripheral site of AChE, were added to the defined medium. Morphological changes of spinal motoneurones were measured using a statistical quantitative morphometric method, allowing the determination of various parameters such as the number of neurites and bifurcations, the length of neurites, the surface and spreading index. Presence of AChE in the medium (4 units/mL) increases the surface and the total length of neurites and axons without any change in the spreading index. When spinal motoneurones were cultured on AChE coated substrate, neurones rapidly migrate and form clusters. Addition of ecothiopate (10–6m) in the medium, which selectively blocks the catalytic site of AChE, leads to a slight increase in the number of primary neurite and a decrease of the spreading index during the three first days in culture. BW 284C51 (10–5m) which blocks the catalytic site but also affect the peripheral one, significantly reduces the number of primary neurites and increases the number of bifurcations. Fasciculin II, a potent blocker (10–9m) of the AChE peripheral site induces a decrease of both primary neurites and bifurcations with a significant increase of the length and growth velocity of the axon, giving a drastic enhancement of the spreading index. These phenomena are discussed in terms of catalytic and non‐catalytic function of AChE, including the involvement of the enzyme in adhesive processes, occurring during growth and differentiation of spinal motoneurones.
Peptidergic innervation and the nicotinic acetylcholine receptor in the primate basal nucleus
Authors: Csillik Bertalan , Rakic Pasko , Knyihár‐Csillik Elizabeth
Published: March 2003

Peptidergic innervation and localization of the neuronal nicotinic acetylcholine receptor (nAChR) was studied in the basal forebrain of Macaca fascicularis in order to provide microstructural proofs for the theory ( Changeux et al. 1992
) that calcitonin gene‐related peptide (CGRP) is responsible for the maintenance of the acetylcholine receptor. Distribution and localization of five neuropeptides, namely substance P (SP), CGRP, neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) neurotensin (NT), and the neuropeptides parvalbumin (PV) and the α‐bungarotoxin‐ (α‐BTX‐) binding protein was studied by means of light‐ and electron microscopic pre‐embedding immunocytochemistry. Immunohistochemical double staining revealed that large cholinergic principal nerve cells in the basal forebrain, corresponding to cell group Ch4 constituting Meynert’s basal nucleus (BNM), and exerting intense choline acetyltransferase (ChAT) immunoreactivity, are synaptically innervated by axons displaying CGRP immunoreactivity. While SP, NPY, PV and CGRP establish dense networks in BNM, innervation by NT and VIP is sparse. Biotinylated α‐BTX visualizes beaded axons that surround dendrites and perikarya of cholinergic principal cells. Electron microscopic organization of the neuropil in BNM is characterized by a glomerular (or rather cartridge‐like) arrangement of axons surrounding dendrites of non‐cholinergic principal nerve cells. At least one of the axons establishing the glomerulus (cartridge) exerts CGRP immunopositivity while α‐BTX‐immunopositive axons, presynaptic to dendrites of principal cells, are attached to the glomeruli (cartridges) from outside. As α‐BTX‐binding indicates localization of the α7 subunit of the neuronal nAChR, the microtopographical arrangement supports the idea that, in a manner similar to that in the neuromuscular junction, CGRP might contribute to the maintenance of nAChR also in BNM. Our results suggest that presynaptic nAChR‐s are involved in the regulation of acetylcholine release from a feed‐forward amplification mechanism of cholinergic principal cells of BNM.
Distribution of L‐type calcium channels in rat thalamic neurones
Authors: Budde Thomas , Munsch Thomas , Pape Hans‐Christian
Published: March 2003

One major pathway for calcium entry into neurones is through voltage‐activated calcium channels. The distribution of calcium channels over the membrane surface is important for their contribution to neuronal function. Electrophysiological recordings from thalamic cells in situ and after acute isolation demonstrated the presence of high‐voltage activated calcium currents. The use of specific L‐type calcium channel agonists and antagonists of the dihydropyridine type revealed an about 40% contribution of L‐type channels to the total

high‐voltage‐activated calcium current. In order to localize L‐type calcium channels in thalamic neurones, fluorescent dihydropyridines were used. They were combined with the fluorescent dye RH414, which allowed the use of a ratio technique and thereby the determination of channel density. The distribution of L‐type channels was analysed in the three main thalamic cell types: thalamocortical relay cells, local interneurones and reticular thalamic neurones. While channel density was highest in the soma and decreased significantly in the dendritic region, channels appeared to be clustered differentially in the three types of cells. In thalamocortical cells, L‐type channels were clustered in high density around the base of dendrites, while they were more evenly distributed on the soma of interneurones. Reticular thalamic neurones exhibited high density of L‐type channels in more central somatic regions. The differential localization of L‐type calcium channels found in this study implies their predominate involvement in the regulation of somatic and proximal dendritic calcium‐dependent processes, which may be of importance for specific thalamic functions, such as those mediating the transition from rhythmic burst activity during sleep to single spike activity during wakefulness or regulating the relay of visual information.
Blockade of neuropsin, a serine protease, ameliorates kindling epilepsy
Authors: Momota Yoshiharu , Yoshida Shigetaka , Ito Jiro , Shibata Masao , Kato Keiko , Sakurai Katsutoshi , Matsumoto Kazumasa , Shiosaka Sadao
Published: March 2003

The behavioural and electrographical abnormalities associated with seizures in epileptic (kindled) mice correspond with those of human epilepsy. In kindled mice, neuropsin was markedly increased in the hippocampus and cerebral cortices. A single intraventricular injection of monoclonal antibodies specific to neuropsin reduced or eliminated the epileptic pattern noted on electroencephalograms and, as a result markedly inhibited the progression of kindling. Therefore, neuropsin appears to be a key protein controlling pathogenic events in the hippocampus, and thus neuropsin inhibitors might be useful for treatment of epilepsy.
The intermediate stage and paradoxical sleep in the rat: influence of three generations of hypnotics
Authors: Gottesmann C. , Gandolfo G. , Arnaud C. , Gauthier P.
Published: March 2003

Paradoxical sleep in the rat, cat and mouse is preceded and sometimes followed by a short‐lasting intermediate stage characterized by high‐amplitude anterior cortex spindles and low‐frequency hippocampal theta rhythm. Several neurophysiological arguments suggest that the intermediate stage corresponds to a brief functional disconnection of the forebrain from the brainstem. This paper is devoted to the review of quantitative and qualitative influences of three generations of hypnotics on the intermediate stage–paradoxical sleep couple. Barbiturates, first‐generation hypnotics, extend the intermediate stage at the expense of paradoxical sleep. Three benzodiazepines are compared, two with a short half‐life (triazolam and midazolam) and one with a long half‐life (diazepam). They also decrease sleep occurrence latency and increase the intermediate stage at the expense of paradoxical sleep, except for midazolam, which increases both the intermediate stage and paradoxical sleep at low dose. Zolpidem and zopiclone, hypnotics of third generation, decrease paradoxical sleep but the intermediate stage never substitutes for paradoxical sleep. The results are discussed in relationship to the functional aspects of this turning‐point period of sleep.
A role for BDNF in early postnatal rat vestibular epithelia maturation: implication of supporting cells
Authors: Montcouquiol M. , Valat J. , Travo C. , Sans A.
Published: March 2003

The early development of the inner ear is largely determined by two members of the neurotrophic family: brain‐derived neurotrophic factor (BDNF) and neurotrophin 3 (NT‐3). Little information is available on the role of these neurotrophins during the late stages of vestibular development in the rat which take place during the first postnatal weeks. At this period where terminal synaptogenesis and maturation occur, we have investigated the expression and the activity of BDNF, the most important neurotrophin in the vestibular system. Using different experimental approaches, we show that BDNF is released by vestibular epithelia on postnatal day 3 (P3) and continues to have a trophic effect on vestibular neurones in vitro. Immunocytochemistry coupled to confocal microscopy revealed a remarkable evolution in BDNF localization during later stages of development. Whereas BDNF is present in both supporting cells and hair cells at P3, its distribution gradually changed and is highly compartmentalized within the upper part of supporting cells at P8 and P15. In parallel, we observed the presence of a truncated form of the BDNF receptor in sensory hair cells. These results suggest an original role for supporting cells, which could be involved in the release of BDNF during the late stages of synaptogenesis in mammalian vestibular epithelia. In particular, BDNF could participate to the set up of the calyx, a specific nerve structure surrounding type I vestibular hair cells.