260 Abstracts / Journal of Physiology - Paris 99 (2006) 245–278

energy transforming system in the inner mitochondrialmembrane. Besides, until now no data are available aboutany interrelationship between protein phosphorylationand Ca2+-induced opening of the ‘‘permeability transitionpore’’ (PTP). The aim of the present study was to investi-gate protein phosphorylation in brain mitochondria andto find out whether they belong to systems that could be reg-ulated by phosphorylation/dephosphorylation, modulatedcalcium as a second messenger. Experiments were per-formed on isolated rat brain mitochondria (RBM).

Phosphorylation of 94, 66–67, 43, 28–30, 26, 17, 6.5 and3.5 kDa proteins has been observed in mitochondria. Ca2+

regulatory effects on phosphorylation of these proteinswere investigated in Ca2+-EGTA buffers. Protein phos-phorylation was found to be modulated by Ca2+ in thephysiological concentration range (10�8–10�6 M freeCa2+). Phosphorylation of the 17 and 21 kDa proteinswas increased within the 10�7–10�6 M free Ca2+ range,whereas an opposite effect was observed for the 3.5 kDa.These effects were strengthened in the presence of calmi-dazolium, an antagonist of calmodulin. It was suggestedthat Ca2+ in physiological concentrations, as a second mes-senger, can control phosphorylation of the 3.5, 17 and21 kDa phosphoproteins in RBM.

Participation of protein kinase-dependent phosphoryla-tion was investigated in the phenomenon of PTP. Dephos-phorylation of the 3.5 kDa peptide (which was identified assubunit c of F0F1-ATP synthase) and enhanced 32P incor-poration into the 17 and 21 kDa proteins were found underconditions of mPTP opening. These effects were preventedor reduced in the presence of cyclosporin A (CsA), inhibi-tor of PTP. It was shown that the drop of M (registratedwith TPP+-selective electrode under the same conditions)was correlated with a dephosphorylation of the 3.5 kDapeptide and an increased phosphorylation of the 17 and21 kDa proteins. CsA prevented mPTP opening and dropof M. Based on these data we suggested that phosphoryla-tion/dephosphorylation of proteins in RBM was connectedwith mPTP opening. It should be reminded that CsA isconsidered to block PTP by interacting with mitochondrialcyclophilin. It is well known, however, that this complex ofCsA and cyclophilin suppress also calcineurin. Calcineurinis calmodulin binding protein, representing a high percent-age of total brain protein. Several CaM-binding proteinswere earlier reported to be present in mitochondria. Usingboth polyclonal and monoclonal antibody against calci-neurin we discovered that brain mitochondria contain cal-cineurin. Taking into consideration that mitochondriacontain PKA and PKA anchoring proteins, which bindboth PKA and calcineurin, it would be logical to suggestthat regulation of mitochondrial protein phosphorylationand modulation of this phosphorylation by Ca2+ mightbe exert through PKA and calcineurin signalling system.

Supported by RFBR N 03-04-48213 and N 02-04-49701.

doi:10.1016/j.jphysparis.2005.12.045

Modulation of synaptic integration by nitric oxide in a

neuronal network of rat visual cortex

Nicolas Le Roux, Muriel Amar, Philippe Fossier

Laboratoire de Neurobiologie Cellulaire et Moleculaire,

CNRS UPR 9040 91198 Gif-sur-Yvette Cedex, France

E-mail address: leroux@nbcm.cnrs-gif.fr (N.L. Roux)

Neurons of the primary visual area continously adjusttheir patterns of discharges in accordance with the environ-mental luminance changes. This adaptation level is modu-lated by information exchanges between primary visualarea and associative area but also by numerous afferences(cholinergic fibers originating from the magnocellularnucleus, serotoninergic fibers issued from the raphe, norad-renergic fibers issued from the locus coeruleus).

The aim of our work is to analyze the effects of variousneuromodulators on a cortical neuronal network in orderto determine the resulting change in the excitability of layerV pyramidal neurons which elaborate the output signalsfrom the cortex. Using rat visual cortical slices, electricalstimulation in layer I evoked a composite synaptic responserecorded in layer V pyramidal neurons patched in wholecell configuration. In this ‘‘physiological context’’, therecorded signal reflects the process of synaptic integrationof excitatory inputs (glutamatergic) and inhibitory inputs(GABAergic) mainly received by the apical dendrite ofthe recorded pyramidal neurone. Briefly, the global evokedsynaptic conductance is further decomposed in excitatoryand inhibitory conductances assuming that reversal poten-tials are respectively equal to 0 mV for excitation and to�80 mV for inhibition. Given the experimentally derivedexcitatory and inhibitory conductance profiles, we canreconstitute the dynamics of the membrane potential.

Immunostaining of neuronal nitric oxide (NO) synthasereveals many positive neurons located between layer II andlayer V with a larger density in layer II/III. To analyse theeffects of the diffusible messenger NO on synaptic integra-tion, we first applied on the cortical slice L-arginin (the sub-strate of NO-synthase) or PAPA-NONOate (an exogenousNO donor). Excitatory and inhibitory conductances wereincreased but the excitability of the pyramidal neuron didnot change. However, L-NAME (a specific inhibitor ofNO-synthase) application induced an increase of neuronalexcitability showing that endogenous NO synthesis maydecrease the excitability of the pyramidal neurone. Thequestion was then to understand how the electrical activityof the network can trigger NO synthesis. We tried a proto-col of stimulation of the cortical network classically used toinduce LTP (long-term potentiation). This protocol consistin theta-burst stimulation (TBS) of layer I followed by therecording of responses to stimulations at low frequency(0.33 Hz) as in control conditions. An increase in excitatoryconductance (by 38%, Gexc) and in inhibitory conductance(by 55%, Ginh) were observed and these effects were main-tained from 15 to 60 min after the TBS. The analysis ofthese results shows that the excitatory inputs are attenuated

Abstracts / Journal of Physiology - Paris 99 (2006) 245–278 261

under these experimental conditions compared to con-trol conditions. Consequently, the pyramidal neuronexcitability was decreased. The inhibition of NO-synthaseprior TBS prevents any change of excitation and inhibi-tion (only 4% increase for Gexc and 1% increase forGinh).

It is concluded that in the neuronal network, NO canmodulate excitatory and inhibitory synaptic transmissionleading to a decrease in the excitability of the recordedpyramidal neurone in our experimental conditions. Thisaction of NO could take part in the modulation of visualcortex output signals.

doi:10.1016/j.jphysparis.2005.12.046

Chemokine fractalkine/CX3CL1 reduces glutamate-mediated excitotoxic death of cultured hippocampal neurons

Cristina Limatola a, Clotilde Lauro a, Myriam Catalano a,

Silvia Di Angelantonio a,b, Cristina Bertollini a,

Davide Ragozzino a,b, Fabrizio Eusebi a

a Istituto Pasteur-Fondazione Cenci Bolognetti &

Dipartimento di Fisiologia Umana e Farmacologia, Centro

di Eccellenza BEMM, Universita di Roma ‘‘La Sapienza’’,

00185 Roma, Italyb Neuromed, 86077 Isernia, Italy

E-mail address: cristina.limatola@uniroma1.it(C. Limatola)

Excitotoxicity is a form of cell death caused by excessiveexposure to glutamate (Glu), contributing to neuronaldegeneration in many acute and chronic CNS diseasesincluding ischemia. In this work we investigated the roleplayed by fractalkine/CX3CL1, a chemokine abundantlyexpressed throughout the CNS, on the survival of hippo-campal neurons exposed to the excitotoxic insult; and weshow that CX3CL1 reduces the Glu-mediated excitotoxic-ity through the activation of the ERK1/2 and PI3K/Aktpathways, being neuron survival abolished or reduced bythe specific inhibitors PD98059 and LY294002, respec-tively. We also report that CX3CL1 protects nerve cellsfrom death not only when administered before Glu appli-cation, reducing the Glu-activated whole-cell current, butalso when administered up to 8 h after excitotoxic stimu-lus. Evidence is also provided that CX3CL1 is releasedfrom hippocampal cells upon stimulation with high dosesof Glu, likely providing an endogenous protective mecha-nism against excitotoxic cell death.

doi:10.1016/j.jphysparis.2005.12.047

Glial soluble factors regulate the activity and expression of

the neuronal glutamate transporter EAAC1: Implication of

cholesterol

Benoit Canolle, Frederique Masmejean, Andre Nieoullon,

Sylviane Lortet

IC2N-CNRS, 13402 Marseille Cedex 20, France

E-mail address: lortet@lncf.cnrs-mrs.fr (S. Lortet)

A coordinated regulation between neurons and astrocytesis essential for the elaboration of the glutamatergic synapseand the control of extracellular glutamate concentration.Here, we investigated the influence of astrocytes on highaffinity Na+-dependant glutamate transport in primary neu-ronal cultures derived from rat embryonic cortices. The rateof glutamate uptake and the expression of the neuronal glu-tamate transporter EAAC1 were lower in neurons grown inthe absence of astrocyte (pure neuronal culture) than in neu-rons grown with few astrocytes (partially purified neuronalculture). Treatment of the pure culture with glial condi-tioned medium (GCM) induced an increase in glutamateuptake rate (increase in maximal velocity). Western blottingexperiments show that at least part of the effect of GCM isattributable to an increase in the total amount of the neuro-nal glutamate transporter protein EAAC1.

Furthermore, we show that cholesterol (10 lg/ml)increased also glutamate uptake activity when addeddirectly to neurons but with no effect on glutamate trans-porter expression.

The effects of GCM on glutamate transport activitywere reduced when cholesterol was removed from GCMby mevastatin treatment of the glial culture (low-choles-terol or LC-GCM) and restored when cholesterol wasadded to LC-GCM. Furthermore, the effects of GCMand cholesterol were not additive, confirming that theyshare a common pathway. Altogether, these results confirmthat cholesterol, present in GCM, regulates glutamatetransport in neurons.

In conclusion, we have provided new evidences for neu-ronal glutamate transport regulation by astrocytes andidentified cholesterol as one of the factors implicated in thisregulation.

Supported by CNRS and the French DGA-DSP (grantnumber 0034052004707501).

doi:10.1016/j.jphysparis.2005.12.048

Extrasynaptic NR2B and NR2D subunits of NMDA

receptors are shaping ‘‘superslow’’ afterburst EPSC inhippocampus

Natasha A. Lozovaya a, Sergei E. Grebenyuk a,

Timur Sh. Tsintsadze a, Bihua Feng b,

Daniel T. Monaghan b, Oleg A. Krishtal a

a Department of Cellular Membranology, Bogomoletz

Institute of Physiology, 01024 Kiev, Ukraineb Department of Pharmacology, University of Nebraska

Medical Center, Omaha, NE 68198, USA

E-mail address: nl@serv.biph.kiev.ua (N.A. Lozovaya)

In conditions of facilitated synaptic release, CA3/CA1 synapses generate anomalously slow NMDA