bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD‐95 and SAP97
Authors: Bassand Patrick , Bernard Anne , Rafiki Amina , Gayet Dimitri , Khrestchatisky Michel
Published: March 2003

The NR1 and NR2 subunits of the N‐methyl‐d‐aspartate (NMDA) receptor are encoded by distinct genes. In the rat brain, four C‐terminal variants of the NR1 subunit (NR1‐1 to NR1‐4) are encoded by a single gene, and are generated by alternative splicing of the C1 and C2 exon cassettes, while four different genes encode the NR2 subunits (NR2 A–D). Functional NMDA receptors result from the heteromultimeric assembly of NR1 variants with distinct NR2 subunits. The NR2B subunit interacts with post‐synaptic density protein 95 (PSD‐95), SAP97 and members of the membrane‐associated guanylate‐like kinase (MAGUK) family of proteins. This interaction occurs through the binding of the C‐terminal tSXV intracellular motif of the NR2B subunit to the N‐terminal PDZ (PSD‐95, discs‐large, ZO‐1) domains of the PSD‐95 and SAP97 proteins. Both NR1‐3 and NR1‐4 also display a consensus C‐terminal tSXV motif. Using the two‐hybrid genetic system in yeast and site‐directed mutagenesis, we compared the binding of the NR2A, NR1‐3 and NR1‐4 tSXV motifs with the PDZ domains of PSD‐95 and SAP97. The main conclusions of the present report are that: (i) while NR2A displays a strong interaction with PSD‐95 and SAP97, the NR1‐3 and NR1‐4 NMDA receptor subunits do not display any interaction despite the presence of tSXV motifs; (ii) the C‐terminal tSXV motif of the NR2A subunit is mandatory but not sufficient for efficient interaction with the PSD‐95 and SAP97 proteins; (iii) as yet unidentified upstream sequences of the receptor subunits determine whether the tSXV motifs will bind to the PSD‐95 and SAP97 PDZ domains; (iv) different tSXV motifs elicit interactions of variable strengths; and (v) residues in positions −3 and −4 modulate the binding affinity of the C‐terminal tSXV motifs. Using immunohistochemistry, we also compared the distribution of the PSD‐95, NR2A and SAP97 proteins in adult rat brain, and we show that in the cortex, hippocampus and cerebellum, there is evidence for colocalization of these proteins.
Muscarinic receptors regulate two different calcium‐dependent non‐selective cation currents in rat prefrontal cortex
Authors: Haj‐Dahmane Samir , Andrade Rodrigo
Published: March 2003

Pyramidal neurons of layer V in rat prefrontal cortex display a prominent fast afterdepolarization (fADP) and a muscarinic‐induced slow afterdepolarization (sADP). We have shown previously that both of these ADPs are produced by the activation of calcium‐dependent non‐selective cation currents. In the present report we examine whether they represent two distinct currents. In most pyramidal neurons recorded with caesium gluconate‐based intracellular solution, a calcium spike is followed by a fast decaying inward aftercurrent (IfADP). The decay of IfADP is monoexponential with a time constant (τ) of ≈ 35 ms. Administration of carbachol (10–30 μm) increases the time constant of this decay by ≈ 80% and induces the appearance of a much slower inward aftercurrent (IsADP). IfADP recorded in control conditions and in the presence of carbachol increases linearly with membrane hyperpolarization. In contrast, the carbachol‐induced IsADP decreases with membrane hyperpolarization. When the sodium driving force across the cell membrane was reduced, IfADP was found to reverse at around −40 mV whereas IsADP remain inward over the same voltage range tested. Finally, bath administration of flufenamic acid (100 μm–1 mm) selectively blocks the carbachol‐induced IsADP without a significant effect on the amplitude of IfADP. These differences in the electrical and pharmacological properties of IfADP and IsADP suggest that they were mediated by two distinct non‐selective cation currents.
Limbic P300s in temporal lobe epilepsy with and without Ammon's horn sclerosis
Authors: Grunwald T. , Beck H. , Lehnertz K , Blümcke I. , Pezer N. , Kutas M. , Kurthen M. , Karakas H. M. , Van Roost D. , Wiestler O. D. , Elger C. E.
Published: March 2003

Limbic P300 potentials can be recorded within the mesial temporal lobes of patients with temporal lobe epilepsy (TLE). To delineate possible mechanisms of their generation and pathological alteration, we analysed limbic P300s in 55 TLE patients with and 29 without Ammon's horn sclerosis (AHS) and correlated their amplitudes with neuronal cell counts in 30 histopathological specimens. Limbic P300 amplitudes were reduced on the side of the epileptogenic focus only in patients with AHS. Moreover, in AHS patients, limbic P300 latencies were prolonged bilaterally; and in patients with left‐sided AHS, amplitudes were reduced bilaterally. Both findings suggest bilateral functional deficits in TLE with unilateral AHS. Limbic P300 areas correlated significantly with neuronal densities of dentate gyrus granule cells but not hippocampal pyramidal cells in the CA1–4 (cornu ammonis) subfields. This finding points to a potential mechanism for the bilateral effects of unilateral AHS as both dentate gyri exhibit strong reciprocal contralateral connectivity.
Perturbation of the synaptic release machinery in hippocampal neurons by overexpression of SNAP‐25 with the Semliki Forest virus vector
Authors: Owe‐Larsson Björn , Berglund , Magnus M. , Kristensson Krister , Garoff , Henrik , Larhammar Dan , Brodin Lennart , Löw Peter
Published: March 2003

We have examined whether the Semliki Forest virus (SFV) expression vector can be used to manipulate the exocytotic machinery in cultured hippocampal neurons. Autaptic responses were recorded in individually identified neurons which overexpressed either a non‐synaptic protein, the transferrin receptor, or the synaptic SNARE protein SNAP‐25 (synaptosomal‐associated protein of 25 kDA). In neurons overexpressing the transferrin receptor, autaptic responses occurred in a similar proportion and had similar amplitudes (12–18 h postinfection) as in uninfected control neurons. With increasing time after the infection, an increasing proportion of the transferrin receptor‐overexpressing neurons showed changes in the shape of the cell body, but the autaptic responses appeared normal as long as recordings could be performed (up to 30 h postinfection). In contrast, in SNAP‐25‐overexpressing neurons, the proportion of responding cells was reduced 12–18 h after the infection, and the amplitude of the autaptic current in responding neurons was also reduced. The sensitivity to exogenously applied glutamate was, however, unchanged. Biochemical analysis showed that 50% of the overexpressed SNAP‐25 was palmitoylated. The levels of two other SNAREs, syntaxin and synaptobrevin (also called vesicle‐associated membrane protein), were not affected. Our results indicate that the SFV vector can provide an effective tool to study the function of proteins participating in neurotransmitter release.
Heparan sulphates upregulate regeneration of transected sciatic nerves of adult guinea‐pigs
Authors: Chau C. H. , Shum D. K. Y. , Chan Y. S. , So K. ‐F.
Published: March 2003

The increased content of soluble glycosaminoglycan‐containing forms in sciatic nerves during recovery from crush injury [Shum & Chau (1996) J. Neurosci. Res., 46, 465] suggests that the glycosaminoglycans modulate the environment for post‐traumatic tissue remodelling and axonal regrowth. To test this, defined amounts of soluble heparan sulphates from bovine kidney or guinea‐pig nerve were introduced into the regenerating environment via silicone conduits that bridged 8‐mm gaps of transected sciatic nerves of adult guinea‐pigs. Controls were bridged using the phosphate‐buffered saline (PBS) vehicle or a chondroition sulphate preparation from whale cartilage. After timed periods of recovery, the animals were assessed for electromyographic signals at the target gastrocnemius muscle to determine the conduction velocity across the bridged nerve. Sections of the bridge were also histologically examined for nerve fibres. Transected sciatic nerves bridged with heparan sulphates or chondroitin sulphate showed earlier stimulated myelination of axons (week 5–6) than PBS‐bridged nerves (week 9). Initial electromyographic indication of reconnection with the target was at week 9 post‐transection. In the course of 20 weeks, transected sections of the bridge indicated similar numbers of unmyelinated axons irrespective of bridge material, but distinctly higher numbers of myelinated axons in heparan sulphate‐bridged nerves than either PBS‐ or chondroitin sulphate‐bridged nerves. At the end of the same period, heparan sulphate‐bridged nerves resumed normal conduction velocities, but both PBS‐ and chondroitin sulphate‐bridged nerves remained at 50% of that of the intact contralateral nerves. These results are the first to demonstrate that supplementation of soluble heparan sulphate to the fluid regenerative neural environment can restore functional, axonal reconnection of the severed nerve with the target muscle.
Real‐time imaging of glucocorticoid receptor dynamics in living neurons and glial cells in comparison with non‐neural cells
Authors: Nishi Mayumi , Takenaka Nobuyuki , Morita Noriyuki , Ito Takao , Ozawa Hitoshi , Kawata Mitsuhiro
Published: March 2003

To investigate the intracellular trafficking of glucocorticoid receptor (GR) in response to various conditions in a single living cell, a green fluorescent protein (GFP) and rat GR chimera construct (GFP‐GR) was prepared. We transiently transfected GFP‐GR into primary cultured rat hippocampal neurons, cortical glial cells, and non‐neural cells, e.g. COS‐1 cells and CV‐1 cells, and compared the dynamic changes in subcellular localization of GFP‐GR in these cells. When GFP‐GR was expressed in the cells, GFP‐GR efficiently transactivated the mouse mammary tumour virus promoter in response to dexamethasone (DEX). The cytoplasm‐to‐nuclear translocation of GFP‐GR induced with 10–7 m DEX, a specific agonist of GR, at 37 °C was completed within 30 min in all cell types used, and the rate of nuclear translocation was dependent on the ligand dose. The translocation of GFP‐GR into the nucleus from the cytoplasm was induced in a ligand‐specific manner, similar to that of the native GR. The disruption of microtubules by colchicine or nocodazole showed no significant effect on the DEX‐induced GFP‐GR translocation from the cytoplasmic region to the nuclear region. The cells were not deteriorated during time‐lapse imaging analysis for 1 h at 37 °C. The present findings suggest that the subcellular localization of GFP‐GR is dynamically changed in response to extracellular and intracellular conditions, and that there are no conspicuous variations in the manner of trafficking of GR among different types of cells in vitro.
Transcriptional repression by the zinc finger protein REST is mediated by titratable nuclear factors
Authors: Leichter Michael , Thiel Gerald
Published: March 2003

The zinc finger protein REST (RE‐1 silencing transcription factor) is a transcriptional repressor that inhibits neuronal gene transcription in non‐neuronal tissues. REST may represent a master regulator of neuronal gene expression. REST contains two repressor domains located at the N‐ and C‐termini of the molecule. To investigate the molecular mechanism of transcriptional repression by REST, in vivo competition experiments were performed. Both repression domains were expressed in the nucleus as fusion proteins with S. japonicum glutathione S‐transferase (GST). The ability of these fusion proteins to block transcriptional repression mediated by the repressor domains of REST was tested. The results show that transcriptional repression by the N‐terminal repression domain of REST could be overcome by expression of a GST fusion protein encoding the N‐terminal, but not C‐terminal repression domain, and vice versa, suggesting that both repression domains have to interact with distinct nuclear factors to exhibit biological activity. The GST‐REST fusion proteins had no effect upon transcriptional repression mediated by the KRAB (Krüppel‐associated box) domain, a strong mammalian repressor domain, or the repressor domain derived from the thyroid hormone receptors α. We conclude that REST has to interact with at least two distinct nuclear factors to inhibit transcription. These factors are distinct from the mammalian corepressor proteins KAP‐1/KRIP‐1 and N‐CoR that mediate repression by the KRAB domain or the thryoid hormone receptor α. Thus, mammalian transcriptional repressors utilize different mechanisms to inhibit transcription by using different kinds of protein–protein interactions.
K+‐dependence of Na+–Ca2+ exchange in type I vestibular sensory cells of guinea‐pig
Authors: Boyer C. , Sans A. , Vautrin J. , Chabbert C. , Lehouelleur J.
Published: March 2003

The properties of the vestibular Na+–Ca2+ exchanger in mammalian type I vestibular sensory cells were studied using fura‐2 fluorescence and immunocytochemical techniques. In the absence of external Na+, the activation of Na+–Ca2+ exchange in reverse mode required the presence of external K+ (K+o) and depended on K+o concentration. Alkali cations Rb+ and NH4+ but not Li+ or Cs+ substituted for K+o to activate the exchange. For pressure applications of 10 mm K+, the contribution of voltage‐sensitive calcium channels to the increase in [Ca2+]i was < 15%. The dependence of the exchange on [K+]o was also recorded when the membrane potential was clamped using carbonyl cyanide p‐trifluoromethoxy‐phenylhydrazone (FCCP) and monensin ionophores. In these conditions, where there was no intracellular Na+, the increase in [Ca2+]i was completely blocked. These physiological results suggest that in reverse mode, Ca2+ entry is driven by both an outward transport of Na+ and an inward transport of K+. The dependence of the vestibular Na+–Ca2+ exchanger on K+ is more reminiscent of the properties of the retinal type Na+–Ca2+ exchanger than those of the more widely distributed cardiac type exchanger. Moreover, the immunocytochemical localization of both types of exchange proteins in the vestibular sensory epithelium confirmed the presence in the vestibular sensory cells of a Na+–Ca2+ exchanger which is recognized by an antibody raised against retinal type and not by an antibody raised against the cardiac type.
Control of neuronal excitability by an ion‐sensing receptor
Authors: Washburn David L. S. , Smith Pauline M. , Ferguson Alastair V.
Published: March 2003

While most central nervous system (CNS) neurons receive the majority of their input through direct synaptic connections, there is evidence suggesting that they are in fact susceptible to modulation by changes in extracellular ionic composition during both physiological and pathophysiological conditions. In many regions of the CNS, there exists an identified extracellular receptor with the ability to sense levels of cations, most notably calcium. Here we report that activation of this calcium receptor (CaR) in neurons of the subfornical organ (SFO), a forebrain circumventricular structure, results in profound effects on neuronal excitability through metabotropic actions on a non‐selective cation channel. Activation of the CaR by NPS R‐467, an allosteric agonist of the CaR, evoked depolarizing plateau potentials ranging in duration from 5 to 30 s. Similarly, 5 mm CaCl2 caused depolarization and increased action potential frequency. NPS R‐467 was found to activate a non‐selective cation channel with a reversal potential of −48 ± 4 mV, and a slope conductance of 2.54 ± 11 nS. This current could also be elicted by spermine, a known agonist of the CaR. CaR‐mediated activation of this channel was dependent upon both G proteins and intracellular Ca2+ signalling, as disruption of these pathways through inclusion of guanosine 5′‐O‐(2‐thiodiphosphate) (GDP‐β‐S) and 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N ′,N ′‐tetraacetic acid (BAPTA), respectively, in the recording pipette prevented activation of the current. Microinjection of CaR agonists into the SFO of anaesthetized rats resulted in a significant, site‐specific elevation of blood pressure (mean area under curve, 141 ± 50 mmHg.s). Together, these results indicate that the CaR can play an important role in transducing the effects of changes in the extracellular ionic composition, and that these effects have implications for the neural control of fluid balance.
Visualization of local afferent inputs to magnocellular oxytocin neurons in vitro
Authors: Jourdain P. , Dupouy B. , Bonhomme R. , Poulain D. A. , Israel J. M. , Theodosis D. T.
Published: March 2003

We recently showed that oxytocin (OT) neurons in organotypic slice cultures obtained from postnatal rat hypothalamus display complex patterns of electrical activity, similar to those of adult magnocellular OT neurons in vivo. Here we used such cultures to investigate the identity and, in particular, the origin of afferent inputs responsible for this activity. Multiple immunostaining with light and confocal microscopy showed that the somata and dendrites of oxytocinergic neurons were contacted by numerous synapses, visualized by their reaction to the synaptic markers, synaptophysin or synapsin. Many were GABAergic, displaying immunoreactivities for glutamic acid decarboxylase or γ‐aminobutyric acid (GABA); others were enriched in glutamate immunoreactivity. Such afferents presumably arose from GABA‐ or glutamate‐immunoreactive neurons, respectively, with distinct and characteristic morphologies and topographies. A few dopaminergic boutons (tyrosine hydroxylase‐ or dopamine‐immunopositive) impinged on OT neurons; they arose from dopamine‐positive neurons located along the third ventricle. No noradrenergic profiles were detected. Despite the presence of choline acetyl‐transferase (ChAT)‐immunoreactive neurons, there were no cholinergic contacts. Lastly, we found oxytocinergic synapses, identified by immunoreaction for OT‐related neurophysin and synapsin, contacting OT somata and dendrites.

Our observations thus demonstrate that inhibitory and excitatory inputs to OT neurons derive from local intrahypothalamic GABA and glutamate neurons, in close proximity to the neurons. They also reveal that OT neurons are innervated by hypothalamic dopaminergic neurons. Finally, they confirm the existence of homotypic OT synaptic contacts which derive from local OT neurons.
Social intelligence in the normal and autistic brain: an fMRI study
Authors: Baron‐Cohen Simon , Ring Howard A. , Wheelwright Sally , Bullmore Edward T. , Brammer Mick J. , Simmons Andrew , Williams Steve C. R.
Published: March 2003

There is increasing support for the existence of ‘social intelligence’[Humphrey (1984) Consciousness Regained], independent of general intelligence. Brothers et al. (1990) J. Cog. Neurosci., 4, 107–118] proposed a network of neural regions that comprise the ‘social brain’: the orbito‐frontal cortex (OFC), superior temporal gyrus (STG) and amygdala. We tested Brothers' theory by examining both normal subjects as well as patients with high‐functioning autism or Asperger syndrome (AS), who are well known to have deficits in social intelligence, and perhaps deficits in amygdala function [Bauman & Kemper (1988) J. Neuropath. Exp. Neurol., 47, 369]. We used a test of judging from the expressions of another person's eyes what that other person might be thinking or feeling. Using functional magnetic resonance imaging (fMRI) we confirmed Brothers' prediction that the STG and amygdala show increased activation when using social intelligence. Some areas of the prefrontal cortex also showed activation. In contrast, patients with autism or AS activated the fronto‐temporal regions but not the amygdala when making mentalistic inferences from the eyes. These results provide support for the social brain theory of normal function, and the amygdala theory of autism.
Localization of synapse‐associated proteins during postnatal development of the rat retina
Authors: Koulen Peter
Published: March 2003

The expression of synapse‐associated proteins (SAPs) was monitored throughout postnatal development of the rat retina using specific antibodies and immunocytochemistry. The distribution of chapsin‐110/postsynaptic density protein (PSD)‐93, SAP90/PSD‐95, SAP97 and SAP102 immunoreactivity was characterized. All SAPs were found to be expressed in the inner plexiform layer (IPL) from birth on or soon after birth. With the exception of SAP97, the IPL labelling changed from a diffuse pattern staining the whole developing IPL to the typical adult punctate synaptic staining in the second postnatal week. Staining in the outer retina was first observed at postnatal day 5 (P5) for all proteins at the onset of outer plexiform layer (OPL) development. All SAPs showed a differential cellular and temporal distribution being either exclusively pre‐ or postsynaptically localized. Except for SAP90/PSD‐95, immunoreactivity was also detected in the nerve fibre layer throughout postnatal development. Possible functions of the early expression of SAPs well before differentiation and maturation of glutamatergic ribbon synapses are discussed.
Neuroprotective actions of peripherally administered insulin‐like growth factor I in the injured olivo‐cerebellar pathway
Authors: Fernandez A. M. , de la Vega A. Gonzalez , Planas B. , Torres‐Aleman I.
Published: March 2003

Exogenous administration of insulin‐like growth factor I (IGF‐I) restores motor function in rats with neurotoxin‐induced cerebellar deafferentation. We first determined that endogenous IGFs are directly involved in the recovery process because infusion of an IGF‐I receptor antagonist into the lateral ventricle blocks gradual recovery of limb coordination that spontaneously occurs after partial deafferentation of the olivo‐cerebellar circuitry. We then analysed mechanisms whereby exogenous IGF‐I restores motor function in rats with complete damage of the olivo‐cerebellar pathway. Treatment with IGF‐I normalized several markers of cell function in the cerebellum, including calbindin, glutamate receptor 1 (GluR1), γ‐aminobutyric acid (GABA) and glutamate, which are all depressed after 3‐acetylpyridine (3AP)‐induced deafferentation. IGF‐I also promoted functional reinnervation of the cerebellar cortex by inferior olive (IO) axons. In the IO, increased expression of bax in neurons and bcl‐X in astrocytes after 3AP was significantly reduced by IGF‐I treatment. On the contrary, IGF‐I prevented the decrease in poly‐sialic‐acid neural cell adhesion molecule (PSA‐NCAM) and GAP‐43 expression induced by 3AP in IO cells. IGF‐I also significantly increased the number of neurons expressing bcl‐2 in brainstem areas surrounding the IO. Altogether, these results indicate that subcutaneous IGF‐I therapy promotes functional recovery of the olivo‐cerebellar pathway by acting at two sites within this circuitry: (i) by modulating death‐ and plasticity‐related proteins in IO neurons; and (ii) by impinging on homeostatic mechanisms leading to normalization of cell function in the cerebellum. These results provide insight into the neuroprotective actions of IGF‐I and may be of practical consequence in the design of new therapeutic approaches for neurodegenerative diseases.
Stimulation of μ‐ and δ‐opioid receptors enhances phosphoinositide metabolism in mouse spinal cord: evidence for subtypes of δ‐receptors
Authors: Sánchez‐Blázquez Pilar , Rodríguez‐Díaz Marta , Frejo María‐Teresa , Garzón Javier
Published: March 2003

The accumulation of inositol phosphates (IPs) induced by agonist‐activated opioid receptors was analysed in mouse spinal cord slices pre‐labelled with myo‐[3H]inositol. Agonists showing selectivity to μ‐opioid receptors, morphine and [d‐Ala2,MePhe4, Gly(ol)5]enkephalin (DAMGO), promoted concentration‐dependent increases in the formation of IPs. The activation of δ‐opioid receptors by the selective agonists [d‐Pen2,5]enkephalin (DPDPE) and [d‐Ala2]deltorphin II produced similar increases in phosphoinositide (PI) metabolism. Pre‐treatment of the slices with pertussis toxin (PTX) blocked the effect of opioid agonists on IP production. The involvement of Gi/Go‐protein (guanine nucleotide‐binding protein) classes in this opioid effect is therefore suggested. The activity of the opioid agonists was reduced by the opioid antagonists naltrexone and naloxone. The antagonist at δ1‐receptors, 7‐benzylidenenaltrexone (BNTX), exhibited greater potency than the antagonists at δ2‐receptors, naltriben methanesulphonate (NTB) or naltrindrole 5′‐isothiocyanate (NT II), in reducing the activating effect of DPDPE on phosphoinositide metabolism. Conversely, NTB and NT II were more potent antagonists of the activity of [d‐Ala2]deltorphin II than BNTX. This work demonstrates the coupling of spinal μ‐ and δ‐opioid receptors to phospholipase C and the generation of IPs. It also provides biochemical evidence for pharmacological subtypes of δ‐opioid receptors in the activation of this signalling pathway.
Involvement of substance P in the anti‐inflammatory effects of the peripherally selective κ‐opioid asimadoline and the NK1 antagonist GR205171
Authors: Binder Waltraud , Scott Caroline , Walker Judith S.
Published: March 2003

We have previously shown that kappa‐opioids have antiarthritic properties. In this study, using two differently acting drugs (the peripherally selective κ‐agonist, asimadoline, and the NK1‐antagonist, GR205171), we have examined possible roles of the neuropeptide substance P (SP) in the pathogenesis and maintenance of experimental arthritis in rats. The anti‐inflammatory actions and the time dependence of these drugs were compared, and concentrations of SP determined in joint tissue. In untreated animals, SP levels in ankle joint tissue increased late in the disease (by day 21) but substantially lagged behind development of clinical disease. Prolonged (days 1–21 or days 12–18) but not early, short‐term (days 1–3) treatment with the NK1‐antagonist GR205171 (1 mg/kg/day i.p.) significantly attenuated joint damage; SP levels showed multiphasic dose dependence over the 21‐day treatment. The data suggest that GR205171 antagonizes the action of SP by presynaptic as well as postsynaptic mechanisms. Treatment with asimadoline (5 mg/kg/day i.p.) produced marked (and sustained) attenuation of the disease with all three time regimes. The effect of asimadoline on SP levels was time dependent: reduction of SP content after 3 days but an increase after 12 or 21 days treatment, paradoxically with clinical improvement in each case. Drug‐induced changes in SP content could follow from changed release or synthesis from either neural or immune cells. The results suggest that both drugs have potential therapeutic value at different stages of inflammatory joint disease.
Activation of the extracellular signal‐regulated kinase 2 by metabotropic glutamate receptors
Authors: Ferraguti Francesco , Baldani‐Guerra Barbara , Corsi Mauro , Nakanishi Shigetada , Corti Corrado
Published: March 2003

Activation of metabotropic glutamate receptors (mGluRs) leads to modulation of a variety of second messenger pathways probably including the mitogen‐activated protein kinase (MAPK) extracellular signal‐regulated protein kinases (ERK). MAPK play a key role in the control of cellular responses to changes in the external environment by regulating transcriptional activity and the phosphorylation state of several cytoplasmic targets. In this study, Chinese hamster ovary (CHO) cells permanently transfected with rat mGluR1a, mGluR2 and mGluR4 were employed as a model to examine the activation of MAPK by glutamate through mGluRs. All three mGluR subtypes rapidly stimulated ERK activation. In particular, mGluR1a and mGluR2 preferentially mediated phosphorylation and activation of ERK2 in a pertussis toxin (PTX)‐sensitive and concentration‐dependent manner. The activation was blocked completely by pretreatment with the antagonist (rs)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) or with the MEK inhibitor PD098059. Furthermore, mGluR1a‐mediated ERK activation was suppressed by the depletion of endogenous protein kinase C (PKC) activity and by the PKC inhibitors staurosporine and calphostin C, but not chelerythrine. When cAMP was elevated in mGluR2‐expressing cells, by forskolin or dibutyryl‐cAMP, slight elevation of ERK activity was observed. However, glutamate‐stimulated ERK activation remained unaffected. In these cells, the phosphatidylinositol 3 kinase (PI3K) inhibitor wortmannin produced a significant, albeit only partial, inhibition of mGluR2‐mediated ERK activation. These findings raise the possibility of a MAPK cascade involvement in glutamate‐dependent neuronal plasticity mediated through stimulation of mGluRs.
Involvement of the subthalamic nucleus in glutamatergic compensatory mechanisms
Authors: Bezard Erwan , Boraud Thomas , Bioulac Bernard , Gross Christian E.
Published: March 2003

The purpose of the present study was to investigate whether the subthalamic nucleus (STN) was implicated in the glutamatergic compensatory mechanisms which have been shown to mask the parkinsonian motor abnormalities at the end of the presymptomatic period in experimental parkinsonism. Using multiunit electrophysiological recordings, we follow changes of activity occurring in the STN and in both the pars externalis and the pars internalis of the globus pallidus of monkeys chronically intoxicated with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), according to a protocol designed to mimic the gradual evolution of dopaminergic neuronal death. STN activity augmented significantly in the course of treatment, even before the first appearance of clinical signs (P < 0.01). This result would indicate that the STN, which increases its level of activity even before the end of the presymptomatic period, is principally responsible for the instigation of glutamatergic compensatory mechanisms which allow the maintenance of the striatal dopaminergic homeostasis.
Positional specificities of retinal growth cones in the mouse superior colliculus
Authors: Llirbat Béatrice , Godement Pierre
Published: March 2003

In the developing retinotectal system, repulsive topographic tectal cues have been demonstrated to contribute to the final mapping. Here, we describe a novel response of nasal axons to growth‐promoting cues expressed by anterior tectal cells. In in vitro experiments, contact of fibres from the nasal (but not temporal) pole of the mouse retina with anterior (but not posterior) tectal membranes leads to their adopting very elongated and filopodial morphologies, and to increase their growth rates. As previously demonstrated, fibres from the temporal pole of the retina are collapsed by posterior tectal membranes in vitro. In addition, a study of retinal growth cone morphologies in vivo, at early stages of target invasion, shows that growth cones of nasal fibres have streamlined morphologies, usually indicative of active elongation growth modes, in the anterior part of the embryonic mouse tectum, and more elaborate morphologies posteriorly. Vice versa, temporal fibres have mainly elaborate growth cones anteriorly, and collapsed growth cones posteriorly.

These experiments demonstrate that nasal retinal fibres respond preferentially to permissive or growth‐promoting cues in the embryonic mouse tectal environment, both in vitro and in vivo. This phenomenon might contribute to ingrowth of retinal fibres in their target area, and to promote the homing of nasal fibres towards the posterior aspect of the tectum, which is their normal target region.
Regulation of the murine NMDA‐receptor‐subunit NR2C promoter by Sp1 and fushi tarazu factor1 (FTZ‐F1) homologues
Authors: Pieri Isabelle , Klein Matthias , Bayertz Christian , Gerspach Jeannette , van der Ploeg Anja , Pfizenmaier Klaus , Eisel Ulrich
Published: March 2003

We have cloned the 5′‐region of the murine N‐methyl‐d‐aspartate (NMDA) receptor channel subunit NR2C (GluRε3) gene and characterized the cis‐ and trans‐activating regulatory elements responsible for its tissue specific activity. By using a native ε3‐promoter/lacZ‐construct & various 5′‐deletion constructs, we compared β‐galactosidase expression in non‐neuronal NIH3T3 cells and in neuronal ε3‐gene‐expressing HT‐4 cells and show that large parts of the ε3 promoter are responsible for the repression of the ε3 gene in non‐neuronal cells. Deletion of exon 1 sequences led to an enhancement of ε3 transcription, suggesting a role of the 5′‐untranslated region in ε3 gene regulation. Sequence analysis of the promoter region revealed potential binding sites for the transcription factor Sp1, the murine fushi tarazu factor1 (FTZ‐F1) homologues, embryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF‐1), as well as for the chicken ovalbumin upstream promoter transcription‐factor (COUP‐TF). Electrophoretic mobility shift assays confirmed specific binding of Sp1, SF‐1 and COUP‐TFI. Whereas point mutation studies indicate that, in neuronal HT‐4 cells, Sp1 is apparently not critically involved in basal ε3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of ε3‐promoter‐driven reporter gene expression upon cotransfection of an SF1‐expression vector, which was reverted by deletion and point mutation of the SF1 binding site.
Decision‐related activity in the macaque dorsal visual pathway
Authors: Thiele Alexander , Distler Claudia , Hoffmann Klaus‐Peter
Published: March 2003

Brain areas at higher levels of cortical organization are thought to be more involved in decision processes than are earlier, i.e. lower, sensory areas. Hence, neuronal activity correlated with decisions should vary with an area's position in the cortical hierarchy. To test this proposal, we investigated whether a change in neuronal activity during error trials depends in a systematic way on cortical hierarchical position. While macaque monkeys discriminated the direction of moving visual stimuli, the activity of direction‐selective neurons was recorded in four extrastriate visual areas: V3A, the middle temporal area, the middle superior temporal area and the posterior part of the superior temporal polysensory area. Neuronal activity was significantly reduced in all areas when the monkeys made errors in judging the direction of stimuli moving in the preferred direction with low and intermediate luminance contrast. The amount of activity reduction was ≈ 50% in all of the visual areas. Thus, the activity on error trials is reduced in early visual processing, independent of the hierarchy in the dorsal visual pathway. The activity reduction depended on stimulus contrast and the direction of the decision relative to the stimulus motion. It was profound and significant in all areas at low stimulus contrast. However, it was nonsignificant at high stimulus contrast. Our data suggest that activity reduction on error trials is due to lack of attention in association with stimulus expectation.
The homeodomain transcription factors Islet 1 and HB9 are expressed in adult alpha and gamma motoneurons identified by selective retrograde tracing
Authors: von Steyern F. Vult , Martinov V. , Rabben I. , Njå A. , de Lapeyrière O. , Lømo T.
Published: March 2003

To study gene expression in differentiated adult motoneuron subtypes, we used fluorescent dextrans for both anterograde and retrograde axonal tracing in adult rat and mouse. Application of these dyes to the cut distal and proximal ends of small extramuscular nerve branches revealed both the peripheral ramifications and the cell bodies of subsets of motoneurons. We show that the soleus muscle is innervated by two nerve branches, one of which contains gamma motor and sensory axons but no alpha motor axons. By retrograde tracing of this branch, we selectively labelled gamma motoneurons. In adult rat, the nerves innervating the soleus and extensor digitorum longus muscles contain almost exclusively axons innervating slow (type I) and fast (type 2) muscle fibres, respectively. We selectively labelled slow and fast type motoneurons by retrograde tracing of these nerves. With immunocytochemistry we show that adult motoneurons express several homeodomain genes that are associated with motoneuron differentiation during early embryonic development. Combining selective retrograde labelling with immunocytochemistry we compared the expression patterns in alpha and gamma motoneurons. The homeodomain transcription factors Islet 1 and HB9 were expressed in slow and fast alpha motoneurons and in soleus gamma motoneurons. Motoneurons in each population varied in their intensity of the immunostaining, but no factor or combination of factors was unique to any one population.
Axoaxonic synapses on terminals of group II muscle spindle afferent axons in the spinal cord of the cat
Authors: Maxwell D. J. , Riddell J. S.
Published: March 2003

The purpose of the present study was to determine if terminals of identified group II muscle spindle afferents participate in axoaxonic synaptic arrangements and, if so, to investigate the transmitter content of presynaptic terminals in these arrangements. Group II muscle afferents supplying the gastrocnemius‐soleus or semitendinosus muscles were identified in adult cats and stained intra‐axonally with horseradish peroxidase. In total, three group II axons were labelled and processed for combined light and electron microscopy. Group II axons gave rise to collaterals which characteristically descended through the superficial dorsal horn and formed relatively sparse terminal arborizations in the dorsal horn (laminae IV and V) and more profuse arbors in the intermediate grey matter (laminae VI–VII). Forty boutons were examined through series of ultrathin sections and all but four were postsynaptic to other axon terminals. Occasionally, more than one axon was presynaptic to a single group II terminal. Immunogold studies showed that all axons in presynaptic apposition to group II boutons contained γ‐aminobutyric acid (GABA) and also that glycine was colocalized in the majority of these axons. This evidence suggests that transmission from group II muscle afferents is under strong presynaptic inhibitory control and that it is mainly the subgroup of GABAergic interneurons with colocalized glycine which mediate this inhibition. Seventeen group II boutons were components of synaptic triads where the presynaptic axoaxonic bouton formed a synapse with the same dendrite as the group II axon. Therefore, a proportion of the interneurons which form axoaxonic synapses with group II axons are also likely to have postsynaptic inhibitory actions on target neurons of group II afferents.
Effects of noradrenaline on frequency tuning of auditory cortex neurons during wakefulness and slow‐wave sleep
Authors: Manunta Yves , Edeline Jean‐Marc
Published: March 2003

This study shows the effects of noradrenaline (NA) on receptive fields of auditory cortex neurons in awake animals; it is the first one to describe the effects of NA on neurons in sensory cortex, in different natural states of vigilance. The frequency receptive field of 250 auditory cortex neurons was determined before, during and after ionophoretic application of NA while recording the state of vigilance of unanaesthetized guinea‐pigs. When NA significantly changed the spontaneous activity (85 out of 250 cells), the dominant effect was a decrease (61 out of 85 cells, 72%). When NA significantly changed the evoked activity (107 out of 250 cells), the dominant effect was also a decrease (84 out of 107 cells, 78%). During and after NA application, the signal‐to‐noise ratio (S/N, i.e. evoked/spontaneous activity) was unchanged, but the selectivity for pure‐tone frequencies was enhanced. When the effects occurring in wakefulness and in slow‐wave sleep (SWS) were compared, it appeared that the predominantly inhibitory effect of NA on spontaneous and evoked activity was present in both states. The S/N ratio was unchanged and the selectivity was increased in both states. However, during SWS, the percentage of cells inhibited by NA was lower, and the effects on the frequency selectivity were smaller than in wakefulness. In contrast, GABA produced similar inhibitory effects on spontaneous and on evoked activity during wakefulness and SWS. Comparisons with previous data obtained using the same protocol in urethane anaesthetized animals (Manunta & Edeline 1997) indicate that the effects of NA were qualitatively the same. Based on these results, we suggest that any hypothesis concerning the role of NA in cortical plasticity should take into account the fact that the predominantly inhibitory effects of NA lead to decrease the size of the receptive field.
Dimethylarginine dimethylaminohydrolase (DDAH) as a nerve‐injury‐associated molecule: mRNA localization in the rat brain and its coincident up‐regulation with neuronal NO synthase (nNOS) in axotomized motoneurons
Authors: Nakagomi S. , Kiryu‐Seo S. , Kimoto M. , Emson P. C. , Kiyama H.
Published: March 2003

As part of a project to identify genes up‐regulated by injury of the motor neuron, a clone encoding dimethylarginine dimethylaminohydrolase (DDAH) was isolated. This enzyme is known to metabolize methylarginines, which are endogenous inhibitors of NOS activity. DDAH may therefore contribute to the control of NO synthesis. The present study demonstrated that both DDAH and nNOS mRNAs are up‐regulated after axotomy in injured hypoglossal motor neurons. The profile of DDAH mRNA up‐regulation in the injured hypoglossal motor neurons paralleled that of NADPH diaphorase staining. While the expression of both DDAH and nNOS was upregulated in motor neurons following nerve injury, the normal distribution of DDAH and nNOS mRNAs in the noninjured central nervous system were distinctly different. We speculate that both genes are involved in the upregulation of NO production following nerve transection, although the role of NO in the process of nerve regeneration is so far unknown.
Spectral sensitivity in hemianopic macaque monkeys
Authors: Cowey Alan , Stoerig Petra
Published: March 2003

We measured the increment threshold sensitivity to 2°, 200‐ms targets presented at a lateral and radial eccentricity of ≈ 20–26° in both visual hemifields of three macaque monkeys whose left striate cortex had been removed 5 years earlier, and in one normal control. As in patients with blindsight, sensitivity of the hemianopic field for blue, green and red stimuli was reduced by as little as 0.5 log units. With increasing light adaptation from scotopic to mesopic to photopic levels, there was a progressive increase in the sensitivity to long wavelengths relative to that for short and medium wavelengths. This shift in relative sensitivity (‘Purkinje shift’) shows that rod and cone mechanisms operate in both the normal and hemianopic fields and that the sensitivity that remains following removal of striate cortex is not mediated exclusively by rods.
Dystrophin and beta‐dystroglycan in photoreceptor terminals from normal and mdx3Cv mouse retinae
Authors: Blank Martina , Koulen Peter , Blake Derek J. , Kröger Stephan
Published: March 2003

Mutations in the dystrophin gene cause muscular dystrophy as well as cognitive impairments, including an abnormal dark‐adapted electroretinogram. To investigate the basis for the ocular phenotype, we analysed dystrophin and the dystrophin‐associated protein β‐dystroglycan in retinae from mdx3Cv mice. This strain has a mutation in the dystrophin gene and abnormalities in the electroretinogram which are similar to those of muscular dystrophy patients. Despite an overall reduction of all dystrophin isoforms and of β‐dystroglycan in retinal tissue from mutant mice, we observed no apparent change in the histotypic layering of the retina, or in the ultrastructure of several specific cell types, including rods and cones. In retinae from wild type and mdx3Cv mice, dystrophin and β‐dystroglycan were concentrated in small extensions of rod and cone photoreceptor terminals protruding into the outer plexiform layer. Beta‐dystroglycan but not dystrophin was also clustered around the inner limiting membrane and the capillary basal laminae. While the labelling pattern around the basal laminae was not altered in the mutant mice, we found that the area as well as the intensity of the dystrophin and β‐dystroglycan immunoreactivity associated with the terminals of rod photoreceptors were severely reduced. The same parameters were much less affected in cone terminals. These results show, that dystrophin and β‐dystroglycan are differentially distributed in the retina, and that a severe reduction of dystrophin has no gross effect on retinal structure, but could influence intraretinal signalling at the level of the photoreceptor terminals. Moreover, the mutation in mdx3Cv mice has a selective effect on rods, providing an explanation for the altered electroretinogram.
C‐fos and c‐jun in the paraventricular nucleus play a role in regulating peptide gene expression, oxytocin and glutamate release, and maternal behaviour
Authors: Da Costa A. P. C. , De La Riva C. , Guevara‐Guzman R. , Kendrick K. M.
Published: March 2003

In sheep, birth leads to the induction of maternal behaviour through brain oxytocin release. Associated with these events is an upregulation of oxytocin, opioid and corticotrophin‐releasing hormone (CRH) gene expression, as well as that of the immediate early gene c‐fos in the paraventricular nucleus (PVN) of the hypothalamus. We investigated the role of c‐fos dimerizing with c‐jun in controlling the induction of maternal behaviour, altered peptide gene expression, and oxytocin and amino acid release in this region at birth. Fluorescence‐labelled antisense oligodeoxyribonucleotides (ODNs) against c‐fos/c‐jun were infused bilaterally in the PVN, via microdialysis probes with 100 kDa cut‐off membranes, and were incorporated into 50–60% of the cells. Compared with the control (scrambled) sequences, they significantly reduced basal concentration of glutamate (to 31.7% of baseline after 10 h) and prevented birth‐induced release of aspartate. In addition, antisense treatment reduced the birth‐induced increase in oxytocin concentration in the PVN, but not in blood. Although all the animals were fully maternal, the antisense treatment did reduce the peak expression of two components of maternal behaviour: low‐pitched bleats; and lamb sniffing. Finally, in situ hybridization histochemistry revealed that the antisense treatment significantly reduced the birth‐induced upregulation of c‐fos, oxytocin, CRH and preproenkephalin mRNA expression in the PVN, whilst not affecting that of arginine vasopressin. These results suggest that c‐fos/c‐jun transcription factors play a role in the birth‐induced upregulation of oxytocin, CRH and preproenkephalin gene expression, as well as on glutamate and oxytocin release in the sheep PVN.
The neuropeptide Y Y1 receptor is a somatic receptor on dorsal root ganglion neurons and a postsynaptic receptor on somatostatin dorsal horn neurons
Authors: Zhang Xu , Tong Yong‐Guang , Bao Lan , Hökfelt Tomas
Published: March 2003

Using indirect immunofluorescence, neuropeptide Y Y1 receptor (Y1 receptor)‐like immunoreactivity (LI) was localized close to the plasmalemma of small neurons in lumbar dorsal root ganglia (DRGs) and neurons in the inner lamina II of the lumbar spinal cord of the rat. Using confocal microscopy, colocalization of Y1 receptor‐LI and transferrin receptor‐LI, a marker for endosomes and coated vesicles, was observed in dot‐like structures along the plasmalemma. Under the electron microscope, Y1 receptor‐LI was localized in coated vesicles and endosomes, in the membrane of tubular cisternae, sometimes connected to multivesicular bodies, and in the plasmalemma. These complex distribution patterns may reflect receptor turnover and internalization processes. In the lamina II of the spinal dorsal horn, Y1 receptor‐LI was localized in the plasmalemma of neurons without any apparent association with paramembrane structures, as described above for the DRG neurons. Many dendrites were Y1 receptor‐positive, and some of them made synaptic contacts with unstained axonal terminals. In general, Y1 receptor‐LI was localized in the membrane outside the postsynaptic density. Double‐immunofluorescence staining showed that most Y1 receptor‐immunoreactive neurons in lamina II contained somatostatin‐LI. Both in DRG and dorsal horn neurons, the Y1 receptor thus seems to represent a postjunctional/postsynaptic receptor.